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            "item" : "Esther Pauline Lederer wrote under the pseudonym Ann Landers from 1955 until her death on June 22, 2002.",
            "count" : "17"
          }, {
            "item" : "Series of lectures given by McClintock at the California Institute of Technology.  The notes are partially written in McClintock's hand.",
            "count" : "16"
          }, {
            "item" : "As a public health officer of the Rockefeller Foundation, Gregg served in Brazil from 1919 to 1922 on hookworm and malaria campaigns.  This diary captures Gregg's observations during the first part of his time there and includes photographs, postcards, and letters he sent to various family members.",
            "count" : "16"
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            "item" : "Part of a longer manuscript draft that reflected McClintock's increasingly complex understanding of the suppressor-mutator as represented in her Carnegie Institution reports after 1956.. NOTE: Pagination is not sequential; several page numbers are duplicated. In addition, page numbers become erratic after page 206; subsequent pagination is extrapolated.",
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          }, {
            "item" : "This notebook lacks standard pagination and is only roughly chronological, with the arrangement based largely on experiment number. \"Page\" ranges given in the titles below are totally artificial.",
            "count" : "13"
          }, {
            "item" : "Taken during the first U.S. heart transplant at the Maimonides Medical Center, Brooklyn, New York.",
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          }, {
            "item" : "Published on the centennial of \"Darwin's Origin of the Species,\" Anfinsen's textbook was instrumental in establishing macromolecular structure information as a tool in evolutionary analysis.  The text was highly influential in shaping the perspectives of a generation of researchers who entered science in the decades following its release.",
            "count" : "13"
          }, {
            "item" : "On October 19, 1954, Presley, then a 34 year old mother of two, had her atrial septal defect repaired by Swan. She had a third child several months after the first anniversary of this life-changing surgery. She sent Swan an update each year on that anniversary from 1955 to 1995. Presley died in 1996, over 40 years after Swan repaired the defect.",
            "count" : "10"
          }, {
            "item" : "This notebook provides provides large insight into the sources of intellectual influence, reading, etc. in the fields of microbiology, chemistry, and genetics.  NOTE: Pagination is totally artificial and is based simply on the original order of the pages of the notebook.",
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          }, {
            "item" : "Sabin proved herself as an exacting anatomical investigator with this manual, published the year after she received her M.D.",
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            "item" : "NOTE: \"Genetics and Chemotherapy\" was eventually abandoned by Lederberg and Cavalli-Sforza and was never published.",
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            "item" : "Part of a flurry of activity by McClintock between 1948 and 1950, this follow-up to the long \"memo\" to Rhoades in early 1949 brings McClintock's complete vision of transposition into clearer focus.. NOTE: Page ranges in title are totally artificial and are based simply on the order of the pages of the notebook.",
            "count" : "8"
          }, {
            "item" : "Response from Lederberg is written on bottom of page.",
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            "item" : "Meeting:Conference of Coordinators of Regional Medical Programs, September/October 1968; Meeting Place:Arlington, VA",
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            "item" : "Meeting:Conference on Regional Medical Programs, January 1967; Meeting Place:Washington, DC",
            "count" : "7"
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            "item" : "Articles",
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            "item" : "Notes",
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            "item" : "Articles,Newspaper columns",
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            "item" : "Newsletters",
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            "item" : "Grant proposals,Excerpts",
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            "item" : "Posters,Slides (photographs)",
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            "item" : "Minutes (administrative records)",
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            "item" : "Awards and Prizes",
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            "item" : "Smoking",
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            "item" : "DNA",
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            "item" : "DNA, Recombinant",
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            "item" : "World War II",
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            "item" : "Regional Medical Programs",
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            "item" : "Cell Culture Techniques",
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            "item" : "Genetic Code",
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            "item" : "Regional Medical Programs,Congresses as Topic",
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            "item" : "United States Public Health Service",
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            "item" : "Peak Years and Decline (November 1970 to November 1974)",
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            "item" : "Organizational Period (October 1965 to September 1968)",
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          }, {
            "item" : "AIDS, the Surgeon General, and the Politics of Public Health",
            "count" : "267"
          }, {
            "item" : "After the Discovery: The Transforming Principle's Reception by the Scientific Community",
            "count" : "265"
          }, {
            "item" : "Extension and Expansion Period (October 1968 to October 1970)",
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          }, {
            "item" : "Demise and Legacy (After November 1974)",
            "count" : "209"
          }, {
            "item" : "Redefining the Federal Role in Public Health, Medical Research, and Education, 1949-1960",
            "count" : "189"
          }, {
            "item" : "Reproduction and Family Health",
            "count" : "181"
          }, {
            "item" : "\"Mr. Public Health\": Later Career, 1960-1967",
            "count" : "179"
          }, {
            "item" : "Retroviruses and the Genetic Origins of Cancer, 1970-1993",
            "count" : "168"
          }, {
            "item" : "Sir William: Regius Professor at Oxford, 1905-1919",
            "count" : "152"
          }, {
            "item" : "Congenital Birth Defects and the Medical Rights of Children: The \"Baby Doe\" Controversy",
            "count" : "148"
          }, {
            "item" : "Investigations of “The Narcotic Drug Problem” and further work on the Mental Examination of Aliens, 1923-1932",
            "count" : "134"
          }, {
            "item" : "Embryology and the Organization of DNA in Higher Organisms, 1966-1976",
            "count" : "130"
          }, {
            "item" : "Antigens and Antibodies: Heidelberger and The Rise of Quantitative Immunochemistry, 1928-1954",
            "count" : "129"
          }, {
            "item" : "\"Father of Modern Medicine\": The Johns Hopkins School of Medicine, 1889-1905",
            "count" : "111"
          }, {
            "item" : "Envisioning Viruses: Birkbeck College, London, 1953-1958",
            "count" : "109"
          }, {
            "item" : "From Neuroblastoma to Homeobox Genes, 1976-1992",
            "count" : "107"
          }, {
            "item" : "At the Rockefeller Institute for Medical Research, 1925-1938",
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            "item" : "Reproduced with permission of Elizabeth M. Davis.",
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            "item" : "Courtesy of Katrin DeBakey.",
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          "largest" : "Zz gpoKeF ELLER Tipgy > \\\\JUN.16 1980) © CE OF THe PRESSE      FOOD SAFETY COUNCIL L  1725 K St., N.W.—Suite 306 Washington, D.C. 20006     J. B. Cordaro ° 202-463-7508 Executive Director June 12, 1980  Dr. Joshua Lederberg President  The Rockfeller University 1230 York Avenue  New York, NY 10021  Dear Dr. Lederberg:  I wish to express my appreciation to you for taking some much of your valuable time to participate in our peer review of the report of the Social and Economic Committee of the Food Safety Council. Although the Committee will be using your comments in the discussion for their specific purpose in revising their report, I also found the comments to be very helpful in my job of integrating their report along with the report of the Scientific Committee. As Paul Hopper promised, I will send you copies of the report of the Scientific Committee when it is published on or about June 15th. If I can be of any assistance to you, please do not hesitate to call upon me.  Sincerely,  J. . Cordaro",
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            "item" : "20th May, 1974 Dear Tom, Thank you for your kind words about my article and especially for your reminiscences.  It is really rather surprising how Chargaff, while frequently being witty, has almost always been wrong.  Pirie, though rather different in character, has always been obstinately sceptical.  I had no idea that he said that about the genetic code in 1963 and would be glad to have the reference. As you probably know, I first realised that nucleic acid would have a lot of evolutionary information in 1958. I have always been mildly supprised that I subsequently have done nothing about it but thanks to your efforts and those of several others the matter seems to be in good hands. I have given up travelling for the time being so that I can lead a quiet life, so we are unlikely to meet in the near future unless you come to England. Best wishes, F.H.C. Crick",
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            "item" : "March 6, 1957 Dear Joshua I just wish to say how very much I hope that you accept the offer to come to Harvard and become a member of this department. I think I can very fairly say that the spirit of this department is changing very rapidly and that the President and Dean Bundy will support very strongly our efforts to make this the most alive laboratory of experimental Biology in this country. There is certainly now no need to convince the people here that modern biology is here to stay and that new appointments must be made in that direction. I certainly have had no regrets about coming here -- In fact I'm terribly pleased to be here. When I was raised in the mid-west I was frequently told that Harvard was a frightfully stuffy place and I could never imagine myself wanting to be a part of it. This, however is just not true and it would be very hard to find more genuine people than John Edsall or Kenneth Thimann. I have been and in the future will be even more strongly connected with John Edsall. We now have just received a very large grant of 180,000 dollars to build up a modern laboratory for protein chemistry and it is my intention to concentrate as much as possible upon precise characterizations of the proteins and nucleic acids of viruses and bacteria. We also have an application into the NSF to procure a new Siemens, by far the best EM now [END PAGE ONE] [BEGIN PAGE TWO] available. In the long term, however, I feel the future of this department must be in embryology (in the broad sense), and in those problems which relate to growth. It is the very important whom we get along these lines. We have tried to persuade Aston [?] Mitchison [?] to come here, and despite his first refusals, we have not given up hope. I certainly feel he is the best person we could get, especially as he comes from genetics. There is also the possibility that Moscone will be here, at least for next year. I am finding the Cambridge and the Boston region a very pleasant environment to live in. I know that Madison is very nice but I think this is equally as good, and certainly is some cultural equal music there is no freer place to live. There is also the fact that Harvard is a very great university -- there is something very comforting about being a member of its faculty -- there is a security which very few universities can offer. The University of Chicago is one, but beside it, there are very few. I am quite aware that we have much to gain if you come here -- on the other hand, I would also press that you and Esther would both benefit for I feel most strongly that Harvard and its tradition for learning and liberal ideas is a most splendid place to be. With best wishes to both you and Esther Jim",
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            "item" : "Dr Oftedal 2/22/82 The genetic effects of nuclear war are of much less concern to me than the immediate (and delayed cancer) somatic ones Hiroshima/Nagasaki show this all too clearly. Long discussed UN/AEC studies showed that survivable doses of radiation (say 100-300 rem) barely double the existing genetic load, and should have an impact on at most 1-3 percent on following generations against a nearly 50 percent lethality on those directly exposed. Of course, if millions were to be exposed to only sublethal doses, that would loom as a larger catastrophe; but in almost every imaginable (?) scenario the somatic effects would greatly exceed the genetic ones. J.L.",
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            "item" : "(From Wilfred Razzell)                                     P.O. Box 875                                     Suva, Fiji                                     30/9/80  Dear Josh:  .fi      I suppose it's a natural combination of your sensitivity and inherent kindness; in any case your letter was extremely encouraging -- very much appreciated.  I had spent 8 years in Vancouver turning a 50 man laboratory in the support of the commercial fishing industry (a $250 million/yr. business there) when the cost-cutters in Ottawa decided to cut back and let the industry do it themselves. They won't, can't and didn't, so I left for the job of creating a new international fisheries organization: a lonely business, albeit fascinating, in which I was the focus of considerable uninformed sniping and my every move second-guessed by a Committee of 12 governments.  By April, the political tricks got to me and I just had not the heart to fight off another.       Now that I've rested, I'm not sure I want to take on any large task; perhaps I needed a sabbatical along the way and just kept missing it?  Anyhow, if the job at CSIRO comes my way it would appear to serve the same purpose: renew my interest and abilities (I hope!) in lab research and give me time to re-establish my place as a working scientist - which is what I conclude I enjoy most.       Over the years I have needed a physical outlet, too, the best being near the water: snorkeling and sailing.  Perhaps it provides a good recuperative milieu to dissipate my fears of failure and allow ideas to sort themselves out.       For these several reasons, considering my refresher period will be in the N. American winter, probably, I'd prefer to try California. Since writing to you I've written to Bill Rutter in San Francisco, who recently published on the amylase mRNA and is a close colleague; so once I hear from CSIRO I'll get serious.  Otherwise, your suggestion of Gobind's lab is good.       For these reasons, too, I hesitate to work into the Barrier Reef organization: it requires a stretch of my abilities which I don't feel ready to exert again yet.  No problem on the \"utilization\" side, but a strong feeling that the biologists on the \"conservation\" side will prove as difficult to cope with as they did in Canada -- and run the show to the frustration of everyone else.  But of course I'll write to Sir Gustav in Melbourne and see him as soon as I have the chance, it was again thoughtful of you to discuss my interests with him, and I thank you for the information.       I've enclosed a c.v., list of publications, and names of references.  Should I be unsuccessful in getting to Australia, I'll just have to sort out the problems of getting Evelyn into the U.S.A. -- mostly a matter of delays for her, I think.  Because of her -- that's important to me, as much as work, I realize -- I wouldn't go to Alberta, although I know that Al Patterson's job as Director of the Cancer Research Lab is vacant and they have a good opinion of my ability to do it.       It would obviously be easier on me, or for me, if I were the kind of person to whom work was everything and stressless; I'm not, I now realize, so try to act accordingly.       When I was in Edmonton, I went to the ASM meeting in New York ('67) and there Seymour and I had a chat with Luria (in his suite: President of the Society at the time).  It was a warming and satisfying moment, and I expect when I see you again the same will be true.  I look forward to that, thank you, wherever we may meet.       I did check at the Fiji School of Medicine on your interest in developments since 1957.  It was Dr. Edmonds who started paramedical training.  In 1961 the name was changed to Fiji School of Medicine and various paramedical personnel were renamed as time went on (in 1964 \"medical officers\" was coined to designate those who would have charge of village clinics, for example).  In 1975, a Medical Assistant program was introduced.  Dr. Pathik, the Registrar (I gather) was the man I spoke to.       It was good of you to send me the issue of \"News and Notes\" to bring me up to date on your family.  I gather you met Marguerite when you were both at Stanford; I'm happy for you, Josh, and wish you continued good fortune.       Finally, I must claim I did recall you had gone East -- but after I wrote my letter -- since I remember writing you a note at the time (but not to where!). I notice my memory often works in compartments: when accustomed to one, it is often difficult to re-enter another which previously was equally accessible.  Then, when the entry is achieved after all, innumerable details become readily accessible again!                                            Best regards,                                               Bill .br  .nf (From Wilfred Razzell)                                             P.O.Box 875                                             Suva, Fiji                                             27/08/80  Dear Josh:  .fi      This letter will, I hope, find you in good health and spirits. I regret my travels in and through the West Coast had not included a stopover in Palo Alto, but I seem to have been everywhere else!       As you may remember, after Mary was ill in Alberta I returned to Vancouver to resettle the family and we stayed there until the children were almost through university.  The Canadian government, in a fit of economy and short-sightedness, cut out most of the technological research across the country - and not incidentally, my job.  So I took on the task of guiding the creation of the South Pacific Forum Fisheries Agency; first, from Suva and in April 1979, set up headquarters in Honiara, Solomon Islands.  Once all was organized, and staffed, the Island countries (led by W. Samoa and Fiji) decided they wanted an Islander as the Director, so I resigned from the whole operation. Too much politics has spoiled my enthusiasm for fisheries management.         Mary decided not to leave Vancouver (where my job proposals were minimal) and we are getting divorced.       Being now on my third career -- and trying not to appear too erratic -- I feel free to reconsider what it is I enjoy and want to do.  I've written to Alex Zaffaroni to say I'd like to get back to lab work and you may hear of that.  However, in case prospects in California don't prove to be what I hope, I've applied to CSIRO in Australia, too.  Their Molecular and Cellular Biology Unit wants someone to work on recombinant DNA procedures to look at expression of eukaryotic genes, and now I have the time to skip deliberately into something interesting (and the maturity to keep my nerve -- which I didn't have unfortunately, at Syntex in 1964) I'd consider that seriously.       I've told them I'd obtain the lab training required to bring myself up to date, at my own expense, if they offer me the job (which is rather more 'junior' than I've had, but one which shouldn't be a strain), and it seems like Paul Berg or Bob Sinsheimer might be good people to ask about that -- for a month or so of space as a trainee.  What do you think?       I've also had to take the liberty of referring to you as one who knew of my work on phosphodiesterases and nucleases when I was active in that area , so they may write you if they take my application seriously.  I'm sorry to impose on you again, but most other colleagues are not in the position of being my \"senior\" - and that would matter to CSIRO, I think. (Of course, I realize you can refer in the past tense).       It is appealing to consider relocating in California, but to Australia there are fewer immigration problems for me and my fiancee -- who is a Solomon Islander.       Should I come to California for a job, or study, I'd be very pleased to see you again, if you have the time, to talk about old friends.                                           Best regards,                                           Bill Razzell",
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            "item" : "-A PROGRAM IN GENETICS AND MOLECULAR BIOLOGY  Genetics Department Stanford University School of Medicine  October 1968 - October 1973  Submitted by:  fo ) f L a {  Ne 5 \\\\ was Pea VW Pb ae VO oe Ne PE eet  7     Aoshua Lederberg, Princ",
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            "item" : "Copyright © 1994 by the Genetics Society of America  Perspectives  Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove  The Transformation of Genetics by DNA: An Anniversary Celebration of AVERY, MACLEOD and McCarty (1944)  Joshua Lederberg'  The Rockefeller University, New York, New York 10021-6399  HE publication of AVERY, MACLEOD and Mc- Carty (1944) just 50 years ago marked the opening of the contemporary era of genetics, its mo- lecular phase. The reverberations continue, now dom- inating large sectors of biomedical science and bio- technology, and have established the centrality of genetics in biological thought (LEDERBERG 1959, 1993a).  AVERY ét al. (1944) can be dissected into the follow- ing observations, claims and tacit extrapolations, which may be paraphrased as:  a) Certain bacteria (pneumococci) have clonally in- herited attributes, notably serospecific polysaccharide capsules. These are associated with virulence and can be selected accordingly, by inoculation into mice or by serological reagents.  b) The genetic Anlage of these attributes can be transferred from clone to clone by cell-free extracts: the phenomenon of transformation. The transformed cells faithfully transmit their new phenotype to suc- ceeding clonal generations, as had been established by GRIFFITH (1928) with crude, heat-killed cell suspen- sions.  c) The chemical structure of that transforming prin- ciple is DNA, to the exclusion of protein or other macromolecules.  Founded on these claims, the following radical ideas emerged:  d) Bacteria have discrete, autonomous genes anal- ogous to those of higher life forms (viz. Drosophila).  e) The gene is DNA, and the transformation phe- nomenon affords the first bioassay for genes extract- able in vitro.  f) Accordingly, bacteria might be favored subjects for genetic investigation and eventually for technolog- ical application of molecular genetic science.  * Raymond and Beverly Sackler Foundation Scholar.  Genetics 136: 423-426 (February, 1994)  I recite these principles with some nostalgia: they are precisely how they came across to me as an under- graduate already working on Neurospora in FRANCIS RyAn’s laboratory at the Columbia University Zool- ogy Department in Morningside Heights, New York’s upper West Side. Elsewhere, I have noted how they vectored my own career aspirations into the pioneer- ing of bacterial genetics (LEDERBERG 1987).  Studying in the academic archipelago called New York, I was uniquely well situated to observe and sometimes participate in the debate. The Rockefeller Institute was across town, overlooking the East River near the 59th Street bridge. ALFRED MIRSKY, likewise a senior member there, was a frequent visitor to Columbia to collaborate with ARTHUR POLLISTER. From 1942 on I heard a good deal of the progress in AveERyY’s laboratory. Reprints of the AVERY et al. (1944) article were circulated in the department. | borrowed one from HARRIETT TAYLOR (later EPHRUSSI), a graduate student working on yeast bud- ding kinetics, who would shortly join AVERY’s labo- ratory for her postdoctoral research. My personal  exclamatory notes were “. . . unlimited in its implica- tions, . . . Direct demonstration of the multiplication of transforming factor . . . Viruses are gene-type com-  ”  pounds [sic]... .  While Mirsky was the principal herald, he was also a dogged critic of the claim that DNA, alone, had been proven to be the exclusive chemical substance of transforming activity (MIRSKY and POLLISTER 1946). That was indeed a difficult proposition: AVOGADRO’s number is a formidable protagonist in that contest. My stance was sympathetic to Mirsky’s: I felt that so crucial a claim should not be impulsively engrafted into the corpus of science as if by first intention. More important than doctrinal conversion was that the issue was squarely on the table and could be settled by 424 J. Lederberg  overwhelming experimental analysis. Previous fias- coes had darkened the history of biopolymers: WILLS- TATTER’s claim of enzymatic activity of protein-free preparations and WENDELL STANLEY’s initial claim in 1935 that crystallized tobacco mosaic virus was a pure protein. AVERY himself was an epitome of caution, having had to weather similar skepticism of his con- clusion that pneumococcal polysaccharide, devoid of protein, was a type-specific antigen. The main fruit of the debate was to stimulate a range of further enqui- ries: CHARGAFF on the base composition of DNA and my own on other modes of gene recombination in E. co. And MACLYN McCarty, later joined by ROLLIN HoTcuHkKIss, added much to the repertoire of enzy- matic and analytical refinements for the exclusion of protein from the DNA preparations (McCarty 1946; HOTcHKIss 1979). WATSON and CRICK perhaps owe some debt to MIRSKy’s obstinacy. PAULING, who had collaborated with MIRSsky on protein denaturation, was led to delay entering the marathon for solving the DNA structure (WATSON 1968).  Conceptually, DNA in the 1940s was an unlikely candidate for biological specificity. The root problem was the unavailability of any homogeneous sample of DNA appropriate for detailed chemical analysis. This would have to await studies with tiny DNA viruses, and much help from precisely targeting restriction enzymes. DNA was then believed to be a monotonous structure, perhaps even merely a tetranucleotide, har- kening back to PHOEBUS LEVENE’s analyses. The pro- tein-enthusiasm evoked by the successful crystalliza- tion of enzymes in the 1930s then dominated most biochemists’ attention.  In fact, DNA was more popular at the turn of the century: “A tempting hypothesis, suggested by Math- ews on the basis of Kossel’s work, is that nuclein, or one of its constituent molecular groups, may in a chemical sense be regarded as the formative centre of the cell which is directly involved in the process by which food-matters are built up into the cell-sub- stance” (WILSON 1906, p. 340).  By 1925, WILSON was discouraged and misled by the apparent loss of chromatin (basophilia) in the nucleus of the growing oocyte:  These facts afford conclusive proof that the individuality and genetic continuity of chromosomes does not depend upon a persistence of ‘chromatin’ in the older sense (ie., basichromatin). It is the expression of a morphological or- ganization that is not destroyed by those chemical and physical transformations that lead to a netlike structure and  a change from the basophilic to the oxyphilic condition (WILSON 1925, p. 351).  Just as these words were being written, ROBERT FEULGEN developed the fuchsin-bisulfite cytochemical reaction that offered the first authentic cytochemical indicator for DNA and restored confidence in the  continuity of the DNA content of the chromosome. (CLARK and KASTEN 1983).  The biological interpretation of the pneumococcus transformation was also fraught with uncertainty. DOBZHANSKY, and later BOIVIN, persisted in describ- ing the phenomenon as a “directed mutation,” and it was given overtones of “cytoplasmic inheritance” by SONNEBORN-these were all rhetorical devices in- tended to seal off a vaguely understood phenomenon from the sureties of chromosomal inheritance. Noth- ing was known of chromosomes or genes in bacteria at that time: a certain leap of faith was required to relate the transformation (and therefore, in turn, DNA) to mendelizing genes. For many years, the only marker studied was the capsular polysaccharide. In that setting, even HARRIETT TAYLOR (1951), report- ing from the Rockefeller Institute, remarked, “No bridge can be seen leading over into classical genetics,” and in private correspondence criticized my own ef- forts to do precisely that. Among early comments from geneticists, MULLER’s (1947) was the closest to the mark:  ... the most probable interpretation of these ... pneu- mococcus results then becomes that of [a] type of crossing over, though on a more minute scale . . . [involving] viable bacterial ‘chromosomes’ or parts of chromosomes [pene- trating] the capsuleless bacteria and in part at least taken root there .. . However, unlike what has so far been possible in higher organisms, viable chromosome threads could also be obtained from these lower forms for in vitro observation,  chemical analysis, and determination of the genetic effects of treatment.  In a retrospection over prior hypothetical interpre- tations of the transforming principle, seven alterna- tives could be listed (LEDERBERG 1956):  1, It was a specific mutagen with a special ability to direct a particular gene to mutate in a definite direc- tion.  2. It was a polysaccharide autocatalyst (perhaps as a complex with DNA) that primed an enzymatic reac- tion for polysaccharide synthesis.  3. It was a bacterial virus, which on infecting the bacteria provoked capsular synthesis as a host reac- tion.  4. It was an autonomous cytoplasmic gene or a morphogenetic inducer.  5. It acted at a distance without penetrating the bacterium.  6. It was a fragment of the genetic makeup of the bacterium, the only one to have been tested to that time.  7. It was an element sui generis for which no general conception should be adduced.  Some of these were not logically distinguishable, but were no less strongly held semantic strongholds. The notion that the transformation was indeed a gene transfer by DNA was eventually solidified by new Perspectives 425  work with markers other than the capsule, and espe- cially by the linkage of mannitol fermentation and streptomycin resistance (HOTCHKISS and MARMUR 1954). It was also bolstered by other phenomena of gene transfer, such as conjugal exchange in E. colt (LEDERBERG 1947) and virus-mediated transduction in Salmonella (ZINDER and LEDERBERG 1953). Finally, the monopoly of the pneumococcus on transforma- tion-and this was a notoriously difficult experimental system—was broken by ALEXANDER and LEIDyY’s (1951) report on Hemophilus, so that a stream of other workers could provide mutual confirmation and reinforcement about the biological interpretations.  The debate about DNA chemistry petered out by sheer exhaustion of the critics and by the conceptual plausibility of DNA as gene, introduced by WATSON and CrIcK’s double helix model (1953). HERSHEY and CHaASsE’s (1952) study of the injection of phage DNA into F. coli lent further support to the “DNA only” view; however, this was quantitatively less rigorous than McCarty and HOorcnkIss’ prior work on the pneumococcus. Even after 1953, HERSHEY himself was still referring to something more than DNA as a possibility. It might be said that rigorous proof was concluded only with the enzymatic and chemical syn- thesis in vitro of biologically active DNA (KORNBERG 1960; KHORANA 1969).  AVERY et al. (1944) was originally published in a medical journal of The Rockefeller Institute that was not habitually read by geneticists of that time. This has led some commentators to compare the launching and reception of AVERY e? al.’s claims to the so-called prematurity of MENDEL’s ideas in the last third of the 19th century (STENT 1972; WyaTr 1975). Mendel was little known and for the most part ignored by his contemporaries. But I would argue that the critical reception initially given to AVERY et al. (1944) exem- plifies the critical scientific method at its most func- tional (MERTON 1973), Far from being ignored, the paper enjoyed almost 300 citations between 1945 and 1954 (Science Citation Index 1945-1954), not to mention many more earned by McCarTy’s elabora- tions (1946). The first in GENETICS was LEDERBERG (1947). The Annual Review of Genetics did not exist at that time, but SEWALL WRIGHT (1945) reviewed the work in the Annual Review of Physiology and it was also noted by no Jess than three reviewers (GULLAND, MUELLER and KALCKAR) in the Annual Review of Bio- chemistry that same year. It was so well known during that decade that, as I can tell from my own experience, it was often cited by indirection, without specific ref- erence (¢.g., LEDERBERG and TATUM 1946; LEDER- BERG 1959).  To return, then, to attributions of “prematurity,” this might mean either that the data do not exist to explain all of the paradoxes and challenges of a new  discovery, and the claims then meet critical resistance, or that the audience is incapable of understanding the challenge. The touchstone is plainly the operational reaction. For AVERY et al, (1944), and MCCLINTOCK (1953) as well, this comprised open controversy and active inquiry. For MENDEL, this was oblivion and a long delay before rediscovery. Happily, such examples are few and far between. In the long run of scientific advance, for a work to be ignored is perhaps only slightly worse than to be swallowed whole. A lot of revision looms ahead even for our well established dogmas (LEDERBERG 1993b).  That AVERY and his colleagues failed to win the Nobel Prize has repeatedly been a subject of critical remark. WENDELL STANLEY (1970) openly apologized for not having been more attentive to that lack of recognition, after he had won his own prize in 1946. In 1958, it came to me to plan my own Nobel lecture, the first in the field of genetics since MULLER in 1946. Rather than recite my own work on bacterial recom- bination, I thought it more important to acknowledge how genetics had been totally transformed by these discoveries: this was embodied in the lecture entitled “A view of genetics” (LEDERBERG 1959). AVERY had consummated this research at the very end of his career and died in 1955 before a full round of rec- ognition could be fulfilled. The survivor of that team, MacLyn McCarty, has written a vibrant memoir (1985) that is a model for expert and methodical tackling of very difficult technical problems. It dis- plays the highest ideals of the scientific personality and leaves no doubt of the importance of his role, together with that of his colleagues, in the pivotal discovery of Twentieth Century biology.  Spanning more than a decade of often frustrating effort, that discovery is an outstanding example of the feedback of clinically motivated inquiry to the most basic issues of fundamental biomedical science (BEECHER 1960). Genetics, especially as we explore the human genome, will be fraught with many more like opportunities, and precisely because of their per- vasive applications with commensurate dilemmas. Many institutional arrangements today nurture such transdisciplinary and vertically integrated research, which is often the arena of the most revolutionary advances. Before the federalization of biomedical re- search financing since World War II, The Rockefeller Institute was very nearly the only site where this could have taken root.  BIBLIOGRAPHICAL NOTE  A vast effort of scholarship and erudition on the history of DNA offers easily accessible guides to the primary sources; see: OLBY (1974, 1990), JuDSON (1979), PORTUGAL and COHEN (1977), CLARK and KASTEN (1983), Moore (1985), Sapp (1990), WoLFF and LEp- ERBERG (1944) and WATSON and Tooze (1981). In addition, mem- oirs by Dusos (1976), HoTcukiss (1979), McCarty (1985), CHar- GAFF (1978), KORNBERG (1989), WATSON (1968) and Crick (1988) 426 J. Lederberg  add indispensable personal perspectives. I have referred to primary sources primarily to document or accent particular items under debate.  LITERATURE CITED  ALEXANDER, H. E., and G. Lewy, 1951 Determination of inher- ited traits of H. influenzae by desoxyribonucleic acid fractions isolated from type-specific cells. J. Exp. Med. 93: 345-359.  Avery, O. T., C. M. MACLEop and M. McCarty, 1944 Studies on the chemical nature of the substance inducing transforma- tion of pneumococcal types. J. Exp. Med. 79: 137-158.  BEECHER, H. K., 1960 Disease and the Advancement of Basic Science. Harvard University Press, Cambridge, Mass.  CuHaroarr, E., 1978 Heraclitean Fire. Sketches from a Life before Nature. The Rockefeller University Press, New York.  Ciark, G., and F. H. KASTEN, 1983 History of Staining. Williams & Wilkins, Baltimore.  Crick, F., 1988 What Mad Pursuit. A Personal View of Scientific Discovery. Basic Books, New York.  Dupos, R. J., 1976 The Professor, The Institute, and DNA. The Rockefeller University Press, New York.  GriFFItH, F., 1928 The significance of pneumococcal types. J. Hyg. 27: 113-159.  HERSHEY, A., and M. CHASE, 1952 Independent functions of viral proteins and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36: 39-56.  Hotcnukiss, R. D., 1979 The identification of nucleic acids as genetic determinants. Ann. N. Y. Acad. Sci. 325: 321-342. Horcnkiss, R. D., and J. MARMUR, 1954 Double marker trans- formations as evidence of linked factors in desoxyribonucleate  transforming agents. Proc. Natl. Acad. Sci. USA 40: 55-60.  Jupson, H.F.,1979 The Eighth Day of Creation. Simon & Schuster, New York.  KuHorana, H. G., 1969 Nucleic acid synthesis in the study of the genetic code, pp. 196-220 in Les Prix Nobel en 1968. Impri- merie Royale P. A. Norstedt & Soner, Stockholm.  KorNBERG, A., 1960 The biologic synthesis of deoxyribonucleic acid, pp. 165-179 in Les Prix Nobel en 1959. Imprimerie Royale P. A. Norstedt & Soner, Stockholm.  KORNBERG, A., 1989 For Love of an Enzyme. Harvard University Press, Cambridge, Mass.  LEDERBERG, J., 1947 Gene recombination and linked segregations in Escherichia coli. Genetics 32: 505-525.  LEDERBERG J., 1956 Genetic transduction. Am. Sci. 44: 264-280.  LEDERBERG, J., 1959 A view of genetics, pp. 170-189 in Les Prix Nobel en 1958. Imprimerie Royale P. A. Norstedt & Soner, Stockholm.  LEDERBERG, J., 1987 Genetic recombination in bacteria: a discov- ery account. Annu. Rev. Genet. 21: 23-46.  LEDERBERG, J., 1993a What the double helix (1953) has meant for basic biomedical science. A personal commentary. J. Am. Med. Assoc. 269: 1981-1985.  LEDERBERG, J., 1993b The anti-expert system: hypotheses an AI program should have seen through, in Artificial Intelligence and Molecular Biology, edited by L. HUNTER. AAAI Press, Menlo Park, Calif. (in press).  LEDERBERG, J., and E. L. Tatum, 1946 Gene recombination in Escherichia colt. Nature 158: 558.  McCarty, M., 1946 Chemical nature and biological specificity of the substance inducing transformation of pneumococcal types. Bacteriol. Rev. 10: 63-71.  McCarty, M., 1985 The Transforming Principle. W. W. Norton, New York.  McCLINTOcK, B., 1953 Induction of instability at selected loci in maize. Genetics 38: 579-599.  MERTON, R. K., 1973 The Sociology of Science. Theoretical and Empirical Investigations. University of Chicago Press, Chicago.  Mirsky, A. E., and A. W. POLLISTER, 1946 Chromosin, a desox- yribose nucleoprotein complex of the cell nucleus. J. Gen. Physiol. 30: 117-148.  Moore, J. A., 1985 Science as a way of knowing—genetics. Am. Zool. 25: 1-165.  MULLER, H.J., 1947 The gene. Proc. R. Soc. Lond. Biol. 134: 1- 37.  Oxsy, R. C., 1974 The Path to the Double Helix. University of Washington Press, Seattle.  Otsy, R.C.,1990 The molecular revolution in biology, pp. 503- 520 in Companion to the History of Modern Science, edited by R. C. OLBy et al. Routledge, London.  PoRTUGAL, F. H., and J. S. COHEN, 1977 A Century of DNA. MIT Press, Cambridge, Mass.  Sapp, J., 1990 Where the Truth Lies. Franz Moewus and the Origins of Molecular Biology. Cambridge University Press, Cambridge.  SCIENCE CITATION INDEX, 1945-1954 Ten Year Cumulation. Insti- tute for Scientific Information, Philadelphia.  STANLEY, W. M., 1970 The “undiscovered” discovery. Arch. En- viron. Health 21: 256-262.  STENT, G. S., 1972  Prematurity and uniqueness in scientific dis- covery. Sci. Am. 227; 84-93.  Taytor, H., 1951 Genetic aspects of transformations of pneu- mococci. Cold Spring Harbor Symp. Quant. Biol. 16: 445- 456.  Watson, J. D., and F. H. C. Crick, 1953 Molecular structure of nucleic acid. A structure for deoxyribose nucleic acid. Nature 171: 737-738.  WATSON, J. D., and J. Tooze, 1981 The DNA Story. W. H. Free- man, San Francisco.  Watson, J. D., 1968 The Double Helix. Atheneum Publishers, New York.  Wizson, E. B., 1906 The Cell in Development and Inheritance, Ed. 2. Macmillan, New York.  WILSON, E. B., 1925 The Cell in Development and Heredity, Ed. 3. Macmillan, New York.  Wo rr, J. A., and J. LEDERBERG, 1994 A history of gene transfer and therapy, in Gene Therapeutics, edited by J. A. WOLFF. Birkhauser, Boston (in press).  WRIGHT, S., 1945 Physiological aspects of genetics. Annu. Rev. Physiol. 7: 75-106.  Wyatt, H. V., 1975 Knowledge and prematurity: the journey from transformation to DNA. Perspect. Biol. Med. 18: 149- 156.  ZINDER, N. D., and J. LEDERBERG, 1953 Genetic exchange in Salmonella. J. Bacteriol. 64: 679-699.",
            "count" : "2"
          }, {
            "item" : "Enid Schoettle [stamped, OCT 23 1986] Expert Advice to the Federal Government [mark to note, go beyond the White House. Include OTA . . . Bill Golden, Harvey Brooks are the 2 old timers we might have. We need new blood.] Dear Enid -- I know you have a special interest in this question. I am thinking of pulling together a fairly broad study -- which would be especially timely as we approach Jan 20 1989. If Jim Coleman agrees to significant co-participation I will probably want to do it. Do you have any thoughts on the substance, sponsorship? See you on 11/14 Joshua. [written in left margin, Sam 187]",
            "count" : "2"
          }, {
            "item" : "STANFORD UNIVERSITY MEDICAL CENTER STANFORD, CALIFORNIA 94305 ¢ (415) 321-1200        STANFORD UNIVERSITY SCHOOL OF MEDICINE Department of Genetics  JUN ¢ 1969  Mr. W. A. Koshland President  A.A. Knopf, publishers 501 Madison Avenue  New York, N.Y. 10022  Dear Mr. Koshland  It has just reached my attention that one of the characters in \"The Andromeda Strain\" has certain biographic resemblances to me, and might plausibly be believed to refer to me. At the same time, the novel exhibits some personal history that bears no such correspondance to facts, and casts the character in an uncomplimentary light. ,  I am writing to ask your cooperation in minimizing the personal injury that might follow from this publication, for example in the style of your advertising which stresses the \"documentary versimilitude eee in authentic detail\" of the book. I do not wish, at this time, to call further attention to the matter by any specific statements about it.  The further mutations that the script might undergo in being produced £sx as a movie are a matter of even deeper concern to me. I have written to Mr. Wise; but I would also ask you to use your ven good offices to help assure that this is handled in a respon- sible fashion. .  The book is an interesting one, and deserves to have a wide sale. But I do not think it would detract from its appeal if it were properly fictionalized. I assume that you will reassure me that the possibility of a personal identification (through such details as the academic position of the character!) had simply es- caped your notice.  Yours sincere.          oshua Lederberg  professor of Genetics and Biology Nobel Laureate (for work in bacterial genetics)  LT. J. P. KENNEDY, JR. LABORATORIES FOR MOLECULAR MEDICINE, DEDICATED TO RESEARCH IN MENTAL RETARDATION MOLECULAR BIOLOGY HEREDITY NEUROBIOLOGY DEVELOPMENTAL MEDICINE",
            "count" : "2"
          }, {
            "item" : "STANFORD UNIVERSITY STANFORD, CALIFORNIA",
            "count" : "2"
          }, {
            "item" : "e/n.w ///.  r/itirt AL.  IltH-iiia  hi its relation to L oeahty tshoivintf the nit in her e.vanitiie/1 , ansi the  ratio re/eetert perJOOt) e.va muted.  mtss/n.  S\\\\ pli ilis  iJt.trf XL I.  hi its rctttfiori to Locality;sitoirin<7 the tnHtiber cayi/ii////t7,anet the  ratio rejected per1000eAYtttnttetf.",
            "count" : "2"
          }, {
            "item" : "AIDS lecture November 15, 1988 17/11 Current Crisis in AIDS by C. Everett Koop, MD, ScD Surgeon General of the U.S. Public Health Service U.S. Department of Health and Human Services Presented to the American Council on Life Insurance New York, New York November 15, 1988 It had been but two days since I had last addressed a public audience on the AIDS epidemic. I opened this talk grateful for the opportunity to meet with, and pleased to honor an invitation from my good friend and their president, Dick Schweiker, who as Secretary of Health and Human Services, supported me so well during the difficult time of waiting for my confirmation as Surgeon General. I made reference to the things that I had done with the insurance industry, especially in the matter of smoking. I referred to the fact that smoking was still the leading cause behind the three major killers of American life: heart disease, cancer, and stroke and that it was not just a casual habit, as my most recent report to Congress indicated: nicotine is an addictive drug that operates in much the same way as other addictive drugs, such as cocaine and heroin. The tobacco industry was more upset with my addiction report than with anything else I did in Washington. I made it clear to the insurance folks that I was upset too: that they would knowingly be selling a product whose major ingredient was an addictive substance. I then moved on to AIDS, which was really the reason that I was there. Because these were people involved with life insurance, I called attention to the fact that the AIDS epidemic had been heaviest among those Americans who were outside the traditional system of health care in this country -- not our familiar patients. They didn't have a family physician that had known and cared for them over the years and they were rarely covered by any type of insurance either. The numbers I was talking about might have been as high as 35 million Americans who were beyond the reach or outside the reach of the mainstream American health and social services. Not wishing to be misunderstood, I made it clear that these 35 million individuals did not engage in high-risk behavior and therefore, most of them would not contract AIDS. Nevertheless, the situation with AIDS was still bad; the epidemic showed no signs of abating. I then turned to the small minority, many of them uninsured, who were culturally and socially isolated and who did engage in some form of high-risk behavior and therefore at great risk of catching AIDS, if they were not already infected. Then, I went through the litany of people who were engaged in high-risk behavior and what that really meant to the spread of the AIDS virus. This was the first time that I pointed out that AIDS cases were rising among I.V. drug abusers and falling among homosexual and bisexual males: for example, the latter group comprised 66 per cent of the 1987 caseload, but only 56 per cent of the present caseload, that year. I turned my attention then to another group that was HIV positive and was outside the reach of orthodox medicine -- about 2,000 women, mainly, but not exclusively, prostitutes. This was another group not commonly found on the patient rolls of most practicing physicians. There were as many heterosexual showing up in the monthly AIDS totals as was the case the previous year. That, I called a very ominous sign, and certainly time proved that to be an understatement. That made it possible to move into the sad situation of newborns with AIDS and the disproportionate of numbers of persons with AIDS who were Black or Hispanic. I noted that the black community -- as a community -- was beginning to feel overwhelmed by the AIDS problem. Despite the very small number of Blacks who were at risk, AIDS came on top of an epidemic of Black-on-Black homicide, of drugs, as well as the chronic deficits they experienced in housing, employment, education, and health care. Then I philosophized a little bit on the problems just previously stated and put an optimistic spin on the situation. In AIDS, one of our greatest enemies was fear, and that meant fear of almost everything from the virus to the stigma, to the affects of AIDS on the social compact that has held us together, and of course, the affect of AIDS on the health care system, and the people who make our health care system work. I closed on the note of understanding that the life insurance people, with whom I spoke, understood the predicament we were in and that I looked forward over the next decade working with them to see the end of some of these aforementioned problems. The user of this archive is not to confuse the title of this AIDS lecture with other titles of lecture with a similar ring to them. This presentation to the life insurance community was quite different and spoke to things they understood better than most people. Because, I have enumerated a number of these things in this introduction to this lecture, there will be no index.",
            "count" : "1"
          }, {
            "item" : "< Sey  WESAE 2 . dase  . iN. ~ LX Wy % ys  oon Se 2 eee ; 2y88  REST a",
            "count" : "1"
          }, {
            "item" : "NOV 9 1985  THE UNIVERSITY OF CHICAGO DEPARTMENT OF PEDIATRICS  BOBS ROBERTS MEMORIAL HOSPITAL FOR CHILDREN \\\\ 920 RAST 59TH STREET d CHICAGO * ILLINOIS 60637 se yv |  11/5/65  Dr. Joshua Lederberg we wag Fae a AS OR Stanford University D foe ot } Stanford, California  Dear Dr. Lederberg:  The University of Chicago will celebrate its Seventy Fifth Anniversary during the year 1966. As part of this celebration and, more specifically, in conjunction with the dedication of the new Silvain and Arma Wyler Children's Hospital, including the Joseph P. Kennedy, Jr. Mental Retardation Research Center, the University is planning a Symposium on the Child during the week of August 28 to September 2. It is planned to bring together authorities to examine the present status of knowledge and problems concerning a wide range of aspects of child health and welfare. We hope this symposium will serve to focus attention on the many problems which confront society with respect to children and be productive of a fruitful interchange between many disciplines which are concerned with child care.  ALP lag  Each of the sessions of the program will have a major focus with several speakers invited to present papers of 30-40 minutes reflecting their viewpoints and work as they relate to the general topic. The University will be deeply honored by your participation in this important event. We will, of course, defray any traveling expenses and offer an honorarium of $300 for your participation.  We invite you to contribute a paper on genetic mechanisms for the session on Friday morning, Sept. 2nd, which will be devoted to basic molec- ular biology and genetics. This symposium precedes the International Con- gress on Human Genetics which will convene on the University of Chicago campus the following week.  WYLER CHILDREN’S HOSPITAL - JOSBPH P. KENNEDY, JR. MENTAL RETARDATION RESEARCH CENTER + HOME FOR DBESTITUTE CRIPPLED CHILDREN * LARABIDA JACKSON PARE SANITARIUM  SEVENTY-FIFTH ANNIVERSARY YEAR Page 2  The suggested topic is preliminary and we are anxious to know your own  reactions and suggestions. We will be very pleased if you are able to accept our invitation.  Enclosed is a draft of the program for the symposium.  Sincerely,  S us  George W. Beadle President of the University  Albert Dorfman Chairman of Pediatrics  Encl. 1     C an Conn lw lase AA_21.2_Q_ rm  fF",
            "count" : "1"
          }, {
            "item" : "/  CMa. 28, , (lr 9.30 2, Up. 80 tee San We! Wrofecd why / NO wae Gyglt ly FE po  Qesrelanrce. Mg em funrdu « ervhzut?     Tee WVAu mo nth ther  R. (Ceregn A VF 25   La : 7 f “7 Jf ‘Tb tad! tay a. & j Dryer 4 DO nel ured ~~ r% Arastiuok a nda hde,   (Via er utk . 99 Ueachie ce at La beck irk like he 4,58 tam 2 VAM -  Wek. het, pra bh ha the pay wt ape og —  TB + > bat Chad LH. oD fs 33 fF  y “ , / $7 dé vod ds fe a Bie eZ Wat, te Hrmdnd 0 1 67S: Ay Ts oy  Neg Ab, Coben's of Tpimy 7 ae  aH Utes ce eg,  Moy. m fy ike, ert 4 le | Crap wed, L. Werele, fr prance  mM ores wet Hs whe tog be we th Apaenn Foret   a | is i a SS GY.   ar Bane  wanes +  ie ih Duel  gh ls 3  Rec a 1 nen i iB an i  ae \" His",
            "count" : "1"
          }, {
            "item" : "December 17, 1968 Dear Marshall: Here is a transcription of my notes of a conversation with Dr. Brenner.  I'm sorry that the press of work delayed my completion of it.  And even I had trouble reading my scribbled notes. The transcription also includes Dr. Benzer's recent comments on his work.  Together, I hope they are useful to you in connection with the remarks or report that will kick off the last day of the next Stated Meeting.  I'm sorry neither Brenner nor Benzer could attend.  But then you have that much les introducing to do as chairman. Also enclosed are our Polaroid copies of the slides shown in conjunction with Dr. Gajdusek's talk at an earlier Stated Meeting. Under separate cover I am sending a copy of the tape recordings that include Dr. Gajdusek's presentation at an earlier Stated Meeting.  Wardy Holman included in his package of the two tapes the track and foot count of the start and finish of the presentation. With best Holiday wishes, I am Sincerely, Theodore Melnechuk Director of Communications Enclosures: 2 T. Melnechuk December 16, 1968 Notes of a Conversation on the Morning of October 30, 1968 at the NRP Center Between Professor F.O. Schmitt and T. Melnechuk and guest, Dr. Sydney Brenner of the Medical Research Council Laboratory of Molecular Biology at Cambridge, England I.   Background At the Stated Meeting of NRP Associates in February 1968, Dr. Marshall Nirenberg gave a brief account of the research strategy he thought he might follow in studying the behavior genetics of simple neural systems.  Believing it would be interesting to hear similar accounts from two other eminent molecular geneticists, Drs. Seymour Benzer and Dr. Sydney Brenner, Professor Schmitt invited the latter two investigators to speak at the Stated Meeting in February 1969, with Dr. Nirenberg chairing.  Neither invitee could be present at that time, but each responded with an account of their strategy, and Dr. Brenner included a status report as well. Dr. Benzer's account was in the form of a paragraph in a letter to Professor Schmitt dated December 3, 1968.   I cite the paragraph in full: The work my group and I are engaged in is the isolation of behavioral mutants of Drosophila, particularly in respect to the phototactic response, with the idea of obtaining genetic blocks at the various steps involved.  We look at the mutants for anatomical or developmental defects affecting the nervous system and also at electrical events in the visual system, as well as biochemical aberrations.  Much of the work is still very preliminary, but we have begun to find interesting electroretinogram abnormalities in certain visual mutants and are writing up that work for publication.   I will send you a copy of the paper when it is ready, for your information. It is also possible that Dr.  Benzer will describe his neuro-biological work at a symposium on  \"Control Processes in Multicellular Organisms\" scheduled to be held in New Delhi next March 16-20 by The Ciba Foundation.  In a preliminary program dated March 14, 1968, Dr. Wolstenholme has Dr. Benzer slated to speak on \"Genetics of Brain Function\" at 4:30-5:00 on Thursday, March 20, 1969. Dr. Brenner's comments were made in the course of a two-hour conversation with F. O. Schmitt and T. Melnechuk on October 30, 1968.  Before reporting that conversation, however, it seems helpful to abstract the main points made by Dr. Brenner two days earlier at the Massachusetts General Hospital in giving the Warren Triennial Prize lecture. II.   \"The Future Of Molecular Biology\" Under the title just given, Dr. Brenner made several major points: 1.  Scientists tackle problems that they consider important.  Of these, they tackle those problems they consider solvable.  And problems become solvable through the onset of new techniques, or new conceptions, or both. 2.  Current major problems include the physical mechanisms of: a.  Polypeptide folding b.  Enzyme action c.  Protein repression and induction 3.  The nature of a scientific explanation is different in physics and biology.  In physics, an explanation is ultimately a differential equation.  But in biology, an explanation is ultimately a list -- for example, a program. 4.  Only Max Delbruck expected that molecular biology might discover new principles of physics, according to Gunther Stent's \"That Was the Molecular Biology That Was\" (Science, Vol. 160, pages 390-395, 26 April 1968; a copy is attached as an appendix). 5.  The goal of molecular biology is to be able to compute an organism from a knowledge of its genes. 6.  The Monod-Jacob notion of repression-and-induction is a sufficient key to understanding development. 7.  Just as \"Project K\" -- to know all about the genetics of the biochemistry of E. Coli -- is feasible, so is \"Project Man,\" about whose physiology much is known. 8.  In order to yield as complicated and hierarchically arranged an organism as man, a genetic program must surely have parts, so there is some hope of getting at its logic before getting a thorough knowledge of its details.  By \"part of a program\" is meant such a sub-program, focused on an organ or a function or constituents, as would permit the eye to evolve without concomitant evolution of the kidney. 9.  Molecular Biology is essentially the Chemistry of Natural Selection, so it must eventually deal with the interplay of the genetic program with the accidents of history. III.   Correlative Neurobiology Unfortunately, Dr. Brenner's remarks at the NRP were not tape-recorded.  What follows is an edited transcription of T. Melnechuk's notes, which were not made with this eventual use in mind: Staff.- Drs.  Brenner and Crick now have a staff of 14 scientists.  After a building delay, they will have 28, who will work in three groups, one on molecular genetics, one on cell biology, and the largest on neurobiology.  The technical staff includes technicians trained to photograph anything at scales from the visible down to the EM level. Subject.- Brenner's own work is in neurobiology: not so much molecular -- that is, not on membranes, and not on biochemistry, except where relevant to the genetic problem, -- as on the genetic control and specification of structures in the nervous system: what they are and how they got there.  He wants to know what single genes can do, both structurally and behaviorally. Organism.- He prefers to work at this time on a system with no learning, one that is rigorously specified, controlled from within.  Therefore, he does not work with goldfish, but with nematodes. The small nematode he works with most is 500-700 mu long in \"adolescence\" and 1 mm long when adult.  The specimens are \"identical\", being naturally inbred, for in their 3-day life cycle, each nematode begets 300, so that in a week, one becomes 10^5; all on one Petri dish. Neuroanatomy.--The small nematode probably has less than 500 neurons.  Brenner is cutting serial sections 10 mu thick, using EM with 30 A resolution.  The job is being done broadly first, to get technical facility.  The entire sensory system has been defined, the neurons counted, and the dendrites pictured, but the hook-up of the sensory system to the central nervous system is not yet known.  All the sensory neurons have dendrites like cilia, with microtubules. Work continues on the motor side.  Work on the innervations of the esophagus is half done.  He has identified 92 muscle cells, though not what these cells do.  Each set of 16 muscle cells constitutes a ring.   Such muscle cells modulate like neurons.  They have oblique striations, rather than sarcomeres. The nematode's head, which narrows down, receives projections from 121 cells.  To get a three-dimensional grasp of the neural processes, Brenner prefers getting a mental picture by handling the actual cross-sectional EM pictures.  Neither a cinemicrotome approach a la Livingston nor an analog of the X-ray crystallographer's overlay method work for him; he wants to reconstruct the nervous system in his own mind. Behavior.- Nematodes have a limited behavioral repertoire.  It is difficult to describe their behavior scientifically.   Brenner takes cine pictures and does a frame-by-frame analysis. Of the 200 mutants he has observed, most are behavioral -- usually forms of paralysis.  He picks the mutants by a brute-force method:  he examines a colony, and picks up the cripples with a toothpick. Genetics.- Brenner is doing an extensive genetic analysis.  The behavioral alterations are produced by mutations of 22 easily recognizable genes; this number will go up to about 30.  The genetic map is saturated; 1 gene has 8 mutants.   The X chromosome is mapped, and the autosomes identified as linkage groups.  This work will take 2-3 months more to finish. The next step is to identify the structural defect in each mutant, so that if there is a change, it can be seen.  This will give some idea of what single genes can do, both structurally and behaviorally. Transmitter Biochemistry.- To do the biochemistry of nematode transmitters,  large numbers of animals are needed, so that mass culture techniques are necessary.  Work is almost ready to begin.  The idea is that if one is defective, you have \"the edge of the wedge\".  For example, you could study what the behavior would be if there were no inhibitory transmitter -- which, by the way, GABA is suspected to be in this organism. Brenner named three people who will be working on nematode neurochemistry: Tony Stratton or Stretton, who was with Vern Ingram; Slater or Slayter, and Dennis Bray, both of whom have been with Steve Kuffler. Neurophysiology.- Unfortunately, the particular animal chosen for being appropriate to genetics and electron microscopy is not appropriate for neurophysiological studies.  They are too small for the insertion of electrodes. However, large nematodes exist: the ascarids.   Brenner believes that their basic structure is similar enough to that of the small nematodes so that the only difference is one of scaling up the 5 or 6 levels from the free-living nematodes.  He says that Goldschmidt's old anatomical work is mostly wrong, by the way. Because of their complicated parasitical life cycle, ascarids can't be grown in the laboratory.  But neurophysiological studies can be performed, and the findings presumed transferable to the smaller species. Philosophy.- Brenner said he was not for, indeed was against, the idea of a changeable genome.  Instead, he was for answering the question:  Given the wiring diagram, what else must be known in order to be able to compute the organism's behavior? Next, a deeper problem:  How is it constructed?  The accurate computation of structure in development argues an utterly deterministic, digital program.  The specificity and precision of detail could not result from analog methods, such as gradients of concentration.  But only the genes are digital.  There must, therefore, be accurate gene switching. But the interplay of the long phylogenetic and ontogenetic process with history and its accidents during the prolonged development of elaborate organisms argues that such complicated systems must be subject to additional, new constraints, such as economy and continuity in evolution (as in the case of the persistence of cilia in sensory receptors).  Complicated systems may, therefore, employ analog principles as well as the digital principles of simpler systems. Brenner believes that storage is not in molecules but in cell assemblies, by means of synaptic switching.  Given the switches, their logic, and the plasticity of the complicated nervous system, he wants to know how it works.   He sees the need of an analysis of function space. IV.   Other Lines Permeability.- Brenner considers this question too wide for him. Molecular Biology of Transmitters.- Here, Brenner would like to see the protein receptor molecules isolated, and has two chemists coming to work at this problem in a professional manner.  He wants to work on Miledi's preparation, denervating a muscle and watching the spread of sensitivity.  He thinks too many people are already working on Torpedo. In his terms, he is after \"the machine language of learning.\"  Of the receptor molecule species he would like to know: Is it localized?  If so, how does it get localized?  Where and by what is it made?  How fast?  Does it turn over?  Etc. Neuronal Cell Transplants and Cultures.- Brenner believes that while good work has been done, it has not yet been quantitative.  For example, Jacobson's work on the retina:  how many cells?  1,000?  10,000?  100,000? Brenner would like to learn how many neurons in culture it takes to get functioning synapses.  What does it require to get at least one criterion of change?  What nutrition?  Perhaps the geometrical requirements may be odd: monolayers versus impregnation of collagen sponges? He sees the need for some analogy to the one-step growth curve of Delbruck's work on phage.  He is aiming for a set of cells (an explant, a clone) that can be assembled in vitro.  Then he thinks the proper tool is the EM. Quantitative Neuroanatomy.-  Brenner would like to see more quantitative human morphology.   He would also like to see produced a Handbook of Wiring Diagrams, containing those circuits that have been established critically in various organisms, as by Trujillo-Cenoz in the retina and others in the cerebellum.",
            "count" : "1"
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            "item" : "NEW  FILE BEGINS  Doe #/47  B&B INFORMATION & IMAGE MANAGEMENT 300 Pewee Grorer’s ROULEVARD UPecr MAaRLEORD, MarrLanpD 20772 ° UBA © (301) 249-3115             RMP Arkansas RM 000  JULY/AUGUST REVIEW CYCLE  Application; The Arkansas May 1 application was recommended for approval In the reduced amount of $1,500,000 by the NAC. The funding level was set at $1,425,255. In addition, $100,000 was awarded for the arthritis  proposal (#95).  The July 1 application request is for $816,563. If approved in its . entirety, funding would remain approximately $30,000 under the targeted figure of $2,272,958. Eighteen new proposals are included in the package.  \" Six deal with improving access to primary care services for the medically underserved population (objective 1) #97, 98, 99, 100, TO1 & 102.  Three deal with coordination and/or sharing expensive secondary and tertiary patient care resources and services (objective #2) #'s 103, 104 & 105.  Nine deal with the development of improved health delivery systems and sharing of scarce resources (objective #3) #'s 106 through 113.  Major concerns of staffing have been rectified. The evaluation section has completed its personnel complement by hiring one additional evaluator (3) and three additional monitors (4). M.D. coverage will be available on a 100% basis. No problems with CHP.  MCO 7/15/74          oortee =. sttRE ste AWD HLTH a0 HOvOMEY WW 680, €66°9E - os _. ootfes . YNYNUYXAL 3YVd: WLYNO3N 2 3udHOD a lze‘sz.. |. 878442 2 85842 oe ASL GNY Naa¥9S gotorany, = 280° .882°Sp «sis Oe SG . i 0668S ee OE awa SQNN4 ¥ad0 WI¥aLNnt ‘awodns, «980 (€89°82. 0 seetee OO Ske oe _aswnoo Y9S G3.37199 BIWIIS , $80 Lee’Sp 208K ROS , 8d 99037 4NOD 8000 i26°8p SES SE i Sad SMOWLS AVHFE HOD E807  .. €L9°86 |. S$2642t = 0. $26 Fy2t oe Mas. WY AOISY ONY YLI NENG NVdXS 2800 - GLe°eLe soe tat o | _ . 0e9e2t  . 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NOI9SSY AG SLNSNOdWOD . €t-OT PHSO mene ONILSIN 3d G31S3N038 Te ee BLOT ES VB     e                                  @ ' + e ‘ . + + te meee Oe ne ee cece ee ce een ee ee ae one mie sa one Ps ne er armen. me care one . ~ ee oe et eo . ‘4 a TTT TT agree seme were ae te cence eee cent iene en ne nee eee renee weneee “mae eenene enn ues eee se eee anne eee cere oe tee wees ae ' . * . - TON Sem te ee oe ree men ae ern nn eet ere econ a meen fo samnntinn “oe nemnere © ee eee ome oe - ‘ : a) . = o ~_ ene ee TTT mapeemeseerieen eam ecmemeraccamae eae m= cpanm cme aaa semen en nat 7 Seti cee sca an ee eee tise mee Senne ee ae pecans enn 2 we ee ~ - ’ . . . . . ‘        -gaz'szptn ee ae _ -  606 €9/ Mau Jl suo1genujquos ¢) sq0afoug - - 2 ce cane et et \"OPES Lag SOLTLALQOY B 3427S weuboug - | ~~ | UOES 180 uoraeooL ly 40 a  att me eee 8 RR Rn A A RE RS NTT OE ORE RF ae ee we mee eee ne  . 000 SOOL ee S$} Layqy - ae ce an ae we commen ne ee te ee ‘ wee oe oo we mee eee en ee ee ~ te ee een ee come     SG2°S2p° L$ tsz*oee*t ~~ oos*zz- ~~ ~~ te6*z08*t~ ~~~ sa¥LOL NOTOSY UT em ee gz“ SENSNOdNOD~¥O- ON.     Dstt ae nme meme ne vate ee ee ee cep eee te Rte ce cee wee ee ee oe meee     259°Ce _asstoe. cele eee eee 2st se cee cuneate ea eemnege mnes aervnree _9sanh Ad Advil wousovaind ne 2 OT STG HH TIN ABABNS SONLTLIV BOF 460 _  Be2‘cb steeez tee tg SAN BR 60  L8L‘lv 899895 ee IS oe _. conan ADOT § SDINIT9 3sNn09 wos HOH 260  £058 $8886 oo Oe 888886 konnisaa 90708 sand VOENAHWOD BIS 60  QAE‘Bbs98@s ZS coco bee S¥ad 1d30 AQUaWa unssy wna 060 _  ems egnaymm oo LSaNQAaY co ASanDaYy ttn ee ee ee ee ree meee me ON\" . WLOL LDS YIONT L93yId J AVLIL LNINOdWOD  - dWO0> ANINILNOD GI.     me wees ee eee tree wen ee et me wee  NOISI930 ; NOTLYDOTIV “OO co ne Sms : “= GO-YA 904d ~ oT OOS YSNYEY ozs  rs foe SS aise) 02 7 921 . NOI9SY AB SLNANOdNOD , ‘ . .       RMP _ ARKANSAS  RM 000 52 MAY/JUNE 1974 REVIEW  Request: _ $1,830,751 Committee Recommendation: —_ $1,500,000  Overall Assessment by Individual Reviewers:  The primary and secondary reviewers rated the overall assessment of the Program as average at this point in time.  vt  _ Critique:  This Region had been-considered above average while it was under the direction of Dr. Charles Silverblatt. However, Dr. Silverblatt resigned in February 1974. Reviewers were concerned that the present staff would not be able to significantly expand the Program beyond the current. dollar level of operation. The Search Committee has not yet recommended a replacement for the Coordinator but has appointed Mr. Roger Warner in an acting capacity. (Mr. Warner is one of the four candidates being considered.) He has been with the Program for four years and has demonstrated expertise in the monitoring and evaluation aspects of the Program. The RAG remains intact with relatively little turnover. Relations with the CHP, the Grantee, the Arkansas Health Systems Foundation (EHSDS Program) and the other health provider institutions appeared to be most satisfactory. ;  Programmatic thrusts covering the 10 identified \"umbrella activities\" were considered ambitious. Several new project proposals such as sickle cell, audiology and stroke rehabilitation were questioned as to the possibility of obtaining impact in a one-year period. Twelve other new project proposals were included in the application.  A clear evaluation protocol was not included in the application. More  information in this area is to be expected in the July 1 application. Also, a clearer staffing pattern is expected in the next application.  JULY/AUGUST REVIEW: Estimated request as of May 1974: _ $800,000 MCO 6/4/74  NATIONAL ADVISORY COUNCIL - June 13-14, 1974 Council concurred with Committee recommendation.  DRMP funding decision - $ 1,425,255  MCO 7/2/74",
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            "item" : "UNIVERSITY OF CALIFORNIA, RIVERSIDE APR 11 27        BERKELEY ° DAVIS * IRVINE * LOS ANGELES * RIVERSIDE * SAN DIEGO * SAN FRANCISCO     DEPARTMENT OF BIOLOGY RIVERSIDE, CALIFORNIA 92502     10 April 1987  Dear Josh,  More on the phylogeny of you and your friends. When Betty was reading your letter and my reply, she noticed that I had not corrected my lineage. I was supervised (in his wonderfully informal manner) by Lester Barth, not Arthur Pollister. But again, Arthur had a great influence not only scientifically but also personally. On one memorable occasion, when the campus medical office had concluded I had malnutrition with vitamin deficiency, he took me over to Frank's in Harlem and fed me a huge steak and mound of fried potatoes!  During the years when I was an undergraduate and graduate Student, a large proportion of graduate students worked with Barth. The 1930s were a decade when hopes were high in exper-  imental embryology. It was later that Arthur had all of those talented students.  Betty also reminded me that Donald Lancefield suggested that ra Barry Commoner's chances for a career in science would be improved if he went to Harvard for graduate work rather than stay at Columbia. Barry, like you, did fine work as an undergraduate. He was greatly stimulated by Arthur Pollister.  The case of your going to Yale and Barry to Harvard is sad indeed. It was generally recognized, by those frequent estimates of relative standing beginning in the 1890s, that the old Depart- ment of Zoology at Columbia was the finest in the land for the research of its faculty members and the training provided to its graduate students. In fact the Department continued to be given that rating well after the time it had begun its decline.  Betty also said that I should check my letters for typos before they are sent. She has the unfounded belief that I could catch them and make the necessary corrections. Hah! es  Deo Yon My best, wr oa  s",
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            "item" : "SAND  jo76  17? Fifth Avenue Waterford, Ct. 06385 December 29, 1975  Dr. Joshua Lederberg  Department of Genetics  Stanford University School of Medicine Stanford, California 91,305  Dear Dr. Lederberg,  I had hoped to get in touch with you much sooner and I apologize for the delay. In any case I wish to thank you for your general expression of interest in my activities during the past summer. I was deeply touched by your sincere efforts to spend time with me when so much was going on. Your sensetivity to my needs and your continual willingness to help were truly inspiring.  I am glad to send these pictures and I hope that you will enjoy them.  Also, thank you for sending me a copy of Dr. Robinow's communication with you concerning the use of the Feulgen stain on inclusion bodies.  The study that I did with you has already proven useful in my endeavors this semester in General Genetics and in Developmental Genetics. I anticipate that this study will also prove to be useful in the Microbial Genetics and Developmental Biology courses that I will be taking this coming semester.  Recently I became a University Scholar, thus fascilitating advanced independent work for me. I anticipate to be working with Dr. Hans Laufer here at UCONN on the use of plasmids as tools for producing large amounts of assorted gene products of Chéronomus Tentans. We are interested in studying these products, notably hemaglobin, and need some mechanism for accumulating larger amounts of them. When I have a more complete picture of where my work will concentrate I will elaborate further.  Once again, let me thank you for the wonderful experience that you made nossible for me.  Sincerely,  Wark, Wtedenhaurs  Mark Weidenbaum nett,",
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            "item" : "SCHOOL OF MEDICINE STANFORD UNIVERSITY, STANFORD, GALIFORNIA 94305  JOSHUA LEDERBERG JosePu D. Grant PROFESSOR February 10, 1978 OF GENETICS  Dr. Attallah Kappas Rockefeller University Hospital Rockefeller University  New York, New York 10021  Dear Kapp:  Thank you very much for the arrangements you have undertaken.for the space for Dr. Smith's research project. The certificate of availability was the urgent requirement at this time, to permit the revised grant application to go forward before the deadline. When I visit in March I will check this out further with you and with the other involved people that you mentioned in your letter. I am confident that they can be reassured that Dr. Smith's work will be of very great value to the Clinical Research Center and that it can be done in a way that mitigates the stress on other requirements for  space.  Thank you also for the background material on the Clinical Research Center and the hospital. It has taken me some time to go through it, but I have enjoyed every bit of it. There is certainly an impressive record of accomplishment and a well earned reputation, whicn confirm my belief that the means must be found to sustain this capability. I had a chance to visit with Ed Rall, the intramural director for the National Institute for Arthritis and Metabolic Disease; and he pointed out another indispensable value of this center: to have.a smoothly running structure in-being, to be able to take advantage of new research initiatives and opportunities as they arise with a minimum of fuss and delay. I am also impressed that an able scientist like Anthony Cerami is able to play a major role in the Center through cooperation with his clinical colleagues, although he himself does not have a medical qualification.  Having confirmed such worthy goals, there remains the matter of means: and I will of course be very much interested to discuss concrete proposals that can lay out various options of modus operandi. The NIH site visit re— ported some concern about the efficiency with which the hospital has operated; and I would certainly be interested to know what steps have been taken to respond to that implied criticism. Has the external advisory committee that you indicated might be chaired by Dr. McGhee Harvey been activated: has it been able to submit any reports as yet? Dr. Attallah Kappas -2- February 10, 1978  Kapp -- these are just some principal thoughts that are at the top of my head at the present time. If you wish to make them part of the agenda for our further discussions next month or if you prefer to develop them further by memorandum in the interval: I leave that entirely to your own preference.  Most important of all is the scientific quality of the work reported and I did find this to be very exciting indeed.  The documents left me a bit confused about the status of Dr. German's project, which seemed to me rather vague both in operational and in scientific  terms.  Yours sincerely,       Joshua Lederberg Professor of Genetics  JL:ek-f  bree, Belle.",
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            "item" : "Commencement Address #2 -May 26, 1988 Ethical Imperatives and the New Physician: II. The Physician-Patient Relationship Commencement Address by C. Everett Koop, MD, ScD Surgeon General of the U.S. Public Health Service U.S. Department of Health and Human Services Presented at the Sesquicentennial Commencement Exercises of Albany Medical School Saratoga Springs, New York May 26, 1988 I gave the Commencement Address at Albany more than once and it's a very interesting commencement indeed. I know of no other that is more family oriented and it was such a joy to see proud mothers and father hooding their own children. The students marched in to their seats between two lines of faculty and the faculty exited between two lines of students. The warmth between students and faculty was apparent and the bagpipes didn't hurt a bit either. I congratulated them on 150 years and told them how impressed I was by anything that worked that long, especially that medical school. Early on, I got into the theme of the day by calling their future careers a \"vocation', because of its root term of \"calling\". That is because the practice of medicine is an activity with a firm moral base. I then announced I wanted to spend the time I had that day looking at one of the most transcendent issues of medical practice and sharing my perceptions of what it has meant to me. I mentioned in passing the cycle of addresses I was giving that spring and that they would receive all of them in a bound copy during the summer. Today, however, I would confine my remarks to what is commonly known as the doctor-patient relationship. I began by telling them a little about my background and how long I had been in medicine, but acknowledged that the most impressive change of all, had taken place in the metaphysical experience we call the doctor-patient relationship. It began with the decline and virtual disappearance of medical paternalism - \"the physician-father knows best\". That goes for the \"physician-mother\" as well. I reminded them and hoped that they would understand for themselves that a physician is not a \"provider\" and my patients were not \"consumers\". My terms carry the implications of a personal relationship - something that is confidential and trusting. I urged that whatever that class called them, to invest them with the feeling of personal involvement that has been the hallmark of responsive medical practice for centuries. Today, physicians are more inclined to be open and honest about their uncertainties. That's one of the origins of the \"second-opinion\". This \"new humility\" could be quite becoming. Accepting one's own human fallibility is really a strength, not a weakness and I was happy to see that physicians have come to agree with that point of view. Although, maternalistic and paternalistic medicine may be fast disappearing, it has not yet been replaced with a single ethic with the same simplicity and power. If anything, we're evolving into a \"physician-patient partnership\". In such a partnership, the patient has a right to know, then armed with this knowledge, be in on the final decision-making process regarding therapy. I then discussed some of the pros and cons of the old and the new relationships. But whether one will treat patients as a partner or in loco parentis, one naturally must maintain the professional right and the obligation to manage the patient the way one thinks is best. I pointed out the pitfalls of moving in one direction for over-treatment and in the other for under-treatment. In general, I have found that the \"take charge\" type of relationship with patients usually comes from physicians, more concerned about the \"sanctity of human life\", because they do in fact see themselves as the ultimate guardians of other people's lives. They take that role very seriously. Then, there is the tendency that may appear with that philosophy to do absolutely anything to save the life of a patient, to extend the process of dying under the guise of doing heroic and extraordinary things. Another question that arises in the physician-patient partnership is the inability of some patients to be partners, because they are profoundly dependent. I got a little personal here and revealed that I always tried to gain, as my allies, the parents of each of my young patients. I need more than just their informed consent. I needed their informed love and concern and energy so that we could fight - together - the particular anomaly or disease that was threatening their child. Win or lose, we always retained that sense of partnership. Could that be the reason - totally unanticipated - that I was never once sued for malpractice in a surgical career that spanned 40 years? I then discussed the \"academic ring\" that some of the things I said must have and how these kinds of questions have always been percolating in the consciousness of medical practitioners, which got me into the subject of medical specialization and group practice arrangements. I spoke of the advantages of such, but lamented the fact that the trade-off for such progress was that few physicians today get to know the whole patient, as well as their predecessors did. I introduced the fact that some patients prefer less dependence and more right to privacy and the freedom to pick and choose. This tension between parental medicine and partnership medicine is being tested now across the board in our profession. The sorting out process is far from over. I gave some examples. I closed with assuring my young audience that there is life after medical school and reminded them that many of them would help bring about the changes that lay in the future. I relayed my confidence that the physicians of America will emerge from this time of ferment with a stronger ethical framework within which to practice a type of medicine that serves the needs of informed and involved patients. I quoted my old friend, Dr. Edward Pellegrino of Georgetown who said, \"Some degree of effacement of self-interest is . . . present in every medical oath, that is what makes medicine truly a profession.\" For myself, I said that I hoped that my reputation for straight talk was lived up to that morning, but that it hadn't dampened my enthusiasm for medicine. If I could do it all over again, I would join them at the start of a magnificent career.",
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  "data" : [ [ "row-jxx6~4nc9-qfdt", "00000000-0000-0000-4EBB-8EAF8044E8FA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Arthur J. Walker to Francis Crick, Leslie Barnett, Sydney Brenner, and R. J. Watts-Tobin", "101584910X100", "101584910X99,101584910X98", "1962", "1 February 1962", "In this letter to the Medical Research Council Unit for Molecular Biology at the Cavendish Laboratory at Cambridge University, a layman offers views on the social implications of biochemical genetic research.", "Letters (correspondence)", "Genetics,Environment,Genes,Genetic Engineering", "Public Reactions to the Genetic Code, 1961-1968", "2", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "1 February, 1962 Doctors: F. H. C. Crick L. Barnett S. Brenner R. J. Watts-Tobin The Medical Research Council's Unit for Molecular Biology -- Cavendish Laboratory, Cambridge University Dear Doctors: May I submit a few comments, with all due respect from a layman, in reference to your work on biochemical genetics that has been given recent publicity.  Since reading some two years ago of the 1959 Nobel Prize award to Doctors Ochoa and Kornberg in the United States for their achievements in this field, it has seemed to me that such research has far greater potential and import toward human benefit and betterment than anything else that is now being attempted or being done. Many are the possibilities, most of which have no doubt occurred to you.  First, perhaps, a better knowledge of real fundamentals underlying human nature.  Some control, even, of such human characteristics that are obviously at the root of so much distress in this world -- of war , poverty, family dissension, et al..  From this might follow two main concepts of approach pertinent to the present need: 1.  A Means Available to Man.  To find out if there is some way through effect of chemical, radiation, or other means present on earth, whereby humans could overcome certain traits of character.  That is not to alter the many variations of aptitude and type essential to an interesting and balanced society, but rather to discover a way to create in all people such a spirit of motivation as would remedy the harm that humankind does unto itself. In the light of past genetic knowledge wherein microscopic cell structure and related traits appear unalterably established by predecessors, this does not seem possible.  However an urgent need exists to find if such a conclusion is entirely correct.  The well-known threat of complete catastrophe now posed by knowledge of nuclear armaments makes immediate and intensive exploration of every possible means to eliminate this threat an imperative necessity.  Something, perhaps, that would so permanently alter humans that they would first feel better - a transcendence that most all would want - and then that humans would act better. 2. A Knowledge of Externals to Man. To find out if there are factors external and beyond the control of man that affect humans, with particular regard to the traits of character and control thereof indicated in the first concept. To quote from an article in a prominent American magazine on tidal effects and such: \"Extrinsic Rhythmicality -- It has recently become evident that organisms -- derive information as to the geophysical rhythms from their environment.  Such information must be transmitted by highly pervasive forces hitherto ignored by biologists.\"  Something similar, perhaps, to implications of the pseudo-science, Astrology.  Since some real scientific observation has of late found a connection in particular between positions of planets and condition of the ionosphere, and also a relation to major earthquakes, might not some effect on genetic arrangement and character in humans follow. Should this be found, and further perchance that an improvement in human character could be expected thereby fairly soon, such a knowledge would be of high potential -- particularly if no means be available for humanity to improve its own nature as in the first concept.  If discovery indicates that an ultimate salvation could be coming one might say, 'in the hands of God', then the knowledge thereof should provide a strong antidote for the present world hopelessness.  A reason to hold the line and use every effort of apparent free-will to avoid nuclear disaster until the coming of mans' better character -- \"The Kingdom of Heaven on Earth\" perhaps -- which would by itself ensure a time of permanent peace and enlightenment. In conclusion may I wish you the utmost success in your present research.  Whatever the outcome it is all a contribution in the endless search of true knowledge.  The kind that can be of real benefit to humanity. Sincerely yours, Arthur J. Walker.", "Walker, Arthur J.", null, null, null, "Crick, Francis, 1916-2004 ; Brenner, Sydney ; Watts-Tobin, R. J. ; Barnett, Leslie", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xyki~n3i7-r89c", "00000000-0000-0000-DAD8-862B35945798", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Frank L. Campbell to Marshall W. Nirenberg", "101584910X101", "101584910X102", "1962", "7 February 1962", "This letter from the executive secretary of the National Research Council seeks comments from Marshall Nirenberg regarding an article he has written.  The article, titled \"The Brownstone Tower,\" was eventually published in the Journal of the Washington Academy of Sciences 51, vol. 1 (1961).", "Letters (correspondence)", "Publishing", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of the Archives of the National Academies.", "Copyright may apply", null, null, "7 February Dear Dr. Nirenberg: Enclosed is a draft copy of my column, called The Brownstone Tower, intended for publication in the March issue of the Journal of the Washington Academy of Sciences.  I should be pleased if you would read it carefully and point out any errors you may find, particularly in the first paragraph on page 2, in which I try to describe the nature of your work.  Please do not hesitate to treat it severely because I shall be grateful if you will prevent me from making mistakes. I mentioned the names of your associates and your superiors so that they can share with you whatever recognition may come to you as a result of this story.  I hope you feel that I have been diplomatic in this matter and that I have not seriously distorted any of the facts presented. If you can, please telephone your suggestions so that I can submit the manuscript to the editor of the Journal as soon as possible. Sincerely yours, Frank L. Campbell Executive Secretary", "Campbell, Frank L. ; National Academy of Sciences. National Research Council. Division of Biology and Agriculture", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dwbs-csp4_rm3j", "00000000-0000-0000-E8BD-2F1C18498426", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Brownstone Tower", "101584910X102", "101584910X101", "1962", "7 February 1962", "This article was later published in the Journal of the Washington Academy of Sciences 51, vol. 1 (1961).", "Drafts (documents), Articles", "Genetic Code", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "3", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "7 February 1962 The Brownstone Tower Everybody who receives this Journal is aware of the initials DNA and RNA and of the current phrase “cracking the code\" carried by both nucleic acids that determines what proteins shall be synthesized in living cells. I was curious to find out whether my unrefreshed, antebellum chemistry would enable me to understand what is going on in this extremely active field of chemical genetics. For enlighteament I went to our own National Institutes of Health to see a young man, Dx. Marshall Wirenberg, whose name, not yet in American Men of Science or on the rolls ot the Washington Academy of Sciences, is becoming known nationally and internationally as a pioneer in \"cracking the code,\" Organizationally, Dr. Nirenberg is a member of the staff of the Section on Metabolic Enzymes in the Laboratory of Biochemistry and Metabolism, National Institute of Arthritis and Metabolic Diseases. The chain of command begins with DeWitt Stetten, Jr., Associate Director in Charge of Research, over Leon A. Heppel, Chief of the Laboratory, over Gordon Tomkins, Chief of the Section, over Dr. Nirenberg, who has three professional associates (Heinrich J. Matthaei, Oliver W. Jones, and Samuel H. Barondes) and two leboratory assistants, Physically, Dr. Nirenberg is to be found in the D corridor on the eighth floor, northwest wing of the big Clinical Center. His name appears beside the door of room 13. Arriyging ahead of the time of my appointment, I retreated when I found standing room only in 13, and little of that. The hall, too, was lined with a variety of equipment that could not be accommodated in the laboratory rooms. At noon I met Dr. Nirenberg, and we went to lunch with Carl Brewer in the pleasant dining room of the new office building, No. 3l, -~ 2 which houses the Division of General Medical Sciences, the Division of Research Grants, and extramural program staffs of the several Institutes. At lunch Dr. Nirenberg explained his work to me. As I understand it, he and his associates were the first to report synthesis of a known protein- like substance (polyphenylalanine) in a cell-free medium containing amino acids and a synthetic RNA (polyuridylic acid). This RNA was specific for the polymerization of phenylalanine. Thus it was shown how to go about the business of synthesizing other proteins from other RNAs of known composition and eventually to relate the sequence of amino acids in the resulting protein to the sequence of basic groups in the template RNA; i.e., to decipher the code, which is believed to be universal. Carl Brewer pointed out that the whole story of the development of concepts of the DNA-RNA role in heredity beginning in 1953 was well told in a long article in the New York Times of 2 February 1962 and, of course, many other popular articles have been written about the subject. Dr. Nirenberg, whose work is outlined in the Times story, endorsed it. I recommend it. Being engaged, as I am, in desk work in biology, it was refreshing to be in the presence of one who is in hot pursuit of knowledge, who has more experiments in mind than he can carry out. He is free to develop his research as he sees fit and is really not under scientific direction of those named in the second paragraph. His work is controlled by his own thinking and by results obtained by others working along similar lines, Communication is very important in such a rapidly developing subject, and there is a grapevine that carries the word among the members of the DNA-RNA fraternity. At lunch Dr. Nirenberg was wearing his rern white laboratory coat, a - 3 = symbol of his absorption in his work. I surmised that he might not count the hours he spends in the laboratory. \"True\", he maid, and added that he lives on the campus in an apartment house built for physicians who must be close to the Clinical Senter. Thus if an idea strikes him at home, he can be in the laboratory in a few minutes to try it out. He showed me around his laboratory and nearhy instrument rooms. Electronics baffle me, and I could respond only to a very simple device in the hall, a large heavy thermos jug containing liquid nitrogen in which tubes containing enzymes are suspended. At such low temperatures the activity of enzymes is preserved for months. Dr. Nirenberg was not always destined for biochemistry. He graduated from the University of Florida in 1948 not knowing what he wanted to do. dHe tried more than one occupation and in 1952 took a Master's degree in entomology, also at the University of Florida. His dissertation was on the Trichoptera of Alachua County, aquatic insects of no economic importance. He had taken a minor in biochemistry and decided to work for a Ph.D in that subject at the University of Michigan under James F. Hogg. He took his Doctor's degree in 1957. Since then he has worked at NIH, first on postdoctoral fellowships, then as a member of the staff. The attention he has received lately must be somewhat distracting and he must be reluctant to give up his valuable time to instruct poorly prepared people like me in the purpose, methodology, and significance of his work. Yet he is doing so patiently and cheerfully. We hope that he may have the satisfaction of solving many of the problems now in his mind and will enjoy both the regard of his colleagues and public recog- nition of his achievements.", "Campbell, Frank L.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-zuuz-iijw~huny", "00000000-0000-0000-03AC-37D4A1B534B1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Cell-Free Protein Synthesis: The Association of Viral RNA and E. Coli Ribosomes", "101584910X103", null, "1960", "[1960s?]", "This report is an extension of work on protein synthesis, RNA, and E. coli undertaken by Marshall Nirenberg.  The experimental evidence involving the mixture of viral RNA and E. coli ribosomes shows that \"in a cell-free system approximately 10 per cent of the ribosomes participate in protein synthesis.\"  Many tables and figures are included.", "Reports", "RNA, Viral ; Ribosomes,Amino Acid Sequence", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "24", "pages", "Text", "English", "Reproduced with permission of Robert Haselkorn.", "Copyright may apply", null, null, "nee S CELL-FREE PROTEIN SYNTHESIS: THE ASSOCIATION OF VIRAL RNA AND E. COLI RIBOSOMES. * by R. Haselkorn, V. A. Fried, and J. HE. Dahlberg Committee on Biophysics University of Chicago -l1- The hypothesis that information specifying amino acid sequences in proteins is carried from the gene to ribosomes by messenger RNAt is now supported by an overwhelming body of experimental facts. Following the pioneer work on phage-infected pacteria’, the most convincing proof of the messenger hypothesis came from experiments utilizing synthetic polynucleotides to direct the synthesis of polypeptides in a cell-free system from E. coli’. Some time ago we observed that RNA from several plant viruses stimulated the incorporation of amino acids into protein in the E. collsystem’; that specific protein can be made in this manner has been shown using RNA from the bacteriophage fe°, Several plant viral RNA's have been characterized extensively, and our familiarity with one of them, TYMV-RNA, prompted us to use it to define the complex carrying our protein synthesis in the E. coli system. In this direction, three basic types of experiment have been carried out, all utilizing zone centrifugation through sucrose gradients. In the first, viral RNA and E. coli ribosomes are mixed, fractionated on the gradient, and the RNA located. Secondly, the RNA-ribosome mixture is fractionated, and the individual fractions are assayed for their ability to incorporate amino acids’. Finally, the RNA-ribosome mixture is allowed to incorporate amino acids, then fractionated, and the RNA and newly made protein located. All three experiments give essentially the same result, namely, that in the cell-free system approximately 10% of the ribosomes participate in protein synthesis, and the active complex consists of one 70s ribosome and one molecule of viral RNA. In this paper we present the results of experiments of the first kind. Materials and Methods.   E. coli ribosomes were prepared as described previously\". TYMV was labeled with pe by growing chinese cabbage plants in Hoagland's solution No. 1 for several weeks©, then inoculating with virus, and transferring the plants to the same solution lacking phosphate, other than .1 - 1.0 mc 7? PO) per liter. Six to ten days later the young leaves showing chlorosis typical of TYMV infection were harvested, minced, and the sap filtered through muslin. There followed a centrifugation at low speed to remove large debris, and a ninety minute centrifugation at 40,000 rpm in the No. 40 rotor of the Model L Spinco to pellet the virus. The pellet was dissolved in a small volume of 0.01 M EDTA, and in each cup of the SW 39 rotor 2.Q0ml of this solution were layered on top of 3.0 ml of CsCl solution having a density of 1.30. After 3 hours at 35,000 rpm the virus ( = 1.49) was completely sedimented, while leaf protein, lipid, and polysaccharide contaminants floated, and could be decanted. The virus pellets were taken up in 0.05 M Tris buffer, pH 7.5, and freed of excess CsCl by another high speed centrifugation. Finally, these pellets were taken up in 0.05 M Tris and shaken with water-saturated phenol as previously described’. This procedure routinely afforded RNA containing 10° c.p.m. per mg. Linear gradients of sucrose (5-20%) were formed using a device like that described by Britten & Roberts. After centrifugation the tubes were placed in a jig whose bottom consisted of a rubber Stopper in which a syringe needle was embedded. The plastic tube could be forced down onto the needle, the stopper then forming - 3 - a tight seal to the bottom of the tube. Drops emerging from the needle were collected in Shevsky-Stafford tubes, the volume and absorbancy of each fraction were measured, and then the entire fraction was transferred to ringed planchets, dried, and counted in a thin end-window gas flow counter. Figure 1 shows the result of centrifuging p’°_labeled TYMV for two different times. In each case the half-width of the zone of virus is approximately one-tenth the total column height; presumably most of the broadening of the zone occurs during the layering of material onto the column and loading the rotor into the centrifuge. In the figures presented below, zones occupying more than one-tenth the column height indicate heterogeneity with respect to sedimentation coefficient. Amino acid incorporation was measured in the complete system as described previously’, except that H?- leucine and H-aspartic acid were used in place of the cl _iapeled mixture of amino acids. After incubation for 30 minutes at 26°C., the reaction was terminated by the addition of one- tenth volume of 50% TCA. The precipitates were collected by eentrifugation and washed twice by solution in 0.1 N NaoH containing cold leucine and aspartic acid and re-precipitation with 5% TCA. The final pellet was dissolved on 1.0 ml of hyamine 10-x, taken up in 14 ml of toluene-PPO-POPOP scintillator fluid, and counted in a Packard liquid scintillation counter. Results. Viral RNA attaches to E. coli ribosomes in the ionic environment in which protein synthesis is observed; no factors other than Mgt? appear to be necessary for attachment. This is shown in Figure 2, in which a 0.2 ml zone containing 7° _yabeled TYMV-RNA in 0.01 M Me*t, 0.01 M Tris, ribosomes and P pH 7.5 was analysed on a gradient containing the same ionic composition. In this medium the ribosomes exist as a mixture of Os and 100s particles; the P?_labeled RNA is seen to move faster than 100s, and in fact occupies a region corresponding to 120-150s. The distribution of ribosomes among sedimentation coefficient classes is affected by a number of factors in addition to Mgt? concentration. One of these is the monovalent salt concentra- tion; since we found initially that optimum amino acid incorporation was observed when 0.07 M KCl was present in addition to Met it was of interest to note the effect of KCl on viral RNA attachment’. In Figure 3b, the charge for the gradient was identical to that for 3a; the experiment differs only in the addition of 0.07 M KCl to the gradient itself. First, it can be seen that the sedimentation coefficient of the RNA-ribosome complex is decreased to 80-100s; secondly, the RNA:ribosome ratio of individual fractions is approximately twice that in the gradient lacking KCl. The calculation of the RNA: ribosome ratio for each fraction in Figure 3b is shown in Table 1. This was done by first determining the amount of 737 RNA from the known specific activity of the RNA and the total counts for each fraction, converting to absorbancy units, Subtracting the RNA absorbancy from the total, and dividing the remainder by the extinction coefficient for E, coli ribosomes. The weight ratio of RNA to ribosomes is given in the seventh column, and the molar ratio in the last. For conversion to the molar ratio, the molecular weight of TYMV-RNA was taken as 2.1 x 10° and of 70s ribosomes as 2.f Xx 10°, For fractions 5, 6, and 7 most of the absorbancy is contributed by ribosomes from the 7OS region, rather than from the complex itself. Making a correction based on Similar runs with ribosomes alone, the molar ratio for fractions 5, 6, and 7 is raised to 0.3, 1.0, and 0.4 respectively. Note that radioactivity attributable to complexes is distributed over half the gradient, a spread well outside the limits for a single species (O.1 of the column height, see Fig. 1). Comparison with Figure 3a Suggests that the leading region (130-170s) is comprised of a small class of particles stable in the presence of KCl, while the peak region (80-100s) is comprised of those that have dissociated. Analytical ultracentrifugation of EB. coli   ribosomes in 0.01 M Tris, 0.01 M Mett Shows a mixture of 100s and 7Os particles; in the KCl-containing medium one finds 70s and 50s particles in the ratio of approximately 2:121, Thus the shift in the peak of radioactivity from 150s in Fig. 3a to 80-100s in Fig. 3b can probably be attributed to _6- the dissociation of 100s particles. The RNA:ribosome ratio of 0.5 calculated for fractions 2, 3, and 4 in Table 1 is con- sistent with the interpretation that these fractions contain complexes composed of two 70s ribosomes and one molecule of RNA. Fractions 5, 6, and 7 (80s-100s) should then contain complexes composed of one 70s ribosome and one molecule of RNA, and have an RNA:ribosome ratio of 1.0. Qualitatively this is observed, although the nature of the calculation precludes the assignment of an accuracy better than a factor of two to the ratio for these fractions. The conclusion that these complexes have the suggested composition rests primarily on their low sedimentation coefficient and the comparison between gradients with and without KCl. Systematic variation of the RNA:ribosome ratio in separate gradients makes it possihle to determine the stoichiometry of the interaction between ribosomes and RNA. In Figure 4a, b, and c, three experiments are shown in which the molar ratio of RNA to ribisomes is 0.48, 0.18, and 0.12 respectively. 32 By comparing the distribution of P in such gradients with the distribution observed when viral RNA is run alone under identical conditions, an estimate of the fraction of RNA bound can be obtained. Figure 5 shows a plot of the fraction of RNA bound as a function of the RNA: ribosome ratio. It can be seen that saturation is obtained at a ratio of approximately 0.1. If one measures amino acid incorporation into protein as a function of the RNA:ribosome ratio in the complete system’, the incorporation goes through a maximum at approximately the same ratio, as shown also in Figure 5. - 7 - This result indicates that only those ribosomes which funetion in protein synthesis bind viral RNA, and that by either test only 10% of the ribosomes in the cell-free system are functional. The attachment of viral RNA to ribosomes is reversible. This can be demonstrated in several ways, of which one is illustrated in Figure 6. In this case ribosomes and RNA in 0.01 Mg’t were layered on a gradient containing 1074 M Met, In the latter medium 70s ribosomes are dissociated into 50s and 30s particles. When this occurs the RNA falls off and is found at a position corresponding to 30s, the sedimentation coefficient of free viral RNA. Reversibility can also be shown by the ability of pee -labeled viral RNA to replace cold viral RNA bound to ribosomes. The reversibility is sufficiently complete that the order of addition of labeled and unlabeled RNA to ribosomes does not affect the fraction of label bound; the only pertinent variable is the final RNA:ribosome ratio. It was of interest to determine which of the subunits of the 70s particle contains the binding site(s) for RNA. Accordingly, 50s and 30s particles were separated by centrifugation in a low Mgt t gradient, concentrated, and each mixed with viral RNA in high Mett. Gradient analyses of these interactions are shown in Figure 7a and b. This experiment has been performed four times with different preparations of ribosomes; in each case attachment to 30s particles was observed while in three of the four experiments attachment to 50s particles was observed. The preparation of 50s -8- particles which did not bind RNA was also unable to combine with 30s particles to form 7Os particles although they had been derived from 70s particles’. We have on several other occasions observed irreversible dissociation of 7Os into 50s and 30s; as might be expected, such particles are inactive in the amino acid incorporation system as well as being 32 unable to bind P~--labeled viral RNA. Discussion. The existence of a cell-free system carrying out specific protein synthesis permits us to formulate two separate questions: what is the nature of the machinery in the cell- free system, and what relation does this machinery bear to that operative in vivo? It should be clear that the results cited above provide information concerning the first question only. The first point to be made is that the active complex + requires only Me and such cofactors as are already present in washed EK. coli 70s particles for its formation. This result has already been obtained for poly yt?» 14, 15 and E. coli messenger anal?, The contention that viral RNA requires energy to attach to ribosomes is not supported by our results’>. In 0.01 M Mg?\" B. coli ribosomes exist as 70s and 100s particles. In this milieu the complex of viral RNA and ribosomes moves faster than 100s, and might be comparable to the \"heavy\" 100s particles described by Risebrough et all’, -9- The addition of KCl to the gradients (Fig. 3) demonstrates that in the case of the viral RNA-ribosome complex heavy 100s particles are artifacts in the sense that they are not obligatory for amino acid incorporation. Gilbert has recently shown that KCl dissociates 100s particles in the crude extract of E. coli as well, although in his experiment the activity for protein synthesis continued to move faster than 100s??, The sedimentation coefficient of the viral RNA- ribosome complex, together with the ratio of radioactivity to ultraviolet absorbancy in fractions from the complex region in KCl-containing gradients indicates that the complex contains at most two, and more likely one, 70s ribosome together with one molecule of viral RNA. Published reports indicate that the active complex in reticulocytes in vivo consists of a string of ribosomes connected by a molecule of anal’: 19 It is not yet clear whether a similar situation is found in bacteria, although Gilbert's experiment!° Suggests that. A polysome-like structure appears to be formed by poly U in vitro, but no such structure has yet been demonstrated with messenger or viral RNA. On the contrary, the present work Suggests that viral RNA is incapable of forming such structures in vitro. Regardless of that handicap, polypeptide synthesis can be efficiently directed by viral RNA ian vitro: we conclude that one 7OS ribosome is sufficient machinery to carry out protein synthesis, a conclusion also reached by Gilbert. - 10- Ishihama et al. reported that E. coli messenger RNA attached to 70s particles only when the particles were first dissociated in low Mgt and then re-associated in the presence of the Rnate, We do not find this requirement for viral RNA. ishihama et al. found that messenger RNA could attach to both 30s and 50s particles; we find the same true of viral RNA. Furthermore, an examination of the stoichiometry of association with the ribosomal subunits indicates that a given number of 30s or 50s particles is capable of binding nearly 80% of the RNA bound by the same number of 70s particles. Since S-RNA attaches specifically to 50s particles 9, and messenger attachment is only slightly enhanced by association with 30s particles, it is tempting to suggest another role for the 30s particle: could it be supplying the amino acid polymerase? summary. Plant viral RNA associates with E. coli ribosomes in the absence of cofactors other than Met. The complex thus formed appears to consist of one molecule of viral RNA and one 70s ribosome, and is capable of carrying out polypeptide syn- thesis. In a typical preparation of ribosomes, 10% are capable of binding viral RNA; the same fraction functions in the amino acid incorporation assay. The association of viral RNA with ribosomes is reversed in 107\" M Me’. Both 30s and 50s particles appear to contain site(s) for viral RNA attachment. -ili- We should like to thank J. Ofengand for gifts of ribosomes and supernatant in the early stages of this work; L. Johnson for technical assistance; and E. P. Geilduschek for numerous helpful suggestions. * This work was supported by Grant E-4448 from the U. S. Public Health Service. -12- REFERENCES Jacob, F. and J. Monod, J. Mol. Biol. 3: 318 (1961). 2, Brenner, S., F. Jacob and M. Meselson, Nature 190: 576 1Q. li. le. 15. 14. 15. 16. 17. (1961). Nirenberg, M.W. and J. H. Matthei, these PROCEEDINGS AT: 1588 (1961). Ofengand, J. and R. Haselkorn, Biochem. Biophys. Res. Comm. 6: 469 (1962). Nathans, D., G. Notani, J. Schwartz, and N. Zinder, these PROCEEDINGS 48: 1424 (1962). . Britten, R.J. and R. B. Roberts, Science 131: 32 (1960). This type of experiment was suggested to us independently by W. Gilbert and J. Ofengand. Hoagland, D. R. and D. I. Arnon, Calif. Ag. Expt. Sta. Cire. No. 347 (1950). Haselkorn, J., J. Mol. Biol. 4: 357 (1962). Yankofsky, S. and S. Spiegelman, these PROCEEDINGS 48: 1069 (1962). The ratio of 70s to 50s ribosomes in 0.05 M Tris, 0.07 M KCl, 0.01 M Mgt t is dependent upon temperature and upon concentration, decreasing as the temperature is raised from 2a, to 25°C., and as the solution is diluted, as one expects for an equilibrium of the type A+B C. Green, M. and B. D. Hall, Biophys. J. 1: 517 (1961). Barondes, S. and M. Nirenberg, Science 138: 813 (1962). Spyrides, G. and F. Lipmann, these PROCEEDINGS 48: 1977 (1962). Gilbert, W., J. Mol. Biol., in press. Ishihama, A., N. Mizuno, M. Takai, E. Otaka, and S. Osawa, J. Mol. Biol. 5: 251 (1962). Risebrough, R., A. Tissieres and J. Watson, these PROCEEDINGS 48: 430 (1962). ~ 13- 18. Warner, J., A. Rich, and C. E. Hall, Science 138: 1339 (1962). 19. Warner, J., P. Knopf, and A. Rich, these PROCEEDINGS AQ: 122 (1963). 20. Cannon, M., R. Krug, and W. Gilbert, unpublished results. TABLE 1   Calculation of RNA:Ribosome Ratio ug 4260 4e60 As60 ug ug RNA FRACTION RNA RNA TOTAL RIBO RIBO Ug RIBO” MOLAR SOMES SOMES SOMES RATIO   1 -3  .007 .042 .035 2.0 215 .20 2 4.009 «-«.027'—=««018-—Sssa1.0 40 51 3 -7 016 .052 .036 2.0 35 45 4 1.4 .032 .098 .066 3.7 38 49 5 2.3 .053 .238 .185 10.3 .22 28 6 3.2 .O74 .571 .497 27.6 .12 215 7 3.2 .074 1.285 1.211 67.2 205 06 8 2.7 .062 2.500 2.440 136.0 .02 02 9 1.7 .039 1.590 1.550 86.0 .02 . 03 10. 1.4 .032 .453 .421 23.4 06 08 11. 1.05 .024 .152 .128 7.1 215 .19 12, 1.0 .023 .163 .140 = 7.8 .13 .17 Data from Fig. 3b. Each fraction was 0.4 ml. The extinction coefficient for TYMV-RNA was taken as 23 and for E. coli ribosomes as 18 for 1 mg/ml at 260 mu. See text for details of calculation. - 14 . LEGENDS TO FIGURES Fig. 1. Resolution of the 5-20% linear sucrose gradient. 7° labeled TYMV (S=115) at 35,000 rpm, Centrifugation of P 25°C. for the times indicated. Pig. 2. Attachment of TYMV-RNA to ribosomes. Centrifugation at24,000 rpm for 2 hours at 25°C. 1.0 mg of ribosomes and 4 Le 72 _tabeled RNA were applied to a gradient containing 0.01 M P Tris, 0.01 M Met, Solid line indicates absorbancy, dashed line radioactivity. Fig. 3. Effect of KCl on the RNA-ribosome complex. Centrifugation at 35,000 rpm for 45 minutes at 25°C. 400 pug ribosomes and 20 ug pe -~labeled RNA in 0.1 ml were applied to each gradient. Gradient in (a) contained 0.01 M Tris, 0.01 M Mg'*; in (b) contained 0.05 M Tris, 0.07 M KCl, 0.01 M Met*. Solid line indicates absorbancy, dashed line radioactivity. Fig. 4. Stoichiometry of the interaction between TYMV- RNA and E. coli ribosomes. Centrifugation at 35,000 rpm for   45 minutes at 25°C, Gradients contain 0.05 M Tris, 0.05 M KC1, 0.01 M Mg’. To each was applied 0.1 ml containing 300 ug ribosomes and (a) 110 ug, (b) 42 ug, (ce) 27 pe pe -labeled RNA. Solid line indicates absorbancy, dashed line radioactivity. Fig. 5. Correlation between RNA binding and amino acid incorporation. Open circles were obtained from six -15- separate gradients as in Fig. 4. The same cold RNA was used for isotope dilution in the gradients as was used in the amino acid incorporation assays, filled circles. Fig. 6. Dissociation of the RNA-ribosome complex. Centrifugation at 35,000 rpm for 60 minutes at 25°C, 72 _jabeled RNA in 0.1 ml of 300 ug ribosomes and 7 wg P 0.01 M Tris, 0.01 M Met? was applied to a gradient con- taining 0.01 M Tris, 0.0001 M Met, Solid line indicates absorbancy, dashed line radioactivity. Pig. 7. Attachment of TYMV-RNA to 30s and 50s particles. Centrifugation at 35,000 rpm, 25°C., for (a) 45 minutes and (b) 60 minutes. 0.01 ml containing (a) 250 ug 50s particles and 24 ug RNA and (bd) 180 pg 30s particles and 48 ug RNA was applied to gradients containing 0.05 M Tris, 0.05 M KCl, and 0.01 M Mgt,           am nee es     1000 ;— 2 500K 9.0   Volume , ml Fig.| +500 + 400 4300 & — 200 + 100           us O9z ‘Adunquosqy   mi. ? Volume Fig.2 Absorbancy ,26Ompz                                                       T | | _ a 4.0 re\" 4 — 1500 1 4 30+ 1 ! rot — 1000 20 ky Ly § u — 500 LO - te Lo - em, _— l | | 40 7 p TL + 1500 —_ 3.0 + r-Fa Pye I 7 — 1000 ra i 2.0 t- i i ; § L rH red 2 4 500 OF ' md -a al Qbeneeeseeemand a ee l | I |.0 20 30 4.0 5.0 Volume , ml Fig.3 C.p.m.   — 1000   prrcerre-             Aw ogg ‘Aounqosqy Fig.4 lO 20 3.0 40 5.0 Volume , m! PUNO YNY sod UoloD14 8     g yl       I S = pe | © Oo wn jOs0d409uj aUIONE7 - gH wdo -0.5 -1.0 log RNA/Ribosome Ratio -15 -2.0 Fig. 5     — !000   3.0 --         | | Oo O ai = mlwogz ‘Aounquosqy 9.0 3.0 40 2.0 |.O Volume , ml Fig.6               i I o om, @ TE cr o x?) c? £ Ww —_ Ss L OQ A a 2 | | | : AN | | oO OQ Oo ° Oo\" 9 OQ fe) Oo nm N - \") AN - Tlu ogg ‘founquosqy Thu Q9z@ ‘kounquosqy Fig.7", "Haselkorn, Robert ; Dahlberg, James ; Fried, V. A.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5azu-jkgr~5xjm", "00000000-0000-0000-5F00-953AA5EDB956", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "On the Coding of Genetic Information", "101584910X104", null, "1963", "1963", "This collaborative article is the product of the Cold Spring Harbor Symposium of 1963.  By the time of this meeting a great deal of information about the code and the translation process had been obtained, but the fine structure of the codon base sequences remained to be clarified.  The authors discuss the efficiency of synthetic polynucleotides, coding ratios, the current \"code word dictionary,\" and synthesis of nucleotides.  Tables and figures are included.", "Reports", "Genetic Code,Nucleotides,Amino Acid Sequence,RNA, Messenger ; DNA", "Translating the Code of Life and the Nobel Prize, 1962-1968", "43", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "ON THE CODLUG OF GENETIC INFORMATION By M. W. Nirenberg, O. W. Jones, P, Leder, BR. F. G. Glark, Wo S. Sly and &. Pestka, Rational Institutes of Health, Bethesda, Md. In Press Cold Spring Harbor Symposium 1963 dhe process of exaressing gayectic fodormwcconu wy Sdlgmaig, Giaateo ecida in proper sequence during protein eynthesia usually requires the RHA polymerase catalyzed synthesis of a strand of BHA complomentary to DHA. Recent experiments reported at this Symposium end elsewhere suggest that cRNA (mescenger RHA) synthesized in vivo is complementary to only one of the two strands of DNA (Robison and Guild, 1963; Marmur, 1963; Spiegelman, 19633 Wood and Berg, 1963). The mRWA becomes beuad to ribosomes, perhaps forming a polysomal aggreate and the amino acids may be carried to these sites and ordered in corvect sequence by specific transfer BNA spacies, Tt is possible that wkN/. codewords are read from a fixed point by nucleotide sequences in trensfer BiA complementary to those in mRNA codewords. Thus coding arrere during proteis synthesis may be minimized by the requirement for correct recognition at three successive steps; that is at the DRA mRNA, sRNA-f:rensfer RNA (or other intermediate), and amino acid- transfer ENA» activiting enzyme levels. Little is known about the mechaniens which impart speci/Acity at the lest two steps. Some Factors Infliencing the Mes © Bffict 9. Pol ieotidea The effect:s of base composition, catalytic ability, molecular waight ? end secondary si:ructure upon the measenger activity of synthetic polynucleo- tides will be considered at this time, __ The messenger activity of synthetic polynucleotides may be related to ite molecular weight. In E. coli extracts, poly u containing more than 100 uridylic acid residues per chain has Sreater template ectivity then smaller chains (Matthael et al 1962), but olfgo A fractions conteining as few as 9-10 adenyiic acid residues per chain recently have been found by Jones eat al. (2963) to direct polylysina synthesia, Also, in yeast extract® oligo U af reovren eheota Tenetts th rattunte Sterna _ ee te ee Meee See ee eae cd ve ee ee ‘ee fee “   cnucleaotides may be degreded by nucleases move ropidly than polynucleotides and thus appear to be les ficient as templates for protein synthesis, RNA chain-length aust be considered when comparing template activities of different BNA fractions. Also, secondary structure of RNA ereatley influences its messenger activity. When poly U is mixed with poly A, double- and triple- stranded helices are forned vhich are completely inactive in directing polyphenyl- clenine synthesis (Nirenberg and Matthaei, 1961). Oligo A also forms helices with poly U, and the extent of inhibition of polyphenylelanine synthesis can be correlated with oligo A chain-length and oligo A-poly U helix stability (Nirenberg et 91,1963}. In addition, Singer et al,{1963), have investigated a series of copolymers containing varying amounts of U and G and have found that guanine-rich polymers containing a high degree of oxdexed secondary structure (perhaps due to G-¢ interacticns} elso are inactive as templates for protein synthesis. These results suggest that RNA with a high proportion of helical structure may have Little template activity for protein synthesis. Recent experiments have whom that poly U-poly A helices do not bind to ribosomes, and for this reason may be unable to direct protein synthesis (Cukier and Nirenberg, unpublished results). It also is possible that small, localized areas of ordered structure may serve as periods in protein synthesis, ft is difficult to compare directly the messenger efficiencies of different polynucleotide prepat rations because the efficiency is modified by molecular size and secondary structure. However, if the average chain length and secondary structure of different RNA preparations are assumed to be a = £5 approximately equal, the data of Yeble I suggest that nucleotide content may not influence greatly the overall template efficiency ef mRNA. Poly U, poly UC, poly ACG and poly UACG contain 1, 8, 27, and 64 triplets respectively, and the preparations of these polynucleotides shown in Table I have been found to direct 1, 4, 9, and 10 emino acids, respectively, inte protein, The essential point is that approximately the same totel quantity of emino acids were directed into protein by each polynucleotide. Although these data must be interpreted with cere beceuse the same factor may not limit the ineorporation rate of each amino acid, they suggest thet the polynucleotide preparations may have approximately equal template efficiencies and that most nucleotide sequences may be able to sede for emine acids. Although nonsense sequences mey exist, thus far, none have been demonstrated definitively. Volynucleotides conteining #11 base combinations now have been used to direct protein synthesis in E, coli extracts, A qualitative summery of these data is presented in Table 2. Only those polynucleotides containing the ttiniousn bases necessary to direct an amino acid into protein ere shown. For example, phenylalanine is directed inte protein by poly U and other U con- taining polymers; however, since other bases are not required, phenylalanine is listed only under poly U0. Poly U, poly A, and poly C direst phenylalanine, lysine and proline, respectively, into protein. Polylysine synthesized in Ee cali extracts under the direction of poly A hag been found to contain 3-15 lysine residues per chain (Jones, Yaron, Seber, Heppel and Nirenberg, une published results}. No wessenger activity has been demonstrated fer poly G (Natthaed. et al , 1962), but the highly ordorad structure of poly G might mask template activity. However, @ polysuclestide composed enly of hypoxanthine whe (poly I) with less secondary structure than poly ©, still hae not been found to direct amino acids into protein. Since hypoxanthine can replace @ in RMA code words, the 2-amino group of G does not appear to be essential for coding amino acids (Basilio et al, 1962, Niernberg and Jones, unpublished data). Bach polynucleotide composed of 2 different bases has 8 triplets, but no polymar has been found to direct more than 6 different amino ecids into protein. Poly UC is unique in that ic codes for only 4 amino acide, even though ali UC triplets appear to function se codawords (see Coding Ratio Section). It ie imortant to note aleo that polynucleotides containing only two different bases direct with grest specificity almost all amino acids into protein. These findings undoubtedly reflect basic molecular characteristics of both the recognition procese and the general nature of the code. The Coding Batic A series of poly AC and poly UC preparations with different proportions of bases were synthesized and thelr activities in Stimulating cell-free amino acid incorporation into protein vere determined, As chown in Fig. 1, poly AC directs the incorporation into protein of proline, histidine, threonine, asparagine, glutamine and lysine at linear rates for 15-20 minutes. Reactions were terminated after 10 minutes of incubation, while the rates of incorporation were still linear. In Table 3 ie presented en example of the deta obtained for each of the five poly AC preparations tested. The theoretical proportions of the abn four doublet and eight triplet parmitationa cxzpscted in randomly-orderad poly AC (containing by analysis, 47 parcent A and 523 percent ©) are shown 4n the first and sacond columns respectively. in the third and fourth columis are shown the pumoles of esch cl4 waminc acid directed into protein by this polyner. A total of 1685 yumoles of amino acids were dixected into protein, and the roiastive proportions of each amino acid incorporated, in percent, ara shown in the last coluan, The 4 doublet parsutations do not contain anough specific information to code for the 6 amino acids incorporated, whereas the information content -@ the § triple: words is adequate. The percent incorporation of lysine, asparagine, glutamine and histidine agrees wel) with triplet codeword fre- quencies, but not with doublet frequencies. If all triplets were read, some amino acids would respond to 2 or more codewords, for 6 amino acids would then be coded by & words. In such cases, che sum of the triplet frequencies would have to be coupsred with the corresponding amino acid incorporation data. For axample, 1£ CAA and CCA both coded for one amino acid, the sum of their frequencies is 24.9 percent, which cannot be distinguished from the frequency of the doublet CA (aleo 24.9 percent). Therefore this experimental approach may allow determination of the coding ratio for some, but not all, avino acids. Analyoia of a series of polynucleotides with varying base-ratios per- mits comparisons to be made with greater accuracy. The expected statistical relationship between codeword frequency and polynucleotide base-ratio are presented graphically in Pige. 2 and 3. Theoretical frequencies in percent of doublet and triplet codewords are shown on the ordinate ani the base-ratio ia shown on the abseissa. Nucleotide sequence 16 arbitrary, and eath curve representa only one of the three possible sequence permutations. As nored before, the sum of the frequencies of the triplets AAC and ACC equale the frequency of the doublet AC. Thus the AC curve represents either the doublet 40, or the sun of the two triplets AAC plus ACC. Also show are the obsarved ci4. amino acid incorporation data, Sach point represents « different poly 4G preparation with the indicated base-ratio. As show in Fig. 2, the ob- sarved incorporetion of c*. nistidine agrees well with the theoretical fre- quency of the triplet ACC and differs markedly from both the AAC triplet and 4&2 doublet curves. Tha data also demonstrate that the ebserved incorporations of both cl - asparagine and cM. slutamine ogres well with the fraquencies of AAC triplets. In contras:, the incorporation of cl. threonine te sinilar to the expected frequencies of either the doublet AC, or the two triplets, AAC pluo ACC. Therefore, threonine appears to be coded eather by a doublet of by two triplets, and it is not possible to differentiate batween thece altematives on the basis of these data. in Fig. 3 are presented the template activities of poly AC preparations for c*. proline and cl. 1ysine. ‘the experinantally obtained incorporation data indicate that proline is coded either by the doublet cc or by the two triplets ccc and cca. cl. rystne appears to be coded by the triplet AAA. ming Agi Tocorporation Directed by Roly UC, The data of Fig. 4 show thet proline is directed into protein either by the doublet CC er by the eum of the two triplets coc end cu. c!*-phenytelenine eppaate to be coded either by the doublet uU or by the tuo triplets UUU ané WUC. it 4e important to note that if the codewords corresponding to thece amino scids are triplets,      =]~ both CCG and CCU would code for proline and both UUU and UUG would code for phenylalanine, In Fig. 5 are shown the poly UC-directed serine and leucine incor- poration data. Both serine and leucine appear to be coded either by the doublet UC, or by the two triplets BUC and UCC, Coding of serine or of leucine by one, rather than 2 triplets is not indicated. It is important to note that if serine is coded by triplets, one triplet would have to contain 2 U residues and the other 2 C residues, Triplet words for leucine also would contain either 2 U or 2 C residues, These experiments strongly suggest that histidine, asparagine, glutamine and lysine are coded by triplet words and that the RNA cade cannot be com- posed only of doublets. Threoutne, proline, phenylalanine, serine and leucine were found to be coded either by multiple triplets or by doublets, These gata are summarized in Table 4. A mixed doublet-triplet cede cannet be excluded on the basis of the available data; however, a uniform code con- taining only triplets would appear more probable. The Current Codeword Dictionary. Assuming for the present that all amino acids are coded by triplets, current approximations of RNA codewords may be summarized as shown in Table 5, Nucleotide sequence is arbitrary. Flity of the 64 possible triplets have been assigned, Almost all amino acids can be coded by polynucleotides containing 2 different baces, Since polynucleotides containing 3 bases direct protein synthesis as efficiently as polymers containing only 2 bases, it seems probable that most 3 base words are recognized. Tentative assignments are given for such words, It seems clear that most amino acids are coded by multiple words, Furthermore, multiple words corresponding to one amino acid often differ in base composition by only 1 nucleotide, These observations also suggest that nucleotide sequences in multiple words often may be identical, A triplet code may be constructed wherein correct hydrogen bonding between 2 out of 3 nucleotide pairs may, in some cases, suffice for coding, or alternatively, a base at one position in the triplet sometimes may pair optionally and correctly with 2 or more bases, Tt should be noted that a triplet code of this type in some respects would bear a superficial re- semblance to a doublet code and would be in accord with all of the dats available, The coding data obtained thus far clearly indicate that most nucleotide sequences can code for amino acids with great specificity. Weisblum et al (1962) have reported that multiple species of leucine transfer RNA recognize different codewords in synthetic polynucleotides; however, additional data presented at this symposium by Benzer and by von Ehrenstein and Gonano suggest that codeword specificity in directing leucine incorporation may be greater with synthetic polyaucleotides than with natural mRNA. It is important to emphasize the possibility that raudomly-ordered synthetic polynucleotides may test the cell's potential to recognize codewords, and that the entire potential May not be utilized in vivo, except perhaps during mutation. Thus oRNA synthesized by a cell may not cantain as many codewords as randoaly-ordered polynucleotides.   £ 1 fe LO Ted § OVS OY ©2   Who, OY have sir lar etructure   Wor example phenylalanive, iyvogine end irepiocphan are derived from Shitovie acid, and isoleucine, valine end teueine are evrnthegized froin raela butysae, BNA codewords correspon ning to thege amino acide a> Such comparigons evgg¢est thay a facile of amino acids w: BY Peo wytioes a family of codewords whoae membera contuia alrailar bases Althe ie!   all amine acida in ible patiera, enough additional examples 3 may be clus! é Ge tonay do: -lopmen'! of the code. ar the recagnition of aucleotides in ccd « eS to warrant ‘he suggestion that guch releiionshing reflect either the evota- le Ca words hy «ine acide. The latter hag been oroposed by Woese 1963) and alae is ¢ cussed by Weinstein in this aynmporurn. “10 OLEGODEOXYSHYMIDYLATE DIRECTED POLYLYSINE SYNTHESES The chemical synthesis of oligodeoxynuciectides by the method of Khorana and his associates (1961, 1962) and the demonstration of an ollgo- deoxynucleotide-dependent synthesis of polyribonucleotides, catalyzed by RNA polymerase (Furth et al, 1961, Stevens, 1961, Chamberlain and Berg, 1962, Falaschi et al, 1963), provided an opportunity to study their ability to stimulate cell-free amino acid incorporation. Since poly A serves as a template for polylysine synthesis (Gardner et al, 1962), oligo aT Coligodeoxythymidylate) has been used to direct poly A, and subsequent polylysine synthesis, as follews: 1) atp Oli SOL RIEVaTE —y» Poly A + PP   2) Lysine POLY ALL 2 wwe ee eee ee ee & Polylysine E. coli Extracts, etc, > uy In addition, natural DNA and poly U have been shown to direct polylysine synthesis, Poly A was synthesized in RNA polymerase oligo dT reaction mixtures (stage I) as described in the legend accompanying Fig. 6, and then components supporting amino acid incorporation into protein (Stage IZ) as in the legend of Fig. 7, were added, After further incubation, incorporation of cl 4.Lysine into polylysine was determined by precipitation with a TCA - tungstate solution (Gardner et al, 1962). The data of Fig. 6, show that cl4.AMP incorporation was dependent upon the addition of oligo dT13.14 (13-14 nucleotides per chain) to stage I reaction mixtures, and that ch 4eayp incorporation was proportional to the   succeed wut a BG ose, AT Wheapt wetedetes hey om aces ae eS gees Seem amore gee a one BUSI OF ORR Oe EAM WAUMMI gun TAGs? Gh wath GE ZOOS PME AGS Ga ake   14 product synthesized in the pre- fhe average chain length of the ¢ sence of oligo aT 3.14 was determined by deproteinizing the reaction mixtures, rencving the co! agp by paper chromatogreshy, hydrolyzing the lf “product in 0.2 N BOR and separating the aucieotides by paper chromatography. Tha radio- acciwity of adenceine, sdenosine-3' ¢2'}-S'odiphoepkate am’ adenosine-3' {(2’jeo gunephosphnte was Seund to be 339, 308 and 22,900 counts por minute, respectively. ‘Shus, oli to ag 330}4 stimulated tha synthesie of poly A of average chain Length fe70 pA Cadanylate) residues. These data confirm similar results obtained by Furth et al (1961) and Felescht et al (1963). Falaschi et al (1963) also demonstrated that elign af chaine are aot alongated by the addition ef (pA) residues to the free 3-hydronyl enda of olige df chains, and hava obtained avidence which suggests that oligedeazy- aucleotides serve as templates rather then primera, Although cus BNA polymerase praparations wore purified 100-150 foid (Chesberlain snd Berg, 1962}, we have detested unprimed mucleotide incorporation under other conditions. Yurther qzyme purification will be necessary to datermine unequivocally whether oligedecoxymiclastides function only aa texplatee in this systen.   After incubating stage I feactfion mistures at 37°, ategs IX components were added ag deceribed in Ebe 14 legend sccompanying Fig. '7 . Wo ducrease in C” -lysine incorporation vas found im the absasace of oligo dT, whereas the addition of 1.2 mumoles of i4 (pdf) rvasidues in oligo oF 3.15 stiuuleted C’ olysine incorporation at a   gi En 240% ae ¥   138, & & ry % cy   Tn ay ORO yh   nye 236 a &   ey sg a» nd FE We te 8 Cee) cae 1 fet at 3. & Pub h 2 hy, & a ae W4   RENT     om & pe     & a eo ay 2 at     at fou re sie a s ee Sardge { a v a fe ard re ¥: or ors   sates 2D * 38 2 ante Bane oa t ee a & Baw oF z   Es moa gd 2 be & iad 43   Se ae     a ; % oe us ot be ob v3   a &   a” Paahat haale of % a Be oe ya   &:   1 Bey ce 4   @ PESTGIR, oe fe   tion tn aed: Stash POP = et & B2E BE a   a & a 1a . a s ere 4 <   Salenot ea comple iy     mae, coke ho tm Ce eR ge Poe Sten WG OUR CO BIg 2 Mean bis PGE 9 Fe} s ey 9 per SS 3 @ & ket ie {chamberhain and Bexg, 19623 ¢ MA cCapendeank synthesis of poly A fcom AEP (in the absence of UTP, GIP and CTP). Under these conditions, Ss = 2s, 2 s, Ge. - * fe poly A syuthesiged under the divsckion of calf thyms BUA stinuiated ct “ lysine iasorppazatzion, Pa’ “Ss i chromategsaphy of polylysine as described in the degend accompatiing Fig. 8, permits separation of lysine peptides of different chain icagths. Peptides containing appromiwately eleven or more lysine gesiduc: cemain at the origin, whereas the mobilities of smaller peptides o nye %, \\\\ a Ca ey te oe %, se 1m + ave ar followss lysine » die 7 trhe ? tetrae > penta > hexae > hepta«> . : ca om oe. 14 : ecte- > nonay » deca-lysine., As shown in Fig. 8, most of the G°”epraducs syiihesized in the presence of oligo Ge 3014 remained ak the ovisin after 2 ‘ 14 chivmatogiuphy. Digastion with trypsin converted the C”\"eproduct almost qu.atitatively to peptides hich migrated with frce, dix, She, and tetzae jysine chneacteristics, . 16 . «4 Za supatate experiments the €” «product was eluted from the origin, . : , . fe 1& . Both aliquots were chromatographed as before, Gne C°\"*spot having the characterlutice mebilicy of free lysine was found following aeid hydzolysis. . . : i, . . Stans Atcer digastion with trypsin, C” “eproducts with the expected mobilities of 4 . 14 free, die, trle, and tetra-lysine were found, ia addition che C= & 3 8 polylysine which remained at the origin was shova to contain carbexyl- ty ee oa . : I elysine residues by a hydzaginolysis method (Akabori et al, 1952). terminal ¢ Effect of Molecular Weicht upon the Activity of Oligo dt, Falaschi ct ai. (1963) denonstrated that oligo d? ebains containing less thaa 4 residues REPS REECLS AKABORE, S., K. OHNO, and K. NARITA. 1952. On the hydvaginolysis of proteins and peptides. Bull. Chem. Soc. Japan, 253 214-228, BASILIO, C., A. J. WAHBA, P. LENGYEL, J. F. SPEYER and S$. OCHOA. 1962, Synthetic polynucleotides and the amino acid code, V. Proc. Nat, Acad, Sci., U.S., 483 613~616, CHAMBERLAIN, M. and P. BERG, 1962. DNA directed synthesis of RNA by an enzyme from E. coli. Proc. Nat. Acad. Sci. U.3., 483 81-94. FALASCRE, A., J. ADLER and H. G, KHORANA. 1963. Chemically synthesized deoxypolynucleotides as templates for RNA polymerase, J. Biol. Chem. (in prass). FURTH, J. J., 3. HURWEZTZ and M, GOLDMANN. 1961. ‘The directing role of DNA in RNA synthesis. Biochem. Biophys. Res. Com., 42 362-367. GARDNER, R.S,, A.J. WAHBA, C, BASILYO, 8, S. MELLER, P. LENGYEI, and J. F, SPEYER, 1962. Synthetic polynucleotides and the amino acid cede, VIZ. Proc. Nat. Acad. Sci. Us8., 48: 2087-2093, JONES, 0. W., EB. E. TOWNSEND, H. A. SOBER and L. A. HRPPEL. 1963. Effect of chain length on the template activity of polyribonucleotides. Biechem, (in press). KHORAMA, H. G. and J. P VIZSOLYI. 1961. Studies on polynucleotides VIII. J. Am, Chem, Soc., 83: 675-685. KHORANA, H. G,. and J. P. VIZSOLYI. 1962. Studies on polynucleotides XIX. J. Am. Cham. Soc., 84: 414-619, KRAKOW, J. S. ard 8, OGHOA. 1963. ENA polymerase of Azotobacter vinelandii, 2. Proc. Nat. Acad. Sci. U.S., 493 88-94, Refervaacas -2< MARCUS, %., A. K. BRETTHAVER, B, HN. BOCK and 8. 0, RALVORSON, 1963, The effect of poly U eise on the incorporation of phenylalenina gn the cell-free yoast system. Proc. Mat. Acad. Sei. U.8.,5 {in press). MABMUR, J. 1963. Biological and phyoical properties of bacillus bacterfcphegs HA. This Symcetum. MATTEART, J. H., 0. W. JONES , R. G, MARTIN, and M. W. NZREIDERS, 1962. Characteristics end composition of BNA coding units. Pree. Hat. Acad. Sci. U.S., 48: 666-677. HIBENBERG, M. W. and J. H. MATTHARI, 1961. The dependence of cell-frce protein synthesis in E. coi% upon neturally occurring of eynthetic polyribonecleotides. Proc. Mat. Acad. Sci. U. 8., 47 1588-1602. WIRENBERG, M. W., J. H. MATYHART, O. W. JONES, R. C. MARTIH and 8. BR. BARONDES. 1963. Approximation of the genetic code via cellefrce protein synthesis directed by template RRA. Fed. Proc. 22: 55-61, ROBISON, M. and W, RB. GUILD. 1963. Evidence for message reading from a ubique strand of DHA. Fed. Proc., 22: 643. SINGER, 4.F., O.W. JOWES and M. W. NERENBERG, 1963, The effect of secomlary . structure on the template activity of polyribonucleotides. Proc, Nat. Acad, Sci. U.8., 49 392+399. SPIRGELMAN, S$. 1963. Some properties of the genetic transcription mechaniamn, This Symposium, STEVENS, A. 1961 Net formation of polyribonucleoctides with base sorpositiona analogous to DNA. J. Biol. Chem., 236: FC43-PCAS. WALEY, 8.6. and J. WATSON. 1953. The action of trypsin on polylysine. Biocken. Jeegat 328-337. Hetecangns Ge WAlBA, A. Je, B, S. GARDNER, C, BASZLIO, H, S. MELLER, J, FP, SPAYSH ond DP. LENGYEL, 1962. Synthetic polynucleotides and the anine acid cada, Wilt. Proc. Heat. Acad. Sad. ULS., 49: 116-122, WEISS, 8. B. and T, MARAMOTO, 1961. On che participation of DMA fn BRA synthesis, Proc, Nat. Acad. Scl. U.8., 473 694-697. WRESS, 8. B. 4963, Properties of DNAedopendent aynthesiaed BNA. pp. S167. gsfsrastional Macromolecules, Rew York. Aeadenie Praga. WEISBLUM, B., 8, BERZER and B. H, POLLEY, 1962. A shysieal basis for degenexacy in the amino ecid code. Prac. Nat. Acad. Sel. U.8., &&3 1R49-1456 , WOESB, C. 1963, Tha Genstic Code. 4.0.8.0. Reviews, (in presa) WOOD, W.5. amd P, BERG. 1963. Seudies on the “messenger” activisy of BMA synthesised with BHA polymerase, ‘his Syapesiun. TABLE 1, ‘TRIPLATE ACTIVITIES oF 1, 2, 3 AND 4 BASE POLYNUCLEOTIDES       POLYNUCLEOTIDE U uC ACG UACG u 100 AT 7 56 BASE RATIO c - 53 32 13 HOLES-PERCENT OA ° ~ 46 5 6 - - 22 25 POSSIBLE TRIPLETS 1 8 a7 64 cl4anio ACIDS DIRECTED PHE PHE LYS ILEU PRO INTO PROTSIN LU ALA HET LYS SER ARG CYSH ALA PRO SER VAL ARG THR Gk oR GLU-NH, TRY GLU-NH, ASP-flily TYR ASBONIL, HIS PHE HIS PRO LEU SER rota c!4.anmo ACID INCORPORATION (munoles) 3e2k 2.91 2.22 5009   Legend for Zeble 1b. See text for details. Cedeword nucleotide sequences are arbitvary. TABLE 2, SUMMARY OF CODING DATA re POLY U A c G RE NST AE: a       PEE LYS FRO © POLY DA te UG AG AG CS UAS TYR LEV LEV HIS ARG ARG bar LEY SEB VAL ASP NH, GLU ALA AS? TILED CYSH GLU-NA, CRUSNHL, SER ASP Nia TRY THR Asp® 7°         Legead fer Zable 2. ve? Ouky thasa polyanuslootid¢es eontainiag the atntmal wmxher of bases necaasery te atinalate on amino acid ints protein are showa. Amino seido codsd by homepolynucleotides axe net listed sgain under rendomly-orderad polynucleotides. *Predicted ' Reported by Wahba et al (1963) CCHPARZSCH DOSTVGEN AMINO ACTOS INCORPORATED wise site foe SA GODEEGRD FREQUEKCT ES               ated le Te a vi ae a ney a Se ARO a Wd PENN Bs ea SRV WMS D AIOE EE GEG i iF AT Bim d 7 . - 1, . . Thearatiest Codeword GU 'shmino Acids Tauvorpovated fnte Pare steney in Poly an Con Protein . Featyse ; : FREQUENCY OF ceining 47% & and 455% ¢ 34 ; 14 LLMOLES a .20NG ACIDS ae 3 wee ary my Pa oa fT * ae “ eke ROULLATS TRIPLETS C'MAIIHO ACID THCOREOPATED © THOCEPORATED peorcant Parecug aA Ooaak AAS 20 ott TYSIRE 383 20,8 AC 24.9 AAG 13,7 ASPARSB ING 192 13.6 GA 26.9 AGA 13,7 GLUTAMINE 257 6,3 Co 28,1 Gok 1.7 THREONTNE fit 26,3 oo Poe CCA 13,2 BUSTIDING 1s9 54 2 >. to oy 1 . Fotak 160.0 age 13.2 PROLIVS $50 32,6 CAS 13.2 ner eres COG Mod fotei 1685 Total 10.0 Leral 199.0 LESENDS FOR TASLES Legend for Table 3°\" Back caaction mixture contained the following compcnents 3a Final voluee 626,25 al: 0.2 # feic, pl 7.8; 0.01 & maguesiun asctates . «3 - 7 =3 is & 3 20 i mereaptoethancl]; Jn 10\" M AY®; 5 x 10°? & i trae ter cy 2.6 Bi + potaceiun phosphoensclorravate: § ug of evgatallias phosphoancloyruvate Einuase {(Cxiif, Corp, Eicchem, Research); 0.9 2 1077 wc eaninge acd; skotein Quivctberg and Matthsed, 1961) Reaetion wlstures wore incubated   a a hg a 2 - a at 37° for 10 ninmutes. Protein precipetation, vashing, and om parforne? so deceribed by Rirerberg end Metthact (29633, the theoveticsl fracuenvies in percent, of dovblcta end triplers 2 in polyneclestides vera caleulated aa folious: The fseqiensy of tho tripe % int J08 in this poly AG preparation would ba .47 2.47 B .47 2 200 — 10,4 percent, The doublet feaqusney fer CA would be .47 « .93 = 100 = 2h, pSeTGone, The pyroles of each amino acid inecrporated in the abseuce of poly- aueleotide wars: lyedne, 30: asparagine, 54; glutarlne, 493 threonine, 403 histidine, 263 proline, 36. TABLE 4 . SUMMARY OF CODING RATIO DATA 14 CODEWORD   Co -pHINO ACID TRIPLET DOUBLET RISTIDUE ACG - ASPARAG IE CAA - GLUTAMINE AAG - LYSINE AAA - ‘CHPZOR INE CCA + ACA or AL DROLINE GCG + GAC + CUC or CC PRENVLALARIEE BUY + UCU ox UU SERINE cUU + CCU or CD LEUCINE vUC + UCC or UG   % Muclestide sequences are arbitrary, ‘a ASBY OF EMA COOEVORRS   AGTGG {5705 See EIN, Dah,   SSPS FAST ASFARLEG ACID CYSPEIPS GRUTSHES ACTD CLUSERT IRE CEYOTRE BISTLOLAS REOLS0CINE LEOGOINS LYSTER PORTNLALAB INS FROLIRE SERIES THRECIENS TRY PLOSIAN SVEISIRS VALENS   PSM RE al OCT   ese oAe Slee ACA GUA GUS GAA BAG GES ACC DAD BUS bbe GA Boy Ces oe CAS GN AUYy Tey   ues! AGH AUA CoA} aay? AGA AGS Agy' GEA Gas &&S Guu ecu UL CSA uA!   BM SORE FORDE 2 ACG! wect Acg' Gaa!' anc’ Ago! C38 BoE CCA BCG       te; SGA     * Arblerary wucleotide sequence. ' Probable 7 hn oe nip ae WATT PE pt ~ f ‘ oe ae ‘ TABLE ©, RELATIONSHIP BETWEEN AVfTHO ACIDS GP SIMILAR METARCLEO ORIGIN OR STRUCTURE AND THETR ANA CODEMCRDS       AROMATIC DICARBCGHYLIC AMEE SLEU, Val, LSU ANING ACTBS ACTOS ANG AMIDES PAMILY FUE vuU ASP~Ri, BAD TLE WUA DUG ALS UAA 2¥R DUA ASP UG VAL Ou RY UGG GLU AUG ERG UuG ASB BUA uC £06 UY Aat RO te Ae OEE ON ET NE Ao EMAAR 2 I LEE © see: SATIRE a TRS PRS rs SET mera ed     BSLASISN LATURU OLTGS @P CHAT LONGI AWh AteEyIR? OIE ST TAR UE Re PE BON oe vere ee at et ORE! TI LY SEE GA ie TR AE i 14 AQUDC IH G 4 app G ~-LYSEYE TECOPPOOA TION EEOCOREQUG ETO (upmeles} faumoles} OS EO OR EEE Mae Ce RRR On SO BS eh IRS, RRR   (EL Ba ly AE AI eT CATR BE, ie lel Rs Ve ROBE 0.4 Q,.075 A? mersolas oligo dF 1,5 6.237 6-7 i? mungies oligo af 6.8 0.337 798 AZ aynoles olive Poa 13.9 0.406 SN ES TN TOON TPE BRN 3 5 EIS PEER OB ROMY INS SO RAPER EO PUM Eos Leone RRR TURTR TE ESE RE ETS URE 2 tne components of ehe renetdon aiztucas c24 Su the legendi acconganging Fig. $ and 7     Bice tete OF L0 camicce eda iancomporerie: Smee efetetn directed oy poly AC ibese ratic - A, 4° pexcene and @, $3 percent), Reaction mixture components are described in tha Legend of Zable 3. 3 ja 1 Lipentes : <! sot ‘fe 7 ‘ ae Ppoubetienns were efopped an tha timer indicated ty the addittor of * . c a ey we e e. Sh noes com 4% “eet ~ $e 3,9 ml. of LO rarcemt TGA se 3B\". Yho sompleg were heated af 06             mgt eK a = set ¥ oe ¢ eu minures. ther ouch KIN Tinea £1 aoe ta Ct are » ke PA ED - ” _ 7 a mae oe cyt waih GS uersont TOA au 4 iviny muscuremonioa we In            et 1] wu & «yy wm. eo >   e ere    wr a Can     report oO wit ty   ad dade   & Seek   a Sen Coun: “ nay ee PWeArag Ae we 3 cb a            roy sy ¢ tha       i 2G rots ne Bh C   x feble 3. . s bad sean YE igs eas des: a ‘2 a)         Ye", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-r3gf~3hnb-tauh", "00000000-0000-0000-1B90-E20FFE376A9E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Synthetic Polynucleotides and the Amino Acid Code, V", "101584910X105", null, "1962", "February 1962", "This report from the Department of Biochemistry at the New York University School of Medicine examines the agreement found between experimentally determined letters of the genetic code and amino acid replacement methods in nitrous acid mutants of the tobacco mosaic virus (TMV).", "Reports", "Nucleotides,Poly U,Amino Acid Sequence", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "11", "pages", "Text", "English", "Reproduced with permission of Peter Lengyel.", "Copyright may apply", null, null, "SYNTRETIC POLYRUCLEOTIDES AND TRS AMINO ACID cope, v®, aX GARLCS BASELIO’, ALBERT J. WARERS, PETER LENGYEI, JOSEPH F. SPEYER, AND SEVERO OCHOA DEPART AENT Cy BUOQOCBEMISTRY, NEW YORR UNIVERSITY SCHOGL GF MEDICINE Gemmunicated February, 1962, In the preceding paper of this series substantial agreement was found between tie experimentally deCertained letters of the genctic code! “* and mine acid Tenlacements in nitrous acid mutants ef tobacco mesaic vine, i220 fo deaminetion of certain bases, treatmens of nucleic acids with HO, -onda to baze substitutions in the polynucicotide chains end hence te ahangea of thotr nuclectide esquence®®?, Guanine is converted te santhine, adenine te hypecmnthine, and cytosine te uracil, Uracil is nat changed, In the cage of tebaceca mosaic virus RNA, controlied HNO, treatment can lead fo deamination cf a aingle base and, as a result of the base change, to the substitution of 6n cinino acid for another at a specific location in the poly- 6 that the conversion peptide chain of the protein coat. I has been assumed of guanine to xanthine, a base which is not present in nucleic acids, is isvarted {guanine taking the place of xanthine) when the RNA is replicated 80 chat no mutation occurs. Ia a similer way, when adenine is deaminated ts hypoxanthine, which like xanthine {s not normally present in nucleic acids, cuenine 4s suppoged to teke She place of hypoxanthine after replication of the RNA. In this case, however, guanine is substituted for the original adenine (AG replacement) and mutation occurs. Deamination of cytosine is uracil leads directly 20 a C—U replacement. Hypoxanthine can be substituted for guanine, i.e. inosinic acid (2 for qguanylic acid (G), in synthetic polynucleotides with retention of their coding chsrecteristics for, as previously noted*, poly UI (5:1) was equivalent 35 poly UG (5:1) in regard to these amino acids (cysteine, glycine, tryntc= phan, ond valine) which are coded by U= and G-containing letters. In iiae with this observation, treatzon? with HNO, conferred upon poly UA (£:1} the coding characteristics of poly UG (3, o.g. cysteine and vali incorporation was substituted fer isoleucine and tyrosine incerpor- Silon. This is the test tube counterpart of amino acid replacements in HNC2 mutants of tobacco moseic virus, On the other hand, deamination of guanine to xanthine by treatmant of poly UG (5+3} with HNO, sliminated the stimulation of valine incorporation caused by the untreated pelymer. aus, contrary to hypoxanthine, xanthine cannot replace guanine in the ganstic code. An account of these experiments is given in this paper, Preparetions end methods.- These were the same as in previous work! unless otherwise specified. Poly UI (5:1) was propared with Azotohscter polyaucleotide phosphorylase! from a mixture of uridine 5\"=diphosphate snd inosine 5°*=diphosphate in molar ratio 5:3, Its sedimentation coefficient was 6.33. Deamination of polynucleotides. - 720 mg of scdium nitrite wore added to a solution of 6 mg of polymer in 9 mi of 20% acetic acid. After standing at room temperature with occesiona! shaking for one hour (experiment 2, Table 2) or two hours (experiment 1, Table z), the solution was dialyzed for 5-6 aours against distilled water, with several changes, and the polymer re~ covered by lyophilization. In the case of poly UA (521), deamination of the adsaine residues appeared te be neer completion in 35 minutes as judged by extensive loss of the capacity te stimuinte the incorporation of isc- leucine info acid-inecluble products in the Escherichia coli system, Results. Experiments with poly Uk As shown in Tubie 1! poly U! {41} stimulated the incorporaticn of phenylalanine, cysteine, valinc, gly- cine, and tryptophan (experiment 1) end leucine (axperiment 2). Although she sctivity of this polymer was somowhet lower than that of poly UG (5:85 (c%,Tabie 2), the phe/cys, phe/vel, arefgly, phe/try, and pho/lev incr porstior faiiog, given in the last column of the table, were in reasonable agreement with the comesponding ratios for poly UG (5:1) (cf. Table 2 of preceding paper}. This proves that hypoxanthine can replace guanine in the genetic cacie,   UNO 2 treated polymers: The results of oxperimenis wits poly UA, UG, and UC are shown in Table 2. In addition te the changes that can be specifically ascribed to deamination, treatment with HNO» re~ sulted in a pronounced overall decrease in the activity of the polymers, This wae reflected by an 85% and 70% decrease of the capacity of poly UA (5:1) to stimulate the incorporation of phenyalanine after treatment with HNO, for 2 hours (experiment 1) and 1 hour (experiment 2}, respectively. The reason for this decrease in activity is unknown. Nevertheless, the chengs in coding characteristics of poly UA, due to A--I conversion by deo~ anination, was readily epparent. Before deamination, poly UA promoted the 4acorporation of isaicucina, leucine, and tyrosine but not that of cysteine end valinc. Deaminetion largely aliminated tha capacity to stimulate in- cesporetion cf isaleucine and tyzosing and brought forth stimulation af the incorporation of cysteine sad valine. Stiovalation of leucine incorporation was largely retained in agreement with the tinding* that ioucine is ceded by 2UIA and 2UIG (er 2UM, Fable 1) besides Z2UIC letters. Treatment of paly UG (5:3) with HNC, (guantne~xanthine conversion} virtually eliminated all activity of this palymer, Phenyalanine incerpore~ Mon was drastically decreased and valine incorporation was wiped out!®, Thus, in sharp contrast to hypoxanthine, xanthine is unable te substitute for guanine in the genetic code, The experiment with pely UC (5:1) showed retention of phenyalanine (coce letter UUU) and marked Joss of serine (code letter 2U1C) incorporetion activity following treatment with HNO2 for 1 hour (C-~U conversion}, Since the activity toward phenylalanine was retaincd, contrary to the marked drop observed with poly UA, it is Ukely that a non=spectfic decrease in activity, caused by HNO treatment, wes compensated by an increase due to conver- sion of poly UC, which codes for phenylalanine and other amino acids, %0 poly U which codes for phenylalanine only. Discuesion,~ As showa in this paper HNO2 treatment of synthetic poly- nucleotides, used es artificial messengers for protein synthesis in the £s_c9li system, leads to amino acid replacements like those observed in HNO, mutants of tobaccs mosaic virus, However, multiplication cf the virus involves two processes viz, replication of the RNA and transecrip~ tion. of its code inte a polvecuitde sequence, Our model experiments with palyarcieotides relate only to the affect of HNO on the transcription ef the mesycga. fhe finding that hypoxanthine cen replace guanine in amins acid coding is nat surprising tn view of the similarity of thesa two bases with regard to hydrogen bonding, Poly I has becn shown to form DNA-ile, double-stranded helical complexes with poly C2412, the stability of the hypoxanthine~cytesine pair ia of the order of magnitude of that of the adentne-uracil pair o6 the melting out temperature of poly A + U (61° in 9.15 M NeCl-0, 235 M sodium citrate) is only about 10 degrees higher than that of poly 1+ C!3, therefore, hypoxanthine in poly UI triplets 6.9. UUN would pair with cytosine in complementary “adapter” !* griptets (AAC) af cysteine or valine transfer RNA. However, the guenine-cytosine pair, with three hydrogen bonds, is held together more tightly than the hypo<- xanihnine~cytosine pair with only two hydrogen bonds. The lower efficiency of poly Ul es compared with poly UG, noted in a previous section, might be a reflection of this difference. Our observations are in line with the finding!5 that deoxy ITP could replace (with 25% efficiency) deoxy GTP in DNA synthesis by DNA polymerese. The further finding reported in this paper that xanthine cannot replece guanine in coding, although epperentiy net explainable in terms ef hydrogen bonding propertica (an xanthine Is similar to guanine in thig respect}, 4s also in line with the failure of decxyxanthosine tiphospaate to replace deoxy GTP in the DNA polymerces vystem!>, in view of these resulta, reversal of the gusuine—xanthine conversion {guanine taking the place of xenthine) on replication of BNO>\" treuted tobacco moszeic virus RNA, is unlikely, Deamination of guanine is raere likely to yield an RNA that is uneble to replicate (iethal miutatioas}. Modification of the coding charmeteristics of synthetic polynucleotides with agents ofer than HNO; might throw Moht on the mode of action of certain mutagens. Several amino acid replacements in tebecco mosaic virus protein have been brought about by treatment of the virus with bromi- nating and alkylating agenta?*?, However, the relationship between the chasrved replacements and the chemical effects of these mutagens on ths nucleic acid bases is obscure. Sugunary.- Hypoxanthine can replace guanine in amino acid coding for, like poly UG (511), poly UI ¢5:1) stimulated the incorporation of cysteine, glycine, leucine, tryptophan, and valine into acid-insoluble products in the Fe_cohi systam to the same relative extent. Treatment of synthetic polyau- cleatides with nitrous acid modiiied their ceding characteristics as expected from the deamination af adenine to hypoxanthine and cytosine to uracil. Poly UA lost its coding specificity and acquired thet of poly UI, and poly UG beat ite activity to stimulete the incorporation of serine but not thet of phenylalanine. Deamtnetion of gusnins to xanthine, by treatment of poly UG with nitrous acid, wiped out the activity of this polymer te stiraulate valine incerparation, Thus, contrary to hypoxanthine, uanthine cannot vaplacea guanine in amino acid coding. We are indalted to Horece Lozina for ckillful technical assisianca, \"aided by grante from the Netional Institute of Arthritis and Matabeitc Dissases (Grant A-1845) of the U.S. Public Health Service and from the jano Goffin Childs Fund fer Medical Research, The akbreviatioas uzed ia thie paper ere the same as in previcus papers of this series. The standard shoarevistions are used for amino acids, *intemationel Postdoctoral Fellow of the National Institutes of Health, U. 8. Public Health Service, Permanent address: Institute de Quimica Fisiclégica y Patoldgica, Universidad de Chile, Santiago, Chils. +F allow of the Jane Coffin Childs Fund for Medical Research. ILengyel, Po» JeFeSpeyer, and 3.Ochoa, these PROCEEDINGS, 42, 493% (1963). “speyer,JoFe, PeLengyel, C. Basilio, and $3. Ochoa, these PROCEEDINGS, 48, 63 (1962), *Lengyel, Pee JeFo Speyer, C. Basilio, and . Ochoa, these PROCEEDINGS, 48, 282 (1962). *speyor, JF.» Po Lengyel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 000 (1962), *Teugita, As, Protein, Nucleic Acid, Enzyme {Tokys, 6 385 (1961). Gwittmann, H.Ge, Maturwissenschaften, 49, 729 (1961). TE eugita, A., and H, Fraenkel-Conrat, 4,.Mgqi. Bigl,, in press. Sschuster, Ha, and G.Schramm, Z,Netucioysch., 13b, 697 (1958). IGierer, A., and ¥W.Mundry, Nature, 182, 1437 (1958), 301, Schuster (Zp Naturforsch, , 15b, 293 (1960)) has reported that the giycosidic bond betwsen pentose and xanthine in RNOgtreated DNA, is more labile to acid than the pentose-guanino bond in untreated DNA. Were this the case for RNA, hydrolytic lose of xanthine could contribute to some extent to the inectivation of poly UG following treatment with HNO2. This will be investigated with poly uridylicexanthylic acid (poly UX) prepared from uridine 5°-diphosphate and xanthogine 5*=diphosphate with polynu- cleotide phosphorylase, 'IDevis, D.R., and A. Rich, Je Agi Chemin, Soc., 30, 1003 (1958). l2pavis, DR.» Nature, 186, 1030 (2960), 13poty, P., H.Boedtker, J.R. Fresco, R. Haselkorm, end M. Litt, these PROCEEDINGS, 43, 482 (1959). M4Crick, Fo H.C., in “The Biological Replication of Macromolecules\", Symposin Soc, Exptl. Biol,» No. 12 (1958). 15g essman, Mo Jeo. LR. Lehman, J. Adler, S,B. Zimmerman, £. 5S. Simms, and A. Komnberg, these PROCEEDINGS, 44, 633 (1958). TABLE 1 EFFECT OF POLY UI(5: 1) ON AMINOAGID INCORPORATION IN E. COLISYSTEM® Expert- Amino acid Without With Net Ratio* ment poly UI poly UI No. i Phenylalanine 0,18 9.14 8.96 oo Cysteine 0.15 1, 28 1,13 79 Valine 0,22 2. 24 2.03 4.4 Glycine 0.17 0, 56 0. 39 23.0 Tryptophan 0.32 0. 53 0. 39 23,0 2 Phenylalanine 0.10 4.93 4.83 renene Leucine 0. 26 1,5) 1, 25 3.9 *mumoles/mg ribosomal protein. *Ratio of phenylalanine incorporation to that of the amino acid in question. TABLE 2 EPPEGT OF VARIOUS POLYNUCLEOTIDES, BEFORE AND AFTER TREATMENT WI ANO5Z. ON AMINO ACID INCORPORATION IN E, GOI! system* Exgeri~ Ardins &oid Polynucleatide ment VA (523) UG (5:3) US ¢5: 4) Nos Before After Before After Before After HNO2 4HNO2 HNO, HNO: HNOz HNO, 1 Phenylelenine 15.9 2.4 22.4 0,07 mee mouse isoleucine 4:3 2495 “me =~e ome ace Valine a On4 Sak Q one wren z Phenylajanine 12.8 3,6 wwe “em 15.8 14,2 Isoleucine 1,5 0,07 “ae --- cow ee Leucine 1.7? 0, 30 moe -—- —— a Tyroains 2x}, 9232 oman ol cones ame Cysteine g On16 am “oe one orem Valine 9 0,33 ~— = —- oe Serine < “ee 3n3 On7 “values given refer to net incorporation (in mumoles/mg ribosomal protein) after subtraction of smell blank incorporation without polynuclectide,", "Ochoa, Severo, 1905-1993 ; Speyer, Joseph F. ; Wahba, Albert J. ; Lengyel, Peter ; Basilio, Carlos", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5rba-kuhv_y7dv", "00000000-0000-0000-6A84-612A46AC1110", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Cell-Free Protein Synthesis and the Genetic Code", "101584910X106", null, "1962", "[ca. 1962]", "In addition to determining the base composition for fifteen amino acids, this paper by Nirenberg and Matthaei reaches the following tentative conclusions: that the genetic code is degenerate, at least partly universal, and still partly unknown.  The number and sequence of nucleotides, amount of degeneracy and polarity, and chemical nature of nucleotides are also discussed.", "Reports", "Amino Acids,RNA, Messenger ; Peptides,Genetic Code", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "12", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "CELL-FREE PROTEIN SYNTHESIS AND THE GENETIC Code by J. Heinrich Matthaei and Marshall W. Mireaberg National Institutes of Health, Bethesda, Md. Introduction Biochemistry is still largely in its analytical stage; people attempt to get exact knowledge of single reactiona occurring in the intact cell. Therefore, they need simplified systems, containing possibly only compounds involved in the reaction under investigation. However, since protein synthesis involves a whole variety of chemical reactions, biechemistsa had to etert working with crude systems, including extacts from Escherichia coli (a bacterium living in our intestines). These extracts can be obtained after grinding the fresh cells with alumimm oxide, followed by extraction with buffer solution and removing all remaining cells, proteplasts and debris by centri - fugation. One may call these extracts a “suspension of ribosomes* in a solution of enzymes and nucleic acids.“ All smaller molecules are thoroughly removed by dialysis to make it possible to have those   * Ultra microscopical particles consisting of ribonucleic acids, very basic proteins and several enszyme-proteins. compounds added in defined amounts, which are necessary for protein synathesia: 20 amino acids as the building blocks of proteing, ATP and GTP as energy sources for the reactions and salts for a certain fonic environment. Mercaptoethanol serves as a stabiliser which allows to prepare extracts and store these in frozen state without loss of activities requirad. The incor- poration ia measured by using ch amino acids and counting radto< activity in the protein precipitated with trichloroacetic acid. rvey of processes involved in tein thesis All the major processes involved in protein synthasiz seem to occur in this crude system (seq fig. 1): l. The activation of the amino acids, including an energy~ transfer from ATP (Lipmann), a liberation of pyrophosphate (Keller and Hoagland) and esterification of the amino acid to {tts spacies of transfar-ribonucleic acid (RNA) (Holley, LipmanngdZachag, Hoagland, and others). fA from 4 kinds of nucleo-   aide-triphoephates complementary in ite bases to the deoxyriboaucleic acid-strand (DHA) of the gene (Hurwits, Weiss, Stephens, and othera). This messenger-RNA (postulated by Jacob and Moned), carries by means of the sequence of its 4 spectes nucleotides the information from the game to the factories of proteins; there, it determines the specific sequence of soma 2) different amino acids within long polypeptide-chains ef up to several hundred units. The demonstration ef an inhibition of amino acid incorporation iato protein by a DNA- destroying enzyme (DitAase) by Tissieres, Wovelli, and Matthaei and Nirenbergy indicated that “nassanger-RRA\" might be produced in R- coli extracts. Further experiments with 4 “labeled nucleo- tides dons in collaboration with Dr. R. Roberts, have shown that @ messenger-l{ke RMA is only made in the absence of DNAase. How- ever, the direct evidence, a production of specific “messengers” upon the addition of the DMA from individual genes, is not established at the moment. ~Ae 3. he complex-formation between messenger-RNA and some stage in the develolment of a ribosome remains to be studied. Ilsotope-labeled aynthetic messanger-RNA, like polyuridylic acid, which we found to be highly active in stimulating seino acid incorporation, will be very useful for such favestigations on the fate of the \"messenger\". 4. The amino seid-charged transfer-RMA is assumed to recognize and bind to specific places on \"measenger”’-RHA by the clasaical base-pairing mechanism vhich wes discovered in DNA by Watson and Crick (A pairs with U, G with C}. Thus, transfer-RNA carries as an adaptor ipecifidd amino acids to their proper places on messenger- RNA, 30 Chat the amine acids can be linked, in correct sequence, into protein. 5. Peptide-bonds are formed batween tha amino acids that are sequentially arranged along the messenger-RNA-strand. The stepa favelvad are unknown and are being investigated im several Labor- atories. Here egain, the use of pelyuridylic acid coding apecifically for phenylalanine is very helpful, since it allows an enormous reduction of the precursers required for a model-synthesis of an extremely simple polypeptide chain. The assay for “messenger -RNA In order to meke all protein synthesis in our system dependent upon the addition of informational or “messenger’-RNA, we destroyed the DNA with DNAane and incubated the &. coli extracts until all of the endogenous “massenger\"-RNA had been inactivated in some way. This treatment gave us the first \"assay\" system for “massenger\"-RMA. Thies “assey~-aystem\" seamed to copy informational RRA extracted from any organism; from Z. coli as well as from yeast, tobacco mosaic virus (TMV), or Ascites twnors of mice. The synthetic RMA polyuridylic acid directed 100 times as much amino acid {ante protein than was ever observed in a cell-free system. This polymer contains only uridylic acid, one of the 4 kinds of nucleotides found generally in ribonucleic acids, and leads to the polymerization of only one - §& ~ of the 20 proteim-amino acids, phenylalanine. We showed that transfer-RHA carries this amino acid towards polyphenylalanine- synthesis.   This discovary opened the way towards the deciphering of the genetic code. The further preduction and use of synthetic polynucleotides let us find out which groups of nucleotides direct the other emine acida by means of their transfer-RRA- adaptors into their proper places in the apecific prateins. When we had enzymatically synthesized RNA containing two, three of four different species of nucleotides in a random sequence, we could determine which amino acids vould be “coded” by certain combinations of nucleic acid-bases: U, C, A ox G. The resulta ac far definitely established, are seen in Table 1. Ochoa and collaborators have reported similar findings.     TABLE 1 Amino Acid Code word determined Alanine UCG... Arginine uUCG... Cysteins UUG... Glutamic acid VAG... Glycine UGG... Isoleucine UUA... Leveine UUG... FUG... Lysine UAA... (7) Methionine UAG... Phenylalanine uuu... Proline UCC... Serine Wuc... + UG... Tryptophane UGG... Tyros ing WUA,.. Valine BUG... ‘weenie The numberg of nucleotides per coding unit, but mot the sequence, was determined. For this purpose, we expressed the amount of each amino acid incorporated in percent of the phenylalanine, which was directed into protein by the same U-containing polynucleotide. - + We calculated also the probability of any triplet of nuclestides in percent of UUU, coding presawaably for phenylalanine. This must be done an the basis of the determined quantitative maucleotide- composition of the polynucleotides used. So we could correlate the observed incorperation of any amino acid to the triplet with the beat-fitting statistically axpected frequency. These deterain- ations were done with many polynucleotides of varied ratios between the comparing nucleotides and agentually led to the same results. On the basis of these calculations, however, we could not decide whether the coding units might contain uridylic acid residues in addition to those specified. If the number of letters per code word would be larger than three, a proportional increase in the Cadivideal mumber of words coding for the amino acids should be eupected ( = more | degeneracy). The triplet-code ia still not only the simplest, but also the most likely and experimentally uncontradicted conespt. This conclusion comes from beth biocheaical and genetic (Criek) evidencs. The use of different methods in synthesizing RNA of well-defined nucleotide-sequences should allow ua in the near future to get direct evidence for the number and sequence of nucleotides in the eode- words. Then, we might also find other coding units possibly existing in addition to the ones already determined for certain amino acids. Tha code could be more degenerate. The high proportion which U takes in the coding units determined thus far, may disappear where other than random-polynucleotides will be used and show possibly U-less code words in addition. The present selection of partially known code words may se just the result of certain limitations inherant in the mathods used. 2-2 es5 of codi its Poly A base-pairs and forms double~ and triple-stranded helices with poly-U. In this manner it totally inectivates poly-U added to our £. coli system. Poly-C does neither base-pair mor inactivate pely U. This way be a model for “reprassion\" of the synthesis of eertain proteins on the level of RMA. Such repressions occur in cells and if their mechanism ig disturbed, uncontrolled synthesis of proteins might result. Universality of the code? These determinations of RNA coding units in a bacterial =~ 19 « aystem would be more significant, if differant organisms used the same set of coding units, and if each unit would be correlated in avery one to the sma amino acid. YThe genetic code would then be “unfversal\". This universality is favored by already existing observations: 1. Mutations of the THV¥-protesin, studied by “Wittmann in Tibingen ) end Taugita and Fraenkel-Conrat in Berkeley, resulted after treatment of the TMV-RNA with nitroue acid. There occur only two autagenic tranaitiiens of nucleic acid bases by oxidative deamination; U replaces C,or G replaces A in ona single place somehfiere along the RNA-chain. Aa @ eonsequence, mutants occur, in which aingle amino acids replace certain other amino acids found in TMV-protein of the wild type. 12 out of 14 different types of amino acid-replacements can be explained by the basa compositions determined with randem-polynucleotides in the E. soli system. Chencea should have led to less than 33% agreement.   2. another approach, taken first by Lippmann and ¥. zbrenstein, ia to use amino acid-charged transfer-RNA from 5. coli and let it deliver ita amino acids in a celi-free system from another organisa, = il - actually from rabbit-reticulocytes. The amino acids became incorpor- ated into the proper places in rabbit-hemoglobin. Thus, at least part of the code seems to bea universal. Further experiments done in marry laboratcrics at the moment, shall finally answer this question. The general nature of these experiments is to put either messengera for the formation of a specific protein or. amino acid-charged trans fer-RNA into a cell-free aystem prepared from another organism. If the code is universal, transfer-RNA from one and messenger from another specias have to fit for making correct amino acid sequences. A fundamental concept, the transfer of inherited information being stored in certain nucleotide sequences for the ultimate translation into the broad variety of functioning preteins, has found its final experimental proof. Certain findings made during our work on the coding problem, have promoted the research of many laboratories on various other processes involved in protein synthesis. SUMMARY 1. The base-composition of coding units for 15 amine acids was determined by means of random-polynucleotides of different base~composition. 2. 3. 4. 5s » 12 @ The code ia degenerate at least for the amino acids leucine and serine. The informational part of RHA appeared to be single-stranded. At leaat part of the code seema te be universal. Important features of the code are still unkaown: The total number and the sequence of nucleotides in the coding units, the smount of degeneracy, and the polarity and chemical nature of “starting points\", from which the measages apparently (Crick) are read off.", "Nirenberg, Marshall W. ; Matthaei, Heinrich", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xjjz_pf8u~mets", "00000000-0000-0000-CC12-B97D7E62A0A8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "A Symmetrical Pattern in the Genetic Code: The Triplets of Links in the Genetic Material Which Specify the Detailed Structure of Proteins Have Now Been Identified in Sufficient Numbers to Fall into a Regular Pattern", "101584910X107", null, "1963", "19 March 1963", "This article argues that triplets specifying protein structure have been identified in sufficient numbers to make a pattern clear.  The author, a biologist at the National Cancer Institute, Bethesda, Maryland, outlines the characteristics of genetic material, citing Nirenberg's work with poly-U as foundational in understanding the code.  Established patterns, unsettled questions, and predictions are covered.. Last sentence on page 8 reads: \"On the other hand, if the purine-pyrimidine pairs are fundamental there should be a specific transfer RNA for every one of the 32 pairs, if not a separate one for every triplet.\"  This sentence is very light on the original page and is difficult to read on the image.", "Articles", "Amino Acids,Nucleotides,RNA, Messenger ; Codon,Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "16", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "B41[6% A SYMMETRICAL PATTERN IN THE GENETIC CODE The triplets of links in the genetic material which svecify the detalled structure of proteins heave now been identified in sufficient numbers to fall into a regular pattern. Richard V. ick   The author is a biologist in the laboratory of Biology, Netional Cancer Institute, Bethesda, Maryland. Page < R.V. Sek The chemical structure of the genetic mechanism must have several very special characteristics. It must contain large, complex molecules capable of representing @ large amount of information intisir possible alternative configurations. It must be autocatalytic - capable of producing exact replicas of itsel?. It mist do this with great reliability, yet be able to mutate ocecasionally, amd still be autecatalytic in its mutated form. The Watson-Crick model demonstrates how these properties folio from the structure of DHA. But this accounts only for the self-reproduction and mutation of the genetic material. It must also have quite a different property. It must catalyse specifically some material other than itself, which enters into the living chemistry of the organism and results in the phenotypic expression of that \"gene\". If it is a gene for black rather than brown hair, for example, it must snecifically produce some chemical which affects the chain of events which finally results in the production of pigment and its deposition in the hair. The \"one gene-one enzyme\" formulation has led to the expectation that this non-genic product is protein. This second problem is more intricate than the first. The reproduction of the genes was pictured as some sort of mold-snd-cast system, and so it has proved to be. But for a “semplate” to determine the structure of some product fundamentally dissimilar from itself requires a more elaborate apparatus. Proteins are long, umbranched chains, made of twenty different kinds of links - amino acids. The exmct order of hundreds of these links appears to be genetically determined for each of thousands of different proteins. The DNA of the chromosomes also occurs as long, unbranched chains, but with only four different kinds of links - nucleotides. There are two kinds of nucleotides, two purines and two pyrimidines, which differ markedly in size. The specific matching of one puxine with one pyrimidine - edenine with thymine, cytosine with guanine - is the basis for the Watson-Crick model. But how could there possibly be such a matching between the nucleotides and the amino acids, especially in view of their different. numbers? This problem was posed in 1954 by Gamow (1) as a mathematical challenze te Page 3 R.V.Eck biochemistry. For each link in a protein chain being constructed, there are twenty possibilities. How can this be specified by a DNA chain containing only four different kinds of links? Evidently more than one, seemingly at least three mucleotides would heve to combine in the determination of a single aminc acid link, perhaps in some overlapping fashion. This mathematical puzzle remains valid, and several facets of it have been clarified. The DHA produces \"messenger REA\" apparently by some means similar to the Watson-Crick mechanism. RNA contains nucleotides corresponding to ah, with thymine replaced by uracil. The messenger RHA goes go the protein-producing organelles in the cytoplasm and determines the production of specific protein molecules, according to its detailed sequence of nucleotides. {ft does not do this directly, but vie a number of adapter molecules, called “transfer RNA's\". Presumably each amino acid has one or more trensfer RNA which specifically attaches to it. Another part of the transfer RNA attaches to its specific nuclectide “codon\" (triplet?) wherever it may occur in the polyribonucleotide chain (2). Thus each amino acid is held in its proper place while the polymerizing mechanism links it to the two adjacent amino acids ’ determined by the sequence of nucleotides in the information-carrying RNA. The elucidation of a codon pattern should provide evidence on the mechaniam of transfer RNA specificity. How, structurally, dees the specific /RHA secognize its proper codon combination? An ansver to this question is suggested in this paper. The Giscovery two years ago that a non-living system could be made to synthesize an artificial protein, polyphenylalanine, using as messenger RNA synthetic polyuridylic acid (3), has made possible a rapidly developing experimental attack on this problem. The in vitro synthesis of proteins using various synthetic copolymers of two or more ribonucleotides as artificial messenger RNA bas been reported from tvo laboratories. Tables have been published giving groups of three nucleotides (\"triplets\") which have been identified as \"coding for\" the various amino acids (4). There has been much interest and speculation in the mmber of codons and the possible relationships among then. Fasious peiteras have teen opopowwed winleb usue dis aocmmasiave the nieeholeres AM Mee te Re CUT Le Kage 4 R.V-Eek that date, and predict certain others. The discovery of additional triplets has then required that many of these hypotheses be abandoned or much modified. Judging by the rate at which new ones have been discovered it appears that nearly all, if not all 4 x4 xh « 64 mathematically possible triplets will ultimately be identified es codons. Will these appear to be a chactic jumble, or will they fall into some regular pattern? | Some regularity can be seen in the list of published triplets. No more amino acids are usually assigned to a nucleotide combination than there can be alternative rearrengements of it. This supports the expectation that the maximm mmber will not exceed 64. There are only three exceptions to this, two of which may be due to laboratory errors. The third exception will be considered later. It frequently appears that the two or more triplets found to code for the same amino acid have two of their three nucleotides in common. Roberts has used this property to derive a\"ioublet\" code » Which implies that the third nucleotide is irrelevant (5). This, of course, gives only 16 combinations and requires some supplementary explanation to account for the 20 amino acids. It has also been suggested that the code may be partly doublet and partly triplet. The Purine-Pyrimidine Pattern In the most recent lists from the two laboratories there are a total of 49 different triplets, 26 of them reported by both (4). This seems like a large enough proportion of the 64 to outline en over-all pattern, if one exists. The data can be arranged in various ways, and if one tabulates these triplets as in table 1, such a pattern emerges clearly. The pattern is this: All 64 triplets occur. Each amino acid is represented by one or more pairs of triplets which are identical except for one nuclectide. The non-identical nucleotides in each pair are the two purines or the two pyrimidines. For exmmple, ACC and AUC have been found to cede for histidine. GGU codes for tryptophan, and this pattzern predicts that GAU will also be found to ecde for tryptophan. This pattern seems acceptable sterecchemically. Furthermore, it is complete ond earns 7 + at 4 ye qs : sigs 7 a4 % ? oa: self-consistent. “here are ezactly 32 vairs. each using ef leest one seporied triples. Fase 5 R. Vu. Beck All 64 possible permutations are used. Of the 49 reported triplets, four were discarded: two because theve were four amine acids assigned where there could be only three permutations, one because it wes assigned to GUG In conflict with phenylalanine, and only one because it was inconsistent with the pattern presented here. The 19 remaining combinations have been assigned to the various amino acids in such a wey as to completc the pattern symmetrically. For example, arginine has a pair, CCG and CUG, and one unpaired triplet, AAC. The missing triplet could be AAA or AGG. But AAA is already fully “occupied”, and AGG is not. Arginine is therefore assigned the missing AGG. As this process continues, completing all the pairs, the number of remaining alternatives is greatly reduced, but ali the missing triplets can ke accounted for with only one discrepancy as notel. This lest polnat is Illustrated in table 2 in which the sane Jascignuents ere re- tabulated. Here one can readily confirm that each triplet has emctly as many amino acids assigned to it as there can be permutations. Any pattern of this sort is open to the suspicion that it may merely resemble the true pattern. 2% would be pointless to compute a “probability” that it could have occurred “by chance\", because the data obviously fall into some sort of pattern - they are not rendom. Eut there is a suiteble test. One can attemnt to construct similar- appearing patterns of 32 pairs in which AC end GU are paired, or AU and CG. ‘This attempt was made; these patterns cannot be constructed without discarding an umreasonebic number of well-established date. For example, the matching triplet for phenylalanine ~- UUU would have to be UGU or UAU respectively. Both laboratories have identified each of these with three other amino acids. Oniy UCU, as in the proposed pattern, is free to represent phenylalanine. About seven such conflicts developed in each attempt. in the purins-pyrimidine pattern only one triplet assignment had to be discarded in this way, and it was reported from one leboretory only. This appears to be a moderately strong indication that this pattern (which incidentally “mikes sense\" chemically) is not just ea contvived modification of some \"partially doublet” code. eV Bele This test indicates that if there is 2 pattern of this general sort in the 64 possible triplets, the existing 46 data (after excluding the three which arc inconsistent in any case) are more than sufficient to determine that pattern. It appears that this many data could fit only into a true pattern. Previously, when there were too few data it was possible to devise en almost endless number of patterns in which they could be accomodated. Determination of Orter The pattern at this stage depends only on the published tables of triplets, not on date from amino acid \"“metants\", ete. If these additionsl clues are used, a beginning can be made in determining the order of the nucleotides within each triples. In the experiments which have yielded the triplet codons, no order can be determined. Thus, in table 1 \"aac\" means, \"AAG, AGA, or GAA\". In table 2, \"ACG means \"ACG, AGC, CAG, CGA, GAC, and GCA\". In table 3, however, these orders have been assigned, and \"CAA-threonine\" means that exact order, with the provision the; the evilence is not rigorously conclusive, and it might be AAC. If the purine- pyrimidine link is always in the same noasitjon, and if this position were known, the sequence would be detexmined in all these cases where toe remaining tyo are the same (ACA = ACA). The experimentally deternincd sequence AU for *vrosine and GUU for cysteine (6) requires one of the isoleucine codons to be WA ana valine to be UUG if the specie] link is in the middie, or UAU and UGU if it is at the Fight end. it evidently cannot be at the left end. For illustration, it is assumed to be in the middie. Aside from these, the orderings in table 3 are not rigorously determined. Any nucleotide pair might be exchanged with the diagonally corresponding one and still be consistent with table 2. For example, lysine-AAg and isoleucine-UAA might be inter- chenged. However, some possibilities seem much more plausible than others. It seems reasonable to expect that a mutetion will often involve the change of a single link. The amino acids which could xeplace one another in this way might ke expected to be found most frequently as \"allele\" pairs in homologous protein sequences. For examle. we abe Ya alamine can change to serine if one ef ites miciesatides esenges from G te th Baie ean Page 7 R. V.Eck occur in two different pairs. However, if the alanines at C...G were exchanged with serine and arginine at G...C, there would then he no codons of alanine and serine having two letters in common. Since alenine-serine is the most Prequently-cccurring \"allele\" pair (7), the arrangement as shown is much preferred. Similarly, if valine and cysteine were exchanged, the numerous allele pairs val-ala, val-ilu, val-leu, and others would not be producible by single-link \"interchanges\". (This, with the previously-mentioned determination of the sequence valine. = WG, constitutes support for this procedure.) By comparing each possible alternative in this way with a list of about three hundred “alleles” from hemoglobin and other proteins, the tentative arrangement shown in table 3 was derived. There are some other uncertain details in table 3. Different amino acids might have been discarded. For example, glutamine at AGG might have been retained, and arginine at AAG discarded (table 2). Furthermore, glycine at GAG might exchange places with glutamine, if it were retained at AGG. There are, however, only a few such alternatives, and at each choice there seemed some good clue to the selection. Another source of uncertainty is the possibility of experinental error. Of the 32 pairs, five consist of an unreported (predicted) triplet and a triplet reported fram only one laboratory. Any of these might prove to be in error. Even if there were no basis for choice in the alternative positions indicated in table 3, it would contain much information about sequence. For each triplet in table 2 there are one, three, or six possible sequences. Table 3 reduces these to two alternatives at most. This pattern predicts all the amino acids coded by the remaining 19 ordered triplets, suggesting that there may be no \"nonsense\" combinations. This is not a strong inference, hovever. If one of the reported triplets is erroneous its assigned pair might represent \"nonsense\". Or, in a few cases the pairs might be sub-divided, one of the two triplets being \"noasense\". we ge R.V.eok Predictions Concerning Transfer RHA's It is consistent with this pattern that there could te 6! transfer RHA's,one for each triplet. The pattern of 32 pairs suggests, hovever, tha) there may be only 32 transfer RHA's and that each one responds indiscriminately to both of its specific triplets. fhe specificity of the attachment site would reside in: one of the four uvucleotides in one position, a purine or a pyrinidine in another position, and one of the four nucleotides in a third positien. These three determ nants vould cceur in three specific (not necessarily adjacent) pesitions on the mereenger RNA chein. The two triplets of each pair vould presumably te indistinguisbnole to the transfer RRA, which might xecognize the third (middle?) nucleotide oniy by its size. in this sense each codon would consist of a pair of tripleis. One might face selously eall this a “two-and-a-haif-Letter™ code. Gn this iuterpretaiion there woulda be only one wransfer RHA fer aspartic acid, cysteine, { BLuts wine, histidine, methionine, phenylalanine. trvotophan, tyrosine, and valine. There would be two for cach of the ovher amino acids except serine, which would have three. (Barring errors, as mentioned above.) Tye xpeximental determinetion of the number of transfer REA‘s for each amino acid vould ke @ poverful check on the correctness of rhis pattern, and therefore on the validity of the individual repovted triplets. Phe two specific eransfer RAA'ts reported for icucine are consistent with this prediction (2). A strong check vill alee be provided by each subsequens Gisecovery of a triplet Vor the evidence on vhich Roberts based his \"@oublet” cade (5}. “NLS PEtitern as a cede whie’   bale p ~ fete “a ty $9 3 thy doublet ard partly triplet. This coulé te teste:   = yay we Baye Spas Pe 4 fam = aS om wf ee sy af pepe Dana for For NEA con be found for alanine 2 BAVCILNS, A50L0 0A: or vee BO ae, veoh bet + + 1 Yn wt 2 sel ye: ee es 4% couliie, ooh tareontoc, eml only tve for sexine, these will Dy 2: Zz st at, te” 2 a benvener: “doi bleta” + Sang ’ meert fe one oe % 5 a so CO! semoe., On the obher Ran ids ok Poke Ay om Te : ele gga ga oe Ly Cet ee Poder PA Neat aay SE SE ie ee    Fage 9 R.V.Eck There are four crucial tests which could te made with isolated transfer RNA's. Is the transfer RIA of proline which responds to CAC the same as the one which responds to CGC? Similarly for cee ee and AUG; glycine GCG an@ GUG; end leucize UAU and UGU. If these shovld prove to be identical, this pattem vould be validated. Unsettled Questions The finding that leucine as well as phenylalenine (4) is ceded by UUU is of considerable interest. Is it an accident caused by some abnormal condition in the in vitro situation, or is it of fundemental significance? It reises the possibility that pexhaps in the presence of some other source of information not normally present in the artificial situation there might be e second pattern of 32 codons. Perhaps some of the triplets discarded in making this pattsera are other ambiguities of this kind rather than errors. Evidence which seems contrary to this is the finding that the same transfer RNA of leucine responds to UGU and to WU (2). This pattern seems to be good evidence for a triplet code, since in it triplets are necessary and sufficient. Eut if the above-mentioned ambiguities prove to be fundamental, they would require extra information, which might reside in still other links (as an overlapping quadruples code?). If this pattern vere the complete code, any simple type of overlepping should be immediately evident by substituting the triplets for the amizo acids in a few of the known protein sequences. This does not appear to be the case. If it were possible to make regularly crdered synthetic polyuers such as poly- dinucleotides, etc., the question ef vhether the three dlements of each triplet are adjacent in the chain could be settled. Also, such polymers could he used to study the possibility of overlapping codes. I% seams possible that some regular BIA's covld be synthesized from synthetic DHA's using the mechanism reported by Chamberlin and Perg and by Otska et al. (8). Page 10 R.oV.Eck In retrospect, it seems that this simple pattern could have been discovered with fewer clues, and we may wonder vhy it was not found earlier. In the lasttwo years there have been a remarkable number of ad hoc proposals to account for the data currently at hand. When there vere about tvelve triplets identified it was expected by some that the total number would be emctly 20 - one for each amino acid. Later a number of special combinations were considered, such as combining the three nucleotides without regard to order, and others of this sort which were mathematically possible tut structurally unimaginable. Then there was the \"high-U\" code, chemically imlausible but mathematically capable of accounting for the results to that date. Recently it was proposed that there may be some simple pattern in vivo but that some circumstance ds obscuring it in the in vitro experiments. Ry permitting some normally hidden potentialities to be expressed, this would produce too many triplets. All of these were attempts to account for the number 20 and to guess the pattern at a stage when an indefinitely large number of patterns were yet mathematically possible. Por this reason the probabilities were strongly ageinst success in this approach. fhe point of view which led to this pattern was from the opposite direction: Whatever the pattern may te, there are 64 triplets. In the end some of them may be “nonsense”, or even non-existent in nature. Some may prove to be equivalent to others, etc. But whatever those details may te they will consist of some sub-pattern of the 6h mathematically possible triplets. Mou, with a total of 49 different triplets identified, surely the pattern must be visible! I tabulated these triplets in various weys and shortly this detail appeared: Five amino acids had two triplets, with two letters in common. Another had three, the third being unrelated to the first two. Of these six examples four contained the alternatives C vs. U (e.g. aspartic acid-ACG end AUG). It was not surprising that none contained © in such alternatives, since the G polymers had given the most experimental difficulty and most of the combingtions having two G's vere as yet unassigned. From this observation table 1 followed directly and the puzzle practically solved itself. Faege 12 R. VeEck A similar history has occurred in the related problem of overlapping or non- overlapping codes. Gamow suggested that the necessary amount of information might be reduced by seme systematic constraint on the sequences of amino ecids. This could be caused by the same nucleotides serving in more than one triplet simultaneously (1). At that time there was a moderate amount of protein sequence dataaetiable. Several curious overlapping codes were proposed, each of which ws followed enthusiastically end then disproved mathematically, using the then currently available data as it con- tinued to increase in amount. Then Brenner (9) concluded thet all overlapping triplet codes were inconsistent with the data, and the attention of most protein eryptographers was diverted to non-overlapping codes, where it has remained ever since. However, the problem was not approached in its most general form. Brenner's computation, the results of the single step mutations, and the results of Crick et al (10) have been taken to disprove overlapping codes, but this is true only for a certain sub-class of such codes. Furthermore it includes the unstated assumption that there is no other source of genetic information. (The results of Crick et al (10) have also been taken as evidence for a triplet code but this inference is valid only for non-overlapping codes. ) A large number of overlapping codes are still mathematically possible (11), and several are even structurally plausible which involve a regular folding or coiling of the RNA strand so that the non-adjacent nucleotides of the codons assume their specific positions. The substance of the argument against overlapping codes, whether from amino acid sequences or from the single-step mutation data, is that such codes could not contain enough information to account for the observed mumber of variations in protein sequences. However, all proposed ccdes have required, explicitly or implicitly, some additional unknown source of information such as \"commas\", spacers\", \"stepping by threes\", “forbidden combinations\", etc. As long as the nature of that additional mechanism is undiscovered the possibility remains that it could contain enough information to supplement: an overlapping code. Clear evidence of patterns in the constraints on protein scquences would be a basis for an attack on this problem. The amount of protein sesuence vta nov available my be barely sufficient for this (7). In numerical proportion this Page 12 R.V.EBck problem is at a mich earlier stage than that of the nucleotide triplets. Here we required about 45 of the 64 possibilities before the pattern revealed itself. In the protein cryptogream less than two thousand of the potential eight thousand tripeptide sequences have been reported. If, say, three thousand of the eight thousand were \"forbidden\" according to some pattern, could we see that pattern? Perhaps this seemingly obscure problem will seem simple when it is solved. Summary The accumulation of experimental results from the system of Nirenberg and Matthaei has now reached about 75% of the total possible, if the triplet” concept is correct. Considered as a mathematical puzzle this has proved to be sufficient to determine an apparently unique soluticn: The sixty-four combinations of four nucleotides taken three at a time, are resolved into thirty-two pairs. ‘The second member of eachmir is identical with the first, except that in one position a purine is replaced by the other purine or a pyrimidine by the other pyrimidine. Almost all of the reported triplets £it into this pattern, and it predicts which amino acids will be found te correspond to the remaining nineteen unidentified triplets. This pattern accounts for several of the observations concerning regularities inthe data. It partially determines the order of the nucleotides in each triplet and suggests a structurel basis for transfer RNA specificity. Whether the three nucleotides of each triplet are adjacent in the nucleic acid chain, and whether they somehow impose constraints on the possible sequences of amino acids which they determine, is yet to be worked out. 1. 6. Te Page 13 R.V-Bek Bibliography G. Gamow, Rature 173, 318 (195k). B. Weisblum, S. Benzer, and R. W. Holley, Proc. Mat. Acad. Sci. 18, 1h49 (19623.   M. W. Nivenberg and J. H. Matthaei, Proc. Nat. Acad. Sci. 47, 1588 (1961).   O. W. Jones, Jr. and N. W. Nirenberg, Proc. Nat. Acad. Sei. 48, 2115 (1962);   J. Wahba, S. Gardner, C. Basilio, R. S. Miller, F. Speyer, and P. tengyel, ibid. 49, 116 (1963); Two additional triplets were reported at a meeting: J. Abelson, Science 139, 774 (1963). R. B. Roberts, Proc. Rat. Acad. Sci. 48, 897 (1962).   A. J. Wahba, C. Basilio, J. FP. Speyer, P. Lengyel, R. 8S. Miller, and S. Ochoa, Proc. Hat. Acad. Sci. 48, 1683 (1962).   R. V. Eck, J. Theoret. Biol. 2, 139 (1962). M. Chamberlin and P. Berg, Proc. Nat. Acad. Sci. 48, 81 (1962); B. Otaka, H. Mitsul, and S. Osawa, ibid. 48, 425 (1962). S. Brenner, Proc. Nat. Acad. Sci. 43, 687 (1957). F. H. C. Crick, L. Barnett, S. Bremer, ani R. J. Watts-Tobin, Nature, 192, 1227 (1961). R. Wall, Feture 193, 1268 (1962). Re Vek. A symmetrical pattern in the genetic code Alanine cCat# CAGH Leucine UAUF CUURy CUO CuUaG¢ = CRE uaueF ccue discard} Arginine cosy; aAAGH Lysine ASAT AAU! (ACAS cucé AaGt AGA AGU? discard} Asparagine ACASS CAUE Hethionine Aut ANIA = CGU? ACG? Aspartic acid sca¢ Phenylalanine vupitt neg UcuF Cysteine SUG Proline coos = caciyt CU? cues# cece Glutamic acid AAG! Gat Serine coug cAGe cae ARG?  GGD? cow? CEG? cau? Glutamine Aace? § (aced Threonine ACC AACE# (cced GC? discard) Avc#~ acc? discs rd) Glycine GUG?# GAGE Tryptophan Goux} GcGy  GGacr GAU? Histidine ACCH# Tyrosine Auang AUCH ACU? Isoleucine Auuet aaud Yaline quu# ACU? = AGIN? ecu? Jable 1. in this pattem there are 32 psirs having the two purines or the two pyrimidines in a certain position (illustrated as if jn the center). %% includes all 64 possible configueabions of three nuclectides. It eccomiodates 45 of the 49 published wWiplets. Taree are discarded because they are intern- ally inconsistent with the published list (too many amino acids for one trip- let). One is discarded because it is inconsistent with this pattern, 19 remaining triplets ave predicted, as shown. The actual order of the nucleotides is still undetermined, except AUD for tyrosine and GUU for cysteine. *t = Reported by Nirenkerg's group. # = Reported by Ochoa's group. 2 = Predicted by this pattern. “ 2 =. s3. teas, ch wee = cs we yen Bo € a 4& Symmetrical Pacvern in the Genetie Code Reported Predicted Discard AAA Syst}! Cec Prot} GGG Gly WUT Phe%} Leu* AAC Asn Glatt Torey Lys AAG Aref Giut# Lys® AAU Asn# Zlué  teretsf CCA Bist?! Prro%! thr? CCG Alat# Arett Prot fhr# CCU Prot? gay Isu GGA Gly? Arg Glu Ging Gec  Gly# Ala Ser GGU Gly Trytf Glu WA iutf Lent¢ tyref WUC Lew# Phe# sert# WUE Cys Leutt vel? ACG Alaf Aso ser Gln Met Tar ACU Asn# His Tart Tiu Ser tyr AGO Asp# Glut? Mett# Tiu Lys Try CGU Ala# Aref Ser* Cys Val Asn Zable 2. Entries of table 1 rearranced » to euphasize the number of emino acids reported and predicted for each triplet. Theze are as rany entries for cach triplet es there are possible permutations of the three nucleo- tides ~- one, three, or six. * = Reported by Hirenberg's group. # = Reported by Ochoa's group. & symmetrical necters im the genetie code   at nagee naged - ef ten he me a“ APOE ee MAU oop Ly Ae & AGG AGU a acer 2064! ACU   nis af ELS yf ue i HAD Ale pas fa wee ys CAF | cacne CGA cage °   B PULET ER. ser   ccAY     hehe : i ats fy cuaAd the Chics? aS 4 SESS § z i me et COATT GAAS SX GALI bey 2S o gpepsp GAU a CE fe be i a are en : 8S oa oS 4G * Gae eau GCA accede Geof ... GU 2... FR ee eye weigy a fF Gi . eos : ¥ Si quasy A AA# wa = BST as * glu a ra] rere UCA ap = eC UCS val. ENR ° GUC sulk me LEU WA! nS 3 Re   Table 3. The purine-pyrimidine paivs in the genetic code, with orier tentatively determined. In one position of each pair, the ivo purines (A and G), and the two pyrimidines (C end U) ave equivalent, reducing the 64 triplets to 32 \"codons\". Amino acids in capitels have theiz sequences unambiguously determined, assuming that the special position is in the middle. Amino acids in lower esse could possibly belong to the pairs aiagonally opposite, e.g., CAA-gin; ALC-thr, ete. The frequencies of amino acid \"alleles\" suggest the assigrments indicated. + 2 Reported by Nireaberg's group. yo Reported by Ochea's group.", "Eck, Richard V.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ziys~muer-g9uv", "00000000-0000-0000-6977-25AB4B419F3A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Human cDNA Clones for an [Alpha] Subunit of Gi Signal-Transduction Protein", "101584910X109", null, "1987", "August 1987", "This article, one of many using clonal lines of cells, presents experimental findings comparing amino acid and nucleotide sequence in clonal human brain and bovine brain tissue, human monocytes, rat glioma, and mouse macrophages.  Homologies and unexpected diversity provide evidence of relative evolutionary conservation.", "Articles", "Cloning, Molecular ; GTP-Binding Proteins,Amino Acid Sequence,Base Sequence,DNA,RNA, Messenger", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 84, pp. 5115-5119, August 1987 Biochemistry Human cDNA clones for an @ subunit of G; signal-transduction protein (receptors /adenylate cyclase /GTP-binding proteins /brain/mRNA) P. Bray*, A. CARTER’, V. Guo*, C. Puckett, J. KAMHOLZ*, A. SPIEGEL’, AND M. NIRENBERG* *Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, “Metabolic Diseases Branch, National Institute of Diabetes, Digestive and Kidney Diseases, *Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892 Contributed by M. Nirenberg, March 27, 1987 ABSTRACT Two cDNA clones were obtained from a Agt11 cDNA human brain library that correspond to a; subunits of G signal-transduction proteins (where a, subunits refer to the a subunits of G proteins that inhibit adenylate cyclase). The nucleotide sequence of human brain a; is highly homologous to that of bovine brain a; [Nukada, T., Tanabe, T., Takahashi, H., Noda, M., Haga, K., Haga, T., Ichiyama, A., Kangawa, K., Hiranaga, M., Matsuo, H. & Numa, S. (1986) FEBS Lett. 197, 305-310] and the predicted amino acid sequences are identical. However, human and bovine brain a, cDNAs differ significantly from a, cDNAs from human monocytes, rat glioma, and mouse macrophages in amino acid (88% homol- ogy) and nucleotide (71-75% homology) sequences. In addi- tion, the nucleotide sequences of the 3’ untranslated regions of human and bovine brain a, cDNAs differ markedly from the sequences of human monocyte, rat glioma, and mouse macro- phage a; cDNAs. These results suggest there are at least two classes of aj MRNA.   Guanine nucleotide-binding proteins (G proteins) couple receptors for extracellular signals to effectors such as ade- nylate cyclase (1) or CGMP phosphodiesterase (2). G proteins consist of three protein subunits, a, B, and y. a Subunits bind and hydrolyze GTP (1, 2) and display specificity for receptors and effectors. Different proteins, G, and G;, mediate stimu- lation and inhibition, respectively, of adenylate cyclase (where a, and a are the corresponding « subunits). G, and one or more forms of G; are assumed to be present in most mammalian cells (1), whereas the a,-1 subunit of transducin is expressed only in retinal rods (3, 4) and a-2 is expressed only in cones (4). Similarly, a, (a G protein of unknown function) is abundant in brain but not in most of the other tissues that have been examined (5, 6). The nucleotide sequences of cDNA clones for bovine (7, 8), rat (ref. 9; R. Reed, personal communication), mouse (10), and human a, (11, 12) have been reported. R. Reed and coworkers have cloned and sequenced three types of a; cDNA from a rat olfactory epithelium Agtl0 cDNA library (personal communication). Other aj cDNAs from bovine brain (13), bovine pituitary (14), human monocyte (15), mouse macrophage (10), and rat C6 glioma (9) have been sequenced. In addition, the sequences of rat (9) and bovine (16) a, and bovine a,-1 (17-19) and @,-2 (20) cDNAs have been reported. The amino acid sequence homologies of a subunits range from ~40% (a, vs. aj) to ~78% (a,-1 vs. a,-2). In this report, the nucleotide sequence of a human brain a; cDNA is described and is compared with sequences of human monocyte (15), bovine brain (13) and pituitary (14), rat C6 glioma (9), and mouse macrophage (10) a; cDNAs. Two types of a; can be distinguished that differ in 12% of the amino acid   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘advertisement’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5115   ————_> + —2+> > ----—> ——> 36 —> ——-2—_» a —> —2—» — iy, > EM M MMM MM M E fu ; rt Lyf 1, ; 1 q 200 400 600 800 1000 1200 1400 +4 @---3---— + #4 + +2. + —_ + <¢+---8 +¢ + +2 —____—__ + Fic. 1. Restriction fragments of BG-4 and BG21-2 a; cDNAs were subcloned into M13mp18 and sequenced. Each arrow repre- sents a subcloned DNA restriction fragment that was sequenced; arrow shafts composed of dashes represent nucleotide sequences from BG21-2 a; cDNA; those with unbroken shafts represent sequences of BG-4 a; cDNA. The numbers shown with some arrows represent the number of subclones of the same type that were sequenced. The number of nucleotide residues in human brain a; cDNA is shown on the scale. E and M represent sites cleaved by EcoRI and Mbo I endonucleases, respectively. residues and possess markedly different 5’ and 3’ untrans- lated sequences that have been conserved during evolution. METHODS A Agtll cDNA library was constructed by a modification of the method of Huynh et al. (21). Poly(A)” RNA was prepared from basal ganglia dissected from a 1-day-old human female brain and was used for cDNA synthesis. Duplex DNA >800 nucleotide pairs in length was ligated to Agtll arms that had been dephosphorylated, and the DNA was packaged. The resulting library contains 10° cDNA recombinants; 90% of the phage contain DNA inserts. Twenty-five thousand phage and 10° Escherichia coli ¥1090 cells were plated per 150-mm Petri dish. Plates were incubated at 42°C for 2 hr and then at 38°C for 4 hr. Phage DNA was transferred to replicate nitrocellulose filters that were incubated in a solution containing 750 mM NaCl/75 mM sodium citrate, 1 mg of bovine serum albumin per ml, 1 mg of polyvinylpyrrolidone per ml, 1 mg of Ficol! per ml, 50 mM sodium phosphate (pH 6.8), 1 mM sodium pyrophosphate, 50 pg of yeast tRNA per ml, and 20% formamide for 16 hr at 42°C. Two probes, designed to hybridize to highly conserved regions of G-a@ subunit cDNAs (22), were synthesized. One probe, 43 nucleotide residues in length, consisted of 32 species of oligodeoxynucleotides, each containing six to eight   Abbreviations: a, and a;, a subunits of guanine nucleotide-binding proteins (G proteins) that activate (G,) or inhibit (G,) adenylate cyclase: a@,-1, a subunit of transducin, a G protein of rod photo- receptor cells that activates cGMP phosphodiesterase: «,-2, a subunit of transducin, a G protein of cone photoreceptor cells: a,. a subunit of G,, a G protein of unknown function. 5116 Biochemistry: Bray er al. deoxyinosine residues (5’ TCATETGCTTS ACIATIGTA- cTéTTFCCIGATTCICCIGCICC 3’), The other probe consisted of a single species of oligode- oxynucleotide 50 nucleotide residues in length (5° ACCT- TGAAGATGATGGCGGTCACGTCCTCGAAGCCGTG- GATCCACTTCTT 3’). The 5’ terminal hydroxyl groups of the probes were labeled with **P from [P]ATP catalyzed by polynucleotide kinase. Each probe (~1.5 x 10° cpm/ml, 150 fmol/ml) was added to sets of four replicate 137-mm filters and incubated for 16 hr at 42°C. Each filter was washed three times in a solution contain- ing 60 mM NaCl/6 mM sodium citrate and 0.1% NaDodSO, at 23°C for 20 min per wash, then washed once at 42°C in 60 mM NaCl/6 mM sodium citrate and 0.1% NaDodSO, for 3 min, and then subjected to autoradiography. Phage from plaques yielding positive autoradiographic signals with both probes were cloned. DNA inserts were Proc. Natl. Acad. Sci. USA 84 (1987) excised with EcoRI and subcloned into M13mp18, and partial nucleotide sequences of the subcloned single-stranded phage DNA inserts were determined by the dideoxynucleotide sequencing method (23). The complete nucleotide sequence of clone BG-4 was obtained by use of specific synthetic oligonucleotide primers and by sequencing BG-4 DNA frag- ments partially cleaved by Mbo I endonuclease and then subcloned. Nucleotide or amino acid residues were aligned by using the NUCALN or PRTALN algorithms of Wilbur and Lipman (24). All amino acid residue alignments were performed with a K-tuple size of 1, window size of 20, and a gap penalty of 1. Alignments of nucleotide residues in the coding regions were performed with a K-tuple size of 3, window size of 20, and a gap penalty of 7; for 3’ and 5’ untranslated region alignments a gap penalty of 1 was used. RNA for transfer blots was prepared from adult male human cerebral cortex and liver (25). Poly(A)” RNA was     Ser Ala Glu Asp Lys Ala Ala Val Glu Arg Ser Lys Met Ile Asp Arg Asn Leu Arg Glu Asp Gly Glu Lys Ala Ala Arg Glu val Lys 30 AGC GCC GAG GAC AAG GCG GCG GTG GAG CGG AGT AAG ATG ATC GAC COC AAC CIC CGT GAG GAC GCC GAC AAG GCC GCG CCC GAG GTC AAC 90 ce ¢c Te AAG G G A G G Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met Lys Ile Ile His Glu Ala Gly Tyr Ser Glu Glu Glu 60 CTG CIG CIG CIC GGT _GCT GGT GAA TCT GGT AAA AGT ACA ATT GIG AAG CAG ATG AAA ATT ATC CAT GAA GCT GGT TAT TCA GAA GAG GAG 180 T TG G G A GG G coc c c G ¢ c GA c c c 6G A Cys Lys Gln Tyr Lys Ala Val Val Tyr Ser Asn Thr Ile Gin Ser Ile Ile Ala Ile Ile Arg Ala Met Gly Arg Leu Lys Ile Asp Phe 90 TGT AAA CAA TAC AAA GCT GTG GTC TAC AGT AAC ACC ATC CAG TCA ATI ATT GCT ATC ATT AGG GCT ATG GGG AGG TTG AAG ATA GAC TTT 270 Cc CG G Coe G T ¢ c ¢ G c TGC AA C A ACC c c Gly Asp Ser Ala Arg Ala Asp Asp Ala Arg Gln Leu Phe Val Leu Ala Gly Ala Ala Glu Glu — Gly Phe Met Thr Ala Glu Leu Ala 119 GGT GAC TCA GCC CGG GCG GAT GAT GCA Coc CAA CIC TIT GIG CTA GCI GGA GCT GCT GAA GAA --- GGC TTT ATG ACT GCA GAA CIT GCT 357 c ccT AA ¢c ¢ CAG GG esA CA GTCTCAC C G G CAA @e@ccc ar oc GK Gly Val Ile Lys Arg Leu Trp Lys Asp Ser Gly Val Gin Ala Cys Phe Asn Arg Ser Ang Glu Tyr Gln Leu Asn Asp Ser Ala Ala Tyr 149 GGA GTT ATA AAG AGA TTG TGG AAA GAT AGT GGT GTA CAA GCC TCT TIC AAC AGA TCC CGA GAG TAC CAG CTY AAT GAT TCT GCA GCA TAC 447 c c Cc CG Goce Gct Cc CA G G c TaGcc AAG A Cc c ¢c A T c Tyr Leu Asn Asp Leu Asp Arg Ile Ala Gln Pro Asn Tyr Ile Pro Thr Gln Gln Asp Val Leu Arg Thr Arg Val Lys Thr Thr Gly Ile 179 TAT TIG AAT GAC TTG GAC AGA ATA GCT CAA CCA AAT TAC ATC CCG ACT CAA CAA GAT GIT CTC AGA ACT AGA GIG AAA ACT ACA GGA ATT 537 ec ¢c ¢ GcT T A’ G AGT GAC c A G G ACG ccc A G&G ¢ G G ¢ Val Glu Thr His Phe Thr Phe Lys Asp Leu His Phe Lys Met Phe Acp Val Gly Gly Gln Arg Ser Glu Arg Lys Lys Trp Ile His Cys 209 GTT GAA ACC CAT TIT ACT TTC AAA GAT CIT CAT TIT AAA ATG TTT GAT GIG GGA GGT CAG AGA TCT GAG CGG AAG TGG ATT CAT TGC 627 G G A c ¢c c G c A Cc c G T ce ¢c c Phe Glu Gly Val Thr Ala Ile Ile Phe Cys Val Ala Leu Ser Asp Tyr Asp Leu Val Leu Ala Glu Asp Glu Glu Met Asn Arg Met His 239 TIC GAA GGA GTG ACG GCG ATC ATC TIC TGT GTA GCA CTG AGT GAC TAC GAC CIG GTT CTA GCT GAA GAT GAA GAA ATG AAC CGA ATG CAT 717 T G c ¢c a c c ct coc T T G G Cc G G ¢ Glu Ser Met Lys Leu Phe Asp Ser Ile Cys Asn Asn Lys Trp Phe Thr Asp Thr Ser Ile Ile Leu Phe Leu Asn Lys Lys Asp Leu Phe 269 GAA AGC ATG AAA TIG TTT GAC AGC ATA TGT AAC AAC AAG TGG TTT ACA GAT ACA TCC ATI ATA CTT TIT CTA AAC AAG AAG GAT CIC TTT 807 G GCA ¢ Tt ¢c c c G ¢c c c c 6c c G Glu Glu Lys Ile Lys Lys Ser Pro Leu Thr Ile Cys Tyr Pro Glu Tyr Ala Gly Ser Asn Thr Tyr Glu Glu Ala Ala Ala Tyr Ile Gin 299 GAA GAA AAA ATC AAA AAG AGC CCC CIC ACT ATA TGC TAT CCA GAA TAT GCA GGA TCA AAC ACA TAT GAA GAG GCA GCT GCA TAT ATT CAA 897 G G G ccc T G c c Te T G CA GGec A T Case c c 6 Cys Gln Phe Glu Asp Leu Asn Lys Arg Lys Asp Thr Lys Glu Ile Tyr Thr His Phe Thr Cys Ala Thr Asp Thr Lys Asn Val Gln Phe 329 TGT CAG TTT GAA GAC CTC AAT AAA AGA AAG GAC ACA AAG GAA ATA TAC ACC CAC TIC ACA ‘TCT GCC ACA GAT ACT AAG AAT GIG CAG TTT 987 A A G G gcc a c G c G G c co uc ¢ ¢c c Val Phe Asp Ala Val Thr Asp Val Ile Ile Lys Asn Asn Leu Lys Asp Cys Gly Leu Phe term 349 GTT TTT GAT GCT GTA ACA GAT GTC ATC ATA AAA AAT AAT CTA AAA GAT TGT GGT CIC TTT TAA GITTIGCAGICCATGGTAAAATGCATTTTCAAACC 1085 G c c ¢ c c G c.)U6UclCUCG G6 ¢ c c C G GGGCCACCGGQCCCCTGGCEGGATEGGCCACCGCOG AAATGAGTACTTATATATGGATCICIGTAGACTAGAGICTTGCAGCAACACAGAA TGTAATATAAGGCAAATGCATCTGGGACTIGACCAAAGCTIGTICIGITTIGITTTTTTAACTGA, 1204 GAATTTGT. AAGTAACAGAAGGACCTTTICTTAAATGIGACAGATGGTCCTGCAGTIGAAACTGAAGGACAGTGTTAAAGCIGGGCICTAGTATATTGATGATTICIGCATAAGIGIAAATATGCAAAT 1323 GCTCCARACGTAGGGGAGGGGCTTCGCACAGGCCTCCCTGTTTIGARAGCCIGCOCTIGTCIGAGATGCTGGSTAA TGGCCA TGGTACCCOCTICTIGGGCATCIGTICIGCTTITTAACCAT GTATGTATACATGTATTTIATG 1344 TGTICTTIGTICTGTGCATGAGGG Fic. 2. Nucleotide sequence of human brain a, cDNA and predicted amino acid sequence of a; protein. On the third line of each set of lines are shown the nucleotide residues of human monocyte a cDNA (15) that differ from those of human brain a, cDNA, except for the 3° tail. Nucleotide residues 1-1276 correspond to BG-4 DNA. The regions of BG21-2 DNA that were sequenced correspond to residues 1-500 and 959-1344, The underlined nucleotides are the sites of hybridization of the 43-mer and 50-mer *?P-labeled oligodeoxynucleotide probes. The first 10 nucleotide residues found in BG-4 DNA are GTGCCGAAAG, whereas the first 11 nucleotide residues found in BG21-2 DNA are TGCCGAAAGCG. We do not know whether these nucleotide residues are cloning artifacts, and therefore these residues are not shown. Biochemistry: Bray et ai. L B - Origin Fic. 3. Transfer blot analysis of poly(A)~ = RNA (20 wg per lane). Lane L, adult human liver - 38 RNA; and lane B, aduit human brain RNA. The nitrocellulose filter was incubated with a [°*P]- . RNA probe corresponding to the minus strand -1.6 of 3’ untranslated region of BG21-2 a cDNA (nucleotide residues 1062-1344). The dash marks on the left indicate the chain lengths of -0.4 the RNA markers used: 9.49, 7.46, 4.40, 2.37, 1.35, and 0.33 kilobases; on the right, the chain lengths of the radioactive brain RNA bands are shown. isolated by oligo(dT)-cellulose column chromatography (26), fractionated by formaldehyde/agarase gel electrophoresis, and then blotted onto BA85 nitrocellulose membranes (Schlei- cher & Schuell). A probe specific for human brain a; MRNA corresponding to BG-4 or BG21-2 a; cDNA was obtained as follows: human brain a; cDNA was subcloned into the EcoRI site of pGEM-blue 3 (Promega Biotec, Madison, WI). The recombinant replicative form DNA was converted to linear DNA by incubation with Sca I endonuclease; the cleavage site is 43 nucleotide residues past the termination codon in the 3’ untranslated region of a, cDNA. The synthesis of a (?PJRNA transcript complementary to 251 nucleotide resi- dues in the 3’ untranslated region of human brain a; was catalyzed by SP6 RNA polymerase (27). The nitrocellulose filters were prehybridized for 8 hr at 57°C in a solution containing 750 mM NaCl/75 mM sodium citrate, 5 mM sodium phosphate (pH 6.5), 1 mM EDTA, 0.5 mg of bovine serum albumin per ml, 0.5 mg of Ficoll per ml, 0.5 mg of polyvinylpyrrolidone per ml, 0.1% NaDodSO,, 200 ug of yeast tRNA per ml, and 50% formamide. The [>?7P]RNA a;-specific probe was added (1 x 10° cpm/ml, 4 fmol/ml) and the reaction mixture was incubated 18 hr at 57°C. The filter was washed three times in a solution containing 15 mM NaCl/1.5 mM sodium citrate and 0.1% NaDodSO, at 55°C, then washed two times at 65°C for 20 min each wash, and then subjected to autoradiography for 18 hr with an intensifying screen. RESULTS AND DISCUSSION Sequence of Human Brain a; cDNA. A Agtll cDNA library prepared from total cellular poly(A)* RNA from 1-day-old human basal ganglia was screened with two 7?P-labeled oligodeoxynucleotide probes complementary to highly con- H BRAIN        B BRAIN MGCTL»+ H MONOCYTE . R Cé GLIOMA wees M MACROPHAGE «+++ Veeeeens Roteeeee \"K were reece c enn eene Proc, Natl. Acad. Sci. USA 84 (1987) $117 served regions of a subunits of G proteins (22). Fourteen of the 575,000 cDNA recombinants screened gave positive signals with both probes. Part of the nucleotide sequence of each positive clone was determined, which led to the iden- tification of 2 a; cDNA clones, BG-4 and BG-21-2, and 11 «, cDNA clones (11). Both strands of human brain BG-4 a; cDNA and part of BG21-2 cDNA were sequenced (Fig. 1). Most regions of BG-21-2 DNA that were sequenced proved to be identical to the corresponding sequence of BG-4 (with one exception noted in the legend to Fig. 2); however, the chain length of BG21-2 was longer than BG-4. The nucleotide sequence of BG-4 human brain a; cDNA (residues 1-1266) and the additional BG-21-2 sequence (residues 1267-1344) are shown in Fig. 2 and are compared with the recently reported nucleotide sequence of human monocyte a; cDNA (15). The first nucleotide of BG-4 corresponds to the 16th residue in the coding region of human monocyte a;. Nucleotide residues 1-1047 comprise an open reading frame coding for 349 amino acid residues followed by a termination codon and 294 additional 3’ untranslated nucleotide residues. Two-hundred and seventy-seven of the nucleotide residues scattered throughout the coding portion of human brain a, cDNA differ from those of human monocyte aj cDNA (27%) (15). How- ever, 221 of the nucleotide substitutions are silent mutations and 56 result in the replacement of 42 of the 349 amino acid residues compared (12%). Little or no homology was found in the nucleotide sequences of the 3’ untranslated regions of human brain and monocyte a; cDNAs (67% of the residues differ). These results show that the nucleotide sequences of human brain and monocyte a; cDNAs differ substantially and suggest that human brain and monocyte a; mRNAs are tran- scribed from different genes. These results agree well with the findings of R. Reed and his colleagues that rat olfactory epithelium contains three types of a; (personal communication). Transfer Blot Analysis of Human a; mRNA. A [*°P]RNA probe complementary to nucleotide residues 1062-1344 in the 3’ untranslated region of BG-21-2 human brain a; cDNA was incubated with human liver and brain poly(A)* RNA that had been fractionated by gel electrophoresis and transferred to a nitrocellulose filter (Fig. 3). The [*7P]RNA probe was ex- pected to hybridize with human brain a; mRNA correspond- ing to BG-4 or BG21-2 cDNA but not to other species of a-mRNA. Two faint, diffuse bands of radioactive liver poly(A)* RNA were detected with chain lengths of 1.7 and 1.0 kb, and one major and three minor radioactive bands of brain poly(A)” RNA were found with chain lengths of 2.2, 3.8, 1.6, and 0.4 kilobases (kb), respectively. The 3.8-kb a; poly(A)” RNA from human brain is similar in size to the 3.9-kb chain length reported for bovine brain aj mRNA (13). SAEDKAAVERSKMI DRNLREDGEKAAREVKLLLLGAGESGKSTIVKOMKIIHKEAGYSEEECKOYK (70) HB AVWYSWTIOSI TAI TRANGRLKIDFGDSARADDARQLE VLAGAABE- “GPMTARLAGVI KRLAKDSGVQACEHRSHEYOLN oY MM te eee ewes LeeVKRe °Ne os hh. “a+ wan weenes Ae \"SCr +s +Qe MLPED+ Se ‘       150 150 150 HB DSAAY YLNDLDRIAQPNY IP TOQDVLRTRVKT'TGIVE-THF'TPKDLHP KMPDVGGORSERKKWIHCFEGVTAI TRCVALSD (229)   Fic. 4. Amino acid sequence of a; from human brain compared with a; sequences from bovine brain (13), human monocytes (15), rat C6 glioma cells (9), and mouse macrophages (10). The letters represent the single-letter abbrevia- tions for amino acids. The symbol : represents an amino acid residue that is identical to the residue shown for human brain a; — represents a gap. 5118 Biochemistry: Bray et al. BOVINE TGGCCGGCG TCAGGAGGAATTCGAACGCCTG HUMAN CCGGCAGTCCCGAGTGCTTCCCGCAGAGGGCTG--GTGGTG MOUSE CeCAGG* se eeee—--- 0 CoAGeCeene B CATCCAGAAAGAAAGAATTCACCTGTGITTCGAGGCAGCGCGCCG GGAGCGGAGTGGAG TCGGGCGGGGCCGAAGCCGGGCCGTGGGC-G eusccseecsesCe omen eee een ste mec eens eel-—eceeeesscceessaCe GAC TTCGAGGGAGCGGCAGCCAGCTITCGCTICCTGGCACA ATG TAGAT CGGGCGGCGGCGGAGCGGCGGAACGCGGG ATG eeGCescoeeeCGeeAse ATG oeGCaccecoeCGeeAee ATG         EPmw Sam *G-- or eee Fic. 5. Nucleotide residues in the 5’ untranslated regions of bovine brain a@-1 cDNA (13) and human monocyte a;-2 cDNA (15). The symbol - represents a nucleotide residue in rat (9) or mouse (10) a; CDNA that is identical to the residue shown in human monocyte a, CDNA. Rat and mouse nucleotide residues that differ from those of human macrophage cDNA are shown. ATG at the 3’ end of each sequence represents the initiation codon for methionyl-tRNA. These results reveal tissue-specific differences in the expres- sion of human aj-1 mRNA. Comparison of a; Amino Acid Sequences. The predicted amino acid sequence of human brain a; is compared in Fig. 4 with a; amino acid sequences predicted from nucleotide sequences of cDNAs from bovine brain (13), human mono- cytes (15), rat C6 glioma cells (9), and mouse macrophages (10). The predicted amino acid sequence of human brain a; is identical to that of bovine brain a; (13) and differs in only 3 amino acid residues from bovine pituitary a; (14) (not shown). In contrast, human and bovine brain a; differ from human monocyte, rat C6 glioma cells, and mouse a; in ~12% of the amino acid residues. The amino acid sequences of a; from human monocytes, rat, and mouse are closely related (99% homology) and contain a codon for an additional amino acid residue, Gln-117, which is not present in human brain, bovine brain, or bovine pituitary a; cDNAs. These data reveal two types of a; CDNA: a;-1 from human brain, bovine brain (13), and bovine pituitary (14), and a;-2 from human monocytes (15), rat C6 glioma cells (9), and mouse macrophages (10). Thirty-six of the 44 amino acid residues of aj-1 and a;-2 that differ are clustered in two regions: region A (amino acid residues 82-142) with 25 residues that differ and region B (amino acid residues 280-309) with 11 residues that differ. Furthermore, only 55% of the amino acid replacements are conservative replacements (28). Regions A and B contain the greatest diversity in amino acid sequence in the a family of proteins and may contain sites that determine the specificity of G-protein interactions with effectors and receptors, re- spectively (29). The predicted secondary structures of a;-1 and a;-2 based on the parameters of Chou and Fasman (30) differ in inter- esting ways. Amino acid residues 118-124 of a;-1 (the numbering system is that of bovine brain q;-1 shown in Fig. 3) are predicted to form an a-helix that is not present in a;-2. Conversely, amino acid residues 97-100 and 120-123 of      ZKEvmwt MACROPHAGE TGAseesesenTooA- Proc. Natl. Acad. Sci. USA 84 (1987) human monocyte, rat, and mouse a;-2 are predicted to form §-turns that are not present in human and bovine q;-1 protein subunits. The differences in predicted secondary structures are located in a variable region of a proteins that is thought to interact with effector molecules. The amino acid sequence of human q;-1 is identical to that of bovine a;-1, whereas the amino acid sequences of human, rat, and mouse q;-2 differ from one another by 3-8 amino acid residues. These amino acid replacements may have resulted from relatively recent mutations during the last 8.5 x 10’ years because human, bovine, and rodent precursors di- verged from a common ancestor ~8.5 x 10’ years ago (33). However, the amino acid sequences of human and bovine a,-1 differ from the sequences of human, rat, and mouse aj-2 in 36 additional amino acid residues, and the mutations that resulted in these amino acid substitutions must have occurred >8.5 x 10’ years ago. Such considerations lead us to speculate that aj-1 and a;-2 mRNA are transcribed from separate genes that originated by duplication of an ancestral a gene much more than 8.5 X 107 years ago and then diverged over a long period of time by accumulation of mutations. The differences between the amino acid sequences of aj-1 and aj-2 are likely to be functionally significant, since the differences apparently have been conserved during evolution. In some ways the relatedness of a;-1 and aj-2 resembles that of a,-1 (17-19) and a,-2 (4, 20), which exhibit 78% amino acid homology and interact with rhodopsin in retinal rods and opsin pigments in cones, respectively. a; Nucleotide Sequences. Comparison of a; cDNA nucleotide sequences from different organisms (not shown here) provides additional evidence for two types of a;. The nucleotide se- quence of human brain aj cDNA closely resembles that of bovine brain (13) and bovine pituitary (14) a; cDNAs (94% homology); in addition, human monocyte (15), rat C6 glioma (9), and mouse macrophage (10) a;-2 cDNA nucleotide sequences closely resemble one another (87-90% homology). However, aj-1 nucleotide sequences differ substantially from those of a;-2. As shown in Fig. 5, the nucleotide sequence of the 5’ untranslated region of bovine brain aj-1 differs markedly from the corresponding sequences of human monocyte, rat C6 glioma, and mouse macrophage aj-2 cDNAs (33% homology). However, the 5’ untranslated nucleotide sequences of aj-2 cDNAs from human monocytes, rat, and mouse closely resem- ble one another, which suggests that the a;-2 5’ untranslated nucleotide sequences have been conserved during evolution. Comparison of the initial nucleotide residues in the 3’ untranslated regions of a; cDNAs (Fig. 6) shows that human and bovine brain (13) a cDNAs are closely related (92% homology) and that human monocyte and rat a, cDNAs are related to one another (83% homology). However, little or no homology was detected between the 3’ untranslated regions of human brain and bovine brain a cDNAs compared to BRAIN TAAGTTTT -GCAGTCC- -ATGGTAAAATGCATTTTCAAACCAAATGAGTACTTATATATGGATCTCIGTA BRAIN TGAs «Ge eGeeGe~-sAAscccasececnoccscnneceeunaceneessseseCneGeceecceueCns MONOCYTE TGAGGGGCAGCGGGGCCTGGCGGGATGGGCCACCGCCGAAT TTGTACCCCCCAACCCCTGAGGAAGATGG C6 GLIOMA TGAsescese4lecescccsehucccesescccensTeoleCecCTeoeneheTansTGescunesens oe eels oe TT CGG*G*sCTeTGCC*ACCCA*TeT+TGs ¢G*TC*GAGGCCCCA HB GACTAGAGTCTTGCAGCAACACAGAATGTAATATAAGGCARATGCATCTGGGACITGACCAAAGTITGTTCTGTTTTGTT BB shh veescennac cece sescc ences saGeneeTe eels ccccnnecceelssesceues ee ene ohean—cee Fic. 6. Nucleotide sequences at the beginning of HM GGGCAAGAAGATCACGCTCcCcGccTeTTceccc -GececrIrrercererrreerererrrerrercaccrececete the 3’ untranslated regions of human brain aj-1 and RG Pewee seenaCoeTecssceTenesccasevcahsoTocusCeoCessGeleeTeccveCevce human monocyte a;-2 cDNAs (15). The nucleotide MM AA++A+*A+GCeCA+GAAG*GIGAGAGA*A*G«s «ATT + Ts GAGACAAAGC *ACCTGCTAT*C*CG*AG*TTTAAAGAAA sequence of bovine brain aj-1 cDNA (13) is compared HB TTTT---TAACTGAAAGTAACAGAAGGACCTTTCT TAAATGTGACAGATGGTCCTGCAGT-TGAAACTGAAGGACAGTGS BBR eecees MDa mee m ane recelTGr ever csrecceGeceGesceeGesGesseesecceseGeeceatsecces Geeoses with the sequence of human brain a,-1 cDNA, whereas the nucleotide sequences of rat (9) and mouse (10) a;-2 HM TCCCCTCA--~-GCTCCAAACGTAGG-cGAGGccTTeccacaccccTcccTerrreaaccerecccrrercteacat-c  ©DNAs are compared to the nucleotide sequence of RG PH eee ee CCTCGe eee TeGe Te cccGuavcccessGlensescceasevessCoheehesCheeTossscenscGehe MM AAAAAGAA*AAR HB TTAAAGCTGGGCTCTAGTATATTGATGATTTCTGCATAAGTGTAAATATGCAAATGTATGTATACATGTATTTATG BH see ces cee oncans ee oGealGeese eCosceAavceCAscccosncvecasccvsessceseussccsseeses HM CIGGTAATGGCCATGGTACCCCCTT -CTGGGCATCTGTTCTGGTTTT-TAACCATTIGTCTTGTTCTGTGATGAGGG RG Ce eGeGaccevarnes oe eT eee anececcuaCheesGeesccsCocssesevccans oe ere rGeGoenece human monocyte a;-2 cDNA. TAA or TGA at the 5’ terminus represents termination codons for a; cDNAs. The symbol - represents a nucleotide residue that is identical to the residue shown for human brain a;-1 or human monocyte a;-2 cDNA. Nucleotide residues that differ are shown. Biochemistry: Bray er ai. HUMAN TAAGAAGCGAACCCCCAAATTTAATTAAAGCCTTAAGCAC SG   AATTAATTAAAAGTGAAACGTAATTGTACAAGCAGTTAATCACC CecnanrevenvesheGoTAcccccccsveCocccceCescens Poem ener enGesccescceCeceCocesccceecoeGesesea Pear e rane eGeercescrcerescccescceeeeseGGecene    Sescece—oeTese ooscesTTeT        a B R M H CACCATAGGGCATGATTAACAAAGCAACCTTTCCCTT-Ccc B os R M FIG. 7. Nucleotide sequences at the beginning of the 3’ untrans- lated regions of a, cDNAs from human brain (11) and human liver (12). The human nucleotide sequence is compared with the se- quences of a, cDNAs from bovine brain (7) and bovine adrenal medulla (8), which are identical in this region, and with rat C6 glioma (9) and mouse macrophage (10) cDNAs. TAA at the 5’ terminus represents the termination codon for a, cDNA. The symbol - represents a nucleotide residue that is identical to that shown for human a, cDNA; nucleotide residues that differ are shown. human monocyte, rat, and mouse a;-2 cDNAs. The sequence of the first 25 nucleotide residues from the 3’ untranslated region of mouse a; cDNA matches the initial 3’ untranslated sequences of human monocyte and rat a; cDNAs (92-96% homology), but thereafter, the sequences are not related. As shown in Fig. 7, the nucleotide sequences of the 3’ untranslated regions of human, rat, mouse, and bovine a, cDNAs also are highly conserved (90-93% homology). How- ever, the initial portion of the 3’ untranslated nucleotide sequence of human brain a, cDNA (11, 12) is not related to the 3’ untranslated sequences of human qj-1 or a;-2, rat a, (9), or bovine a@,-1 (17-19) or q-2 (20) cDNAs (not shown). The relatively high homologies in untranslated regions of ;-1, aj-2, or a, MRNAs in different species suggest that the untranslated nucleotide sequences are functional and thus have been conserved during evolution. The 3'-terminal untranslated region of bovine brain aj-1 cDNA contains many repeats of (A+T)-rich sequences similar to the consensus sequences TTATTTAT (34) and TTI(G/A)NNNTTTTTTT (35), which have been found in the 3’ untranslated regions of some species of MRNA and have been proposed to function as signals for rapid turnover of mRNA (36). The (A+T)-rich sequences are less frequent in a and a, and have not been found in human, rat, or mouse aj-2 cDNAs. Whether a;-1 mRNA turns over more rapidly than a;-2 mRNA remains to be determined. The nucleotide sequences in the 3’ untranslated regions of B-actin, cardiac a-actin, c-fos, nerve growth factor, and creatine kinase mRNAs from different organisms also have been conserved during evolution (see ref. 37 for discussion). Different forms of actin and creatine kinase with conserved 3’ untranslated regions have been shown to be the products of separate genes that are expressed in different tissues and at different times during development. Data from a cDNAs have revealed an unexpected diversity in a and a, (7-18). Comparison of human brain and human monocyte (15) aj cDNAs suggests that the two types of human a; are transcribed from separate genes. The nucleotide se- quences of a, cDNAs reveal that a; genes are subject to strong selective pressure in the coding region and the 5’ and 3’ untranslated regions. Comparison of amino acid sequences predicted from a; cDNAs suggests that a;-1 and aj-2 proteins may differ in function as well as in tissue distribution and abundance. We thank Dr. Gerald Zon for synthesizing the oligodeoxynucleo- tide probes. This work has been presented by P.B. in partial fulfillment of the Ph.D. requirements of George Washington University (Wash- ington, DC). e 10. 11. 13. 14. 15. 16. 17. 18. 19, 20. 29. 30. 31. 33. 34. 35, 36. 37. Proc. Natl. Acad. Sci. USA 84 (1987) 5119 Gilman, A. G. (1984) Cell 36, 577-579. Fung, B. K.-K., Hurley, J.B. & Stryer, L. (1981) Proc. Natl. Acad. Sci. USA 78, 152-156. Grunwald, G. B., Gierschik, P.\\", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qkq8-hcgn_xfmc", "00000000-0000-0000-AECB-F912B5266DBF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Murine Hox-1.11 Homeobox Gene Structure and Expression", "101584910X110", null, "1992", "July 1992", "Reporting on the nucleotide sequence and expression of homeobox genes in mice during embryonic development, this article anticipates that the Hox-1.11 homeobox protein may be required for the development of some \"tissues in the head and thorax, such as the medulla oblongata, some cranial ganglia or cranial nerves, and tissues that originate from or interact with the cephalic nerve crest.\"", "Articles", "Amino Acid Sequence,Cloning, Molecular ; Genes, Homeobox ; RNA, Messenger ; Molecular Sequence Data", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of the National Academy of Sciences (U.S.).", "Copyright may apply", null, null, "Proc. Natl, Acad. Sci. USA Vol. 89, pp. 6280-6284, July 1992 Biochemistry Murine Hox-1.11 homeobox gene structure and expression (Hox-1.11 nucleotide sequence /embryonic development) DOoNG-PING TAN**, JACQUELINE FERRANTE*, ADIL NAZARALI*, XIAOPING SHAo*, CHRISTINE A. KOZAK, Vicky Guo*, AND MARSHALL NIRENBERG* *Laboratory of Biochemical Genetics, National Heart Lung and Blood Institute, and {Laboratory of Molecular Microbiology, National [nstitute of Allergy and Infectious Diseases, National Institutes of Health. Bethesda, MD Contributed by Marshall Nirenberg, March 31, 1992 ABSTRACT The Hox-1.11 gene encodes a protein 372 amino acid residues long that contains a conserved pentapep- tide, a homeodomain, and an acidic region. The amino acid sequence of the homeodomain of Hox-1.11 is identical to that of Hox-2.8, and the N-terminal and C-terminal regions of Hox-1.11 are similar to those of human HOX2H, which is the equivalent of murine Hox-2.8. The Hox-1.1] gene was shown to reside on murine chromosome 6, which contains the Hox-1 cluster of homeobox genes. One species of Hox-1.11 poly(A)+ RNA approximately 1.7 kb long was detected in mouse em- bryos, which is most abundant in 12-day-old embryos and progressively decreases during further embryonic develop- ment. The most anterior expression of Hox-1.11 poly(A)+ RNA in 12- to 14-day-old mouse embryos was shown by in situ hybridization to be in the mid and posterior hindbrain. Hox- 1.11 poly(A)* RNA also is expressed in the VII and VIII cranial ganglia, spinal cord, spinal ganglia, larynx, lungs, vertebrae, sternum, and intestine.   Mouse chromosomes contain four clusters of homeobox genes that are thought to have originated during evolution by successive duplications of an ancestral Antennapedia— Ultrabithorax (Antp-Ubx) cluster of homeobox genes (1, 2). Both the amino acid sequences of the homeodomains derived from these genes and the order of the genes within each cluster have been conserved during evolution (1, 2). The order of the homeobox genes in a mammalian Antp-Ubx chromosomal cluster of genes is related to the most anterior site of expression of each gene in the embryo, which is successively displaced toward the posterior, starting with the second gene from the 3’ end of the cluster and progressing toward the gene at the 5’ end of the cluster (for a recent review, see ref. 3). However, the expression of many of the homeobox genes overlaps toward the posterior. Some of the Drosophila homeobox genes in the Antp and Ubx clusters of genes (4) function as homeotic selector genes (5), which determine unique parts of the body. Homeotic selector genes also may be determinants of cell compartments—i.e., they may regulate genes that encode molecules that enable cells to mix only with cells in the same compartment. Relatively little is known about the functions of Antp-—Ubx clusters of homeobox genes in mammals. However, recent evidence suggests that a segmental pattern of rhombomeres is generated during the development of the vertebrate hind- brain. Motorneuron nuclei of branchiomotor nerves V, VII, and IX are produced by rhombomeres 2 and 3, 4 and 5, and 6 and 7, respectively. Furthermore, pairs of hindbrain seg- ments match adjacent branchial arches (6). In addition, cells in the hindbrain of the developing chick embryo do not cross rhombomere boundaries (7), which suggests that rhom- bomeres correspond to cell compartments in the developing   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked *‘advertisement”’ in accordance with 18 U.S.C. $1734 solely to indicate this fact. 6280 hindbrain. Krumlauf and Boncinelli and their colleagues (8) have shown that anterior expression boundaries of some Hox genes correspond to rhombomere boundaries and have sug- gested that combinatorial sets of homeobox and other pro- teins that regulate genes may impart unique positional ad- dresses to hindbrain rhombomeres and associated structures in the branchial region of the embryo. In this report we describe the nucleotide sequence$ and the expression of the murine Hox-/.// gene during embryonic development. METHODS AND MATERIALS Hox-1.11 Clones. Part of the homeobox region of the murine Hox-/.// gene (9) (nucleotide residues 1539-1658 in Fig. 2) was used as a template for the synthesis of a 32P- labeled RNA probe (=1.4 x 10? cpm per wg of RNA), which was used to screen an ICR Swiss mouse genomic DNA library in AGem-11 (Promega) for the Hox-/.// gene. Hy- bridization was performed at 65°C in 1 M NaCl/50 mM Tris-HCl, pH 7.6/1% SDS containing 100 wg of yeast tRNA per ml and 3.3-4.4 x 105 cpm at 22P-labeled RNA per ml. Filters were washed (final wash) in 0.1x SSC (1x SSC = 0.15 M NaC!1/0.015 M sodium citrate, pH 7) containing 0.1% SDS at 40°C for 30 min. Escherichia coli C600 hfl cells (BNN 102) infected with an 11.5-day-old Swiss mouse embryo cDNA library in AgtlO (Clontech) were plated, and 2 x 10° plaques were screened with an *°S-labeled RNA probe (specific activity, 1.9 x 10° cpm per wg of RNA) transcribed from a Hox-/.// genomic DNA subclone (nucleotide residues 1494~2220 in Fig. 2). Hybridization was performed in 50% formamide containing 5x Denhardt’s solution (1x = 0.02% polyvinylpyrrolidone/ 0.02% Ficoll/0.02% bovine serum albumin), 0.5% SDS, 175 pg of yeast (RNA per ml, and 4 x 10° cpm of 35S-labeled RNA per ml at 42°C for 20 hr. The final wash was with 0.1 SSC/0.1% SDS at 40°C for 1 hr. DNA Sequencing. Genomic DNA and cDNA fragments were subcloned into pBluescript II KS(+) (Stratagene). The exonuclease II/mung bean nuclease unidirectional deletion method (10) was used to generate genomic DNA or cDNA subclones with overlapping deletions. Both strands of DNA were sequenced by the dideoxynucleotide chain-termination method (11) using Sequenase 2.0 (United States Biochemical) or Taq polymerase DNA sequencing kits and with M13 forward or reverse primers or specific primers. 7-Deaza-dGTP or dITP (United States Biochemical) were used to resolve compressions. An Applied Biosystems DNA sequencer and fluorescent primers or dideoxynucleotides also were used to   *To whom reprint requests should be addressed at: Laboratory of Biochemical Genetics, Nationa] Heart Lung and Blood Institute, National Institutes of Health, Building 36, Room 1C06, 9000 Rock- ville Pike, Bethesda, MD 20892. SThe nucleotide sequences of Hox-/.// genomic DNA and cDNA have been deposited in the GenBank data base (accession nos. M93148 and M93292, respectively). Biochemistry: Tan et al. determine DNA sequences. GCG computer programs were used for sequence analysis. We thank Marvin Shapiro for help in using the DNAdraw program (12) to make Figs. 2 and 3. Northern Analysis. BALB/c mouse embryos 10, 12, 14, 16, or 18 days after fertilization were homogenized in 4 M guanidine thiocyanate/0.1 M Tris chloride. RNA was puri- fied by ultracentrifugation through 5.7 M CsC1/10 mM EDTA (13). Poly(A)* RNA was obtained by oligo(dT)-cellulose column chromatography (13) and then was fractionated by gel electrophoresis (1% agarose/formaldehyde gels; 10 yg of poly(A)* RNA per lane). A 510-base-pair (bp) cDNA frag- ment starting from the 5’ end of the cDNA clone without the homeobox (nucleotide residues 345-821 and 1462-1494 in Fig. 2) was labeled with [32P]dCTP (2.1 <x 10° cpm per yg of DNA) by the random primer method (13). Hybridization was performed at 42°C for 22 hr in 50% formamide containing 5.8 x 10° cpm of the 3?P-labeled cDNA probe per ml, 5x SSPE (lx = 0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA), 5x Denhardt’s solution, 0.5% SDS, and 200 yg of yeast tRNA per ml. The final wash at 65°C was with 0.1x SSC/0.1% SDS. Identification of the Chromosome That Contains the Hox 1.11 Gene. Seventeen Chinese hamster X mouse somatic hybrid cell lines (14) were used to identify the mouse chro- mosome that contains the Hox-/./] gene. Chromosomes from 10 hybrid cell lines were identified by Giemsa-—trypsin banding; mouse chromosomes from other cell lines were identified by isozyme analysis. For Southern analysis, 10 ug of DNA from each line of hybrid cells was incubated with HindIII, subjected to electrophoresis in a 0.4% agarose gel, and then transferred to a nylon membrane. The hybridization solution contained 2.8 x 10° cpm of >2P-labeled cDNA (nucleotide residues 345-821 and 1462—1494 in Fig. 2) per ml, 50% formamide, 6x SSPE, 1% SDS, and 133 yg of dena- tured, sheared herring sperm DNA per ml. The final wash of the filters at 60°C was with 0.1x SSC/1.5% SDS. In Situ Hybridization. BALB/c mouse embryos 12.5 or 14 days after fertilization were separated from parental tissue and frozen as described by Dony and Gruss (15). Sections 10 pm thick were cut in a cryostat at —20°C and collected on slides coated with poly(L-lysine). Sections were fixed and hybridized by modification of the method described by Hogan et al. (16). S-labeled RNA probes without the homeobox (1-2 x 108 cpm/g) were prepared from the 5’ region of the cDNA (nucleotide residues 429-821 and 1462- 1494 in Fig. 2) by incorporation of uridine 5’-[a-@*S)thio]- triphosphate. Slides were washed with 2x SSC/1 mM DTT at 50°C, then with 0.2x SSC/1 mM DTT at 55°C, and finally with 0.2x SSC/1 mM DTT at 60°C (1 hr each wash). RESULTS AND DISCUSSION Two million recombinants from a murine genomic DNA library were screened for the Hox-/./1] homeobox gene by using *?P-labeled RNA synthesized from PCR-amplified, cloned mouse genomic DNA corresponding to the Hox-!./1 homeobox region described previously (9). Three Hox-/./1 clones, AT7, A16, and A33, were obtained. In addition, two million recombinants from an 11.5-day-old mouse embryo cDNA library were screened with a genomic DNA fragment corresponding to most of exon 2 of Hox-/.// genomic DNA, and one Hox-1.11 cDNA clone, 1250 bp long, was obtained. The structure of the Hox-/.// gene and partial restriction maps of Hox-1./1 genomic DNA and cDNA are shown in Fig. 1. The Hox-!.1] gene contains two exons separated by a small intron. Four thousand and forty nucleotide residues of Hox-/]./1 genomic DNA and 1250 residues of cDNA were sequenced. The composite nucleotide sequence of Hox-/.// genomic DNA and cDNA and the deduced amino acid sequence of the Hox-1.11 homeobox protein are shown in Fig. 2. The Hox- Proc. Natl. Acad. Sci. USA 89 (1992) 6281 SEQUENCED REGION   5 EXON 1 EXON 2 5 (Ss) PINTRON | (3) PPHRR RHH PH PP Rip yPBTPHH H PHBSHP BHPP BX ° 2 4 6, 8 \\\\ 10 12 14 14s 2.17 GENOMIC DNA CLONE 7 \\\\ 7 NX sf ‘N 7 YPWMK = HOMEOBOX ‘ / Py Bt pu 1 (AL, 3 S p12 3 & Tc2 cDNA CLONE 1 99 477 513 692 1250 BP Fic. 1. Partial restriction maps of Hox-/.// clones AT7 genomic DNA in AGem-11 and ATc2 cDNA in Agt-10. Exons 1 and 2 in cloned genomic DNA are represented by grey boxes and intron 1 by a narrow open box. The black box in exon 2 of cloned genomic DNA and cDNA corresponds to the homeobox. The location of the 4 kilobases (kb) of genomic DNA that was sequenced is shown above the genomic DNA restriction map. The boxed regions in cDNA Tepresent the coding portion of the cDNA. The black box in exon 1 of the cDNA corresponds to the conserved pentapeptide core, Tyr-Pro-Trp-Met-Lys (YPWMK). Abbreviations for restriction en- zymes are as follows: B, BamHI; H, AHindIll; P, Pst 1; R, EcoRI; S, Sac I; X, Xba 1. 1.11 gene contains a 1116-nucleotide-residue open reading frame that encodes a protein of 372 amino acid residues with a calculated M, of 40,793 and a pI of 5.67. The first ATG in the open reading frame (nucleotide residues 443-445) is assumed to be the initiation codon for protein synthesis; however, only 6 of the 10 nucleotide residues in the putative translation initiation site match the Kozak consensus se- quence for initiation of protein synthesis, GCC (A or G) CCATGG (17). The ATG codon is preceded by two adjacent in-frame termination codons 102 nucleotide residues up- stream of the ATG. Exon 1 encodes 126 amino acid residues with a conserved hexapeptide core, Glu-Tyr-Pro-Trp-Met- Lys, found in some vertebrate homeobox proteins. Similar sequences also are found in homeotic homeobox proteins of Drosophila. Comparison of the nucleotide sequences of Hox-1.11 genomic DNA and cDNA revealed a 640-bp intron with an unusual 5’ splice site, AG | GTCAGT. Only 3% of approximately 400 vertebrate 5’ splice sites examined contain C as the third nucleotide residue from the 5’ terminus of the intron (18); however, the splice branch site and 3’ splice site match the consensus sequences perfectly. Exon 2 encodes a homeodomain (amino acid residues 139-198), which is fol- lowed by a region with 9 acidic amino acid residues out of 12 residues (amino acid residues 214-225). Four potential phosphorylation sites for protein kinase C, one for cAMP-dependent protein kinase A, and six for casein kinase II are shown in Fig. 2. Two putative protein kinase C phosphorylation sites are within the homeodomain: Ser-139, the first amino acid residue in the homeodomain, and Thr-179, the amino acid residue immediately before the putative third a-helix of the homeodomain, which is thought to be the major DNA binding site of the protein. The last amino acid residue in the homeodomain, Thr-198, is a possible phosphorylation site for cAMP-dependent protein kinase A. One putative protein kinase C phosphorylation site is within the acidic domain, and a casein kinase I] site, Ser-227, is the second residue after the acidic domain. The possibility that the activity of Hox-1.11 protein as a regulator of gene expression is reversibly controlled by phosphorylation and dephosphor- ylation of serine or threonine residues deserves further study. The termination codon and polyadenylylation signal (AT- TAAA) are separated by only four nucleotide residues. AT- TAAA was reported to serve as a polyadenylylation signal in 12% of 269 vertebrate cDNAs surveyed, and the polyadeny- lylation activity of ATTAAA was shown to be 77% of the activity found for AATAAA (19). Comparison of the nucleo- tide sequences of Hox-/.// genomic DNA and cDNA showed 6282 Biochemistry: Tan et al. : . AP2_ . : - : . - : : : CAGGAGGCAAACAGGCACTCTCGCCCCCCACCCACTCCCGGGGCATTGCCATCCACACCCACATATATGTATTTTTGCCCTGAAAAAAAGTGTAAATAAAGCCTCGATGGCCCCCAATGA rc GGCGTTCCTTTCTGACTTTTTTGGATCAATCAAACAGACAGTGGCTTCTTTTGATTAAAGCCCAAATTGTCATTGGGCAGAAGCAATCATGTGACAGCCAATTCGGTCCAATTTCAACCT TGTCTCCATGAATTCAATAGTTTAATAGTAGCGCGGTCCCCATACGGCTGTAATCAGTGAAT TAGAAAAAAAACACCCCAGCAGCGATCTTCTATGATAGATTTTPTTTTTCCTTCGCGCT CGCCTTTTTCCTGGGCCTTGCCCCCCCAAAGCCCCTCCAAAAGAGGGAACTTTTCCTCCGAGGGGGCTCCAAGGAGAAGGCCATGAATTACGAATTTGAGCGAGAGATTGGTTTTATCAA MN ¥ E F E R E IT G F I oN TAGCCAGCCGTCGCTCGCTGAGTGCCTGACATCTTTTCCCCCTGTCGCTGATACATTTCAAAGT TCATCAATCAAGACCTCGACGCTTTCACACTCGACACTGATTCCTCCTCCTTTTGA S$ Q PLS}L A E C L TT & F P PV AOD T F QS 5 8 I K T S T LS H S T L IT P PP F CE GCAGACCATTCCCAGCCTGAACCCGGGCAGTCACCCTCGCCACGGCGCTGGCGTTGGCGGCCGCCCCAAGTCGAGCCCCGCGGGCAGTCGCGGCAGCCCGGTGCCTGCCCGCGCCCTGCA Q T TIT P S LN PG S H P RH GA GV GGRP K S&S S$ P AGS RG S85 P A G AL Q GCCGCCTIAGTATCCCTGGATGAAGGAGAAGAAGGCGGCCAAGAAAACCGCGCTGCCGCCCGCCGCCGCCTCCACGGGCCCTGCCTGCCTCGGCCACAAAGGTCAGTCCGGAGACTTGGC P PIE Y P W M KJE K K A A K K T AL PPA AA S T GP ACL GH EK CCCAGGTCTCGGGGACCCTTGTCCCCCTCCGGGCTTCCCTAGGAGCGGTTGTGGGGGAGGGGACCATGAGCTTCGAGGGAAGGGGGAGAGAGAGAGATGCCTGGTGGCGGGTCTCATGCC CCTGTGGTCTCAGGAGTGGGTTTGGTGGAGGGGAAAATAGATCCCGAGTCCCACAATGAGACATATATATTTTGAGGAGGGGGGGCACTTTCCCTAACTTGTGTAATGTAGGATGATTTA TTTGAGTTGGAACTGACCTCCTCTTIGTCTAGTIGTCCTAGAGTTTGGCTTTTTGACAGTAATGAAGAGTGATAGACCGCTCTTGCTCAGCTAAGCAGCTGATGCATTAATTATAAATTGT GTTGTAGCTAATATAAAGTTTGCTCCCGGATGAAGAGGTTGGGGGAAGC CACAGGCAGGAATTTGATGGAGGTGGAAGAGACTGGGCTTCCCCGGGCTGGGCTCCCAGGAAGGGCAGCAC SPLICE BRANCH SITE AATAGCTGCACTGCATCCAGGGGCGGCCATTTTGTTGCAGTTGATCTTTTCTGCTGTATTTATGCTCCAATGGAATAACCCTGCTCGGACCCTTCCACCTICAACTGTATGTGTGTCTCT   TGTTGGTTTCCCTTTCTGCAGAATCCCTGGAAATAGCTGATGGCAGCGGCGGGGGATCCAGGCGTCTGAGAACCGCGTACACCAACACTCAGCTTTTGGAGC TGGAAAAGGAATTTCAT E S L EI AODGS& 6&6&GE&E 8 R R L RT AY T N T QL LE LE K E F     CAACAAGTACCTTTGCAGACCCCGCAGGGTGGAAATCGCCGCGCTGCTGGATTTGACCGAGAGACAAGTCAAAGTGTGGTTTCAGAACCGGAGAATGAAGCATAAGAGGCAAAC: N K Y L ¢C R P R R V ET A AL LD & g ER @Q@ Vv K V W F Q N R RM K H K R Q 2 GCAAGGAGAACCAAAACAGCGAAGGGAAATTTAAAAACCTGGAGGACTCGGACAAAGTGGAGGAAGACGAGGAAGAGAAGTCACTCTTTGAGCAAGCCCTCAGTGTCTCCGGGGCCCTTC Cc K E N Q N S E G K F K N L E_D 8 DK VF EE D FE BE E£ K[SJL F EQaAtLS VS GAL TGCAGAGGGAAGGGTACACTTTTCAGCAAAATGCGCTCTCTCAACAGCAGGCTCCCAATGGACACAATGGCGACTCCCAAACTTTCCCAGTTTCGCCTTTAACCAGCAATGAGAAAAATT LE R EG ¥ TF QQ NAL §& @ QQ A PN GHN GD S$ QT F PV 5 [tr] K N   TGAAACATTTTCAGCACCAGTCACCCACTGTTCCTAACTGCTTGTCAACAATGGGCCAGAACTGTGGAGCTGGCCTAAACAATGACAGTCCCGAGGCCATCGAGGTCCCCTCTTTGCAGG LK H F Q H QS P TV PN CLS TM GOQNC GAGHLNN OD S PE ATI EV P[S]L Q ACTTCAATGTTTTCTCCACAGATTCCTGCCTGCAGCTTTCAGATGCACTGTCGCCCAGCTTGCCTGGCTCCCTGGACAGTCCTGTAGATATCTCAGCTGACAGCTTTGACTTTTTTACAG DF N V F S$ T DS €C L @LS& DA LS PS LPG S$ LD PV D I S A D S F D F F T POLY A SIGNAL T/G CLUSTERS ACACACTCACCACAATCGACCTACAGCATCTGAATTAC TAAGAACATTAAAGCAARACAAAGCTTCACAAAACAAAACGCCTTTGACCAGGTGGCTTTGCCTTCTTTTATTCTCGGGAGTT yY * DT LET}T I DLoOQHLsN   POLYADENYLATION SITE GATTTTCGTTTTAGTTTCTTICTTGATCTACCCCTACTCTCTCAAATGTTGAGGACTTTCCGTTTAATGTTCTCCCCTGACACAGTTTTAAAGCCATCTCTTGCAAATTATGTTGGCGTTC TAAGTGGTTTTTACACAGAACCCAACAAGCTICTATGTGATTTTCCTGAAAAACAAAACAGGAGGCCTGCAAGAAAGTGACCATAAATTGTCTTGTCACTTTCTGTTTATTTTTGTACCA CATTAGGATGCATTGTCATGCGTATTTTTGGTAGAATAAATTCTCCTTTGCTATAAGTAGCTITCTTATTTITITICTICCCCTCTITCTCAAGACTCATACTGATTTCTTATACTTCTTT   TAGTCTAACATGGTAAATAAAAGTCTGGTCACAATTTACTTTTCAATCTTAATATATTTTATTAGGGTGGCATGTTCAAAGCC TGCAACAAACAGCAACAAAGAAGAAAACCAGAAAGTG   CTAGGGCTTCTGGCAAGACTTITITITTITITTITTITITTITTTITTTTTTGGATTTTTCCCCCCTAAGGGAATGAATTTIGGTTTTCAGTGTTGAGACTCAGGCCATAGAGTTACATTAGTA ATAGAAATGGGCTTGGGCAGCCTGAGTTGGCCCTAGCCCAAGCTTTTGAGTGTGGAGAGGAAAAGTGCCTACAAAAACTTCCAGTTCTAGGACGAGTGGAGTGGAATGGAGTTCCCOTTAC CAAGCTGCTAGTTTCTAGGTCAATAACGGAGTTATCAAAAATTCATTTATTTATACACATTIGTTTCAAACATCTACGTTICCAACTCTTCCTTTCCCTCCCCCACACCCCCATTTTCAGT AP4 GTGGTTGATTAATCTCCCAGCTGTGGATGGGTTCAGGAAAAACAGACCTAAAAGACAGAGGAAAACAAAACAAAACAAAACCACGGTTACTAGCTGGGAAATGCATGCCAGGAAGSGCTC Z-DNA TITCCAGCCATCCCAAGATTCTTGCCCTCACCACACACACACACACACACACACACACACACACACACACACAATCGATATTTAATCCTTAAATCTGTTTACATTGCTAATAATAAATCA TTAAAGGTTTGAAGTCAGGTTAGCAAGTGGAGATTT TAAAAGTGTTTTTGCACCCTAGATTATTAATTAACCCAGTTTATTAGACAGAGAAGGAGCACTTTAAATGAAATATCAATAAAA HiNF-A HiNF-A TGTGATCTAGGGTATGTTATTGGGCTGTTCTGGTITTTTTTCCTICTITTGTCTCCTCCCACTATTTATTTATTTATTTATTTATTTATTTCCCTTTCTGGGTTCTATTCTGGTGACTTAC AP2 APYSP1 CGTGAGAAACTGCAGCAACATGTGCGTGGGGGGGGGGGGGCGGTGATTGATGGAAGAACCCAGTTCTTAATAGCAAAAGCTTGGGAATTACAATGAAACCTCGGACTTTATTCAGGGGTT AP2 TOCTCTCTACCTTCCCTTCACCCTCTACTCCCCTAATCCCTCAGCAGCCTCCATATGTGGGGACTGTGGTGGTTTTCAGGCCCCAGGCTCTGAGATCTGAGCAGGGTGTCCGGGCTCTGG CCAGGGCTGCSCAGTTCCTGGATGTGATTCGTCCTAAGCTCATAAATCAAACGCTTTCTATGAATGAGAATGTCATCAAAGAGATCAATTGCAGGAACACATGCACAAATAAAAATCCTC API TTACGTATTTGCCGGGGATCCCCGTCCGAAAGCATTAAGT TAGAAGGCGTTTAGTCATAATTCATTTTTATTGCTCTTTT Proc. Natl. Acad. Sci. USA 89 (1992)         120 240 360 480 13 600 53 720 93 840 126 960 1080 1200 1320 1440 1560 159 1680 199 1800 239 1920 279 2040 319 2160 359 2280 372 2400 2520 2649 2760 2880 3000 3120 3240 3360 3489 36CC 3720 3840 3960 4040 Fic. 2. The nucleotide sequence and the deduced amino acid sequence in single-letter code of the murine Hox-/.1/ gene. The nucleotide sequence is a composite obtained by sequencing 4,040 nucleotide residues of Hox-/.// genomic DNA (clones A16 and AT7) and 1,250 residues of clone ATc2 cDNA, which correspond to nucleotide residues 345-821 and 1462-2234. Numbers on the right correspond to deoxynucleotide or amino acid residues. The conserved hexapeptide, EYPWMK, and the homeobox are enclosed within boxes. Hox-1.11 mRNA synthesis is initiated at nucleotide residue 135. The black inverted triangles correspond to RNA splice sites. The splice branch recognition sequence near the end of the intron is underlined, and the branch site is shown as a boldface letter (nucleotide residue 1424). Putative sites for phosphorylation catalyzed by protein kinase C are shown as white S or T residues on black backgrounds; a black T shown on a grey background (T-198) is a putative phosphorylation site catalyzed by cAMP-dependent protein kinase A. S or T residues enclosed within open boxes are possible phosphorylation sites catalyzed by casein kinase II. The polyadenylylation signal sequence, ATTAAA (2206-2211), is underlined, and the polyadenylylation site (nucleotide residue 2234) is shown in boldface letters. T/G clusters on the downstream side of the polyadenylylation site and six additional polyadenylylation signal sequences are underlined. Some putative binding sites for proteins that regulate gene expression also are underlined. Two polypyrimidine regions and 30 sequential T residues also are underlined (30 T residues were found with Al6 DNA, 20 with clone AT7 DNA). that nucleotide residue 2234 functions as the polyadenyl- ylation site. T/G clusters, which may play a role in the formation of poly(A)+ RNA (for review see ref. 20), were found downstream of the polyadenylylation site. Six addi- tional polyadenylylation signals were found in the 3’ untrans- lated region of the Hox-/.// gene, but we do not know whether they function as alternative polyadenylylation signals. Primer extension experiments (not shown here) revealed one major site for initiation of Hox-1.11 mRNA synthesis at nucleotide residue 135. Further work is needed to determine whether the AATAAA sequence 40 nucleotide residues up- stream of the initiation site for mRNA synthesis functions as a TATA box. The first nucleotide residue of ATc2 cDNA corresponds to nucleotide residue 345 shown in Fig. 2; Biochemistry: Tan et al. Hox-i.il 4 INGEFERE IGF INSQPSLAECLTSFPG eTFOSSSTKASTLESEAMEEEP PE) 56 HOX2H WII Top Pd 59 e) SR SET OR SESE ey Vole US PUAN Pay ae eee seen PE le 47 PFEQTHPSLAPG HERHGAGVGGUIPK SSEIAGSRESISVED Ls ie bala as 1cg Pee AIGA S'S _ORPRSOE EDGYALP PPR SrSL gE Tah 0 od AEST I E 167 ¥ oa ALPP PACHGHK.. 2... ESMELADGSERESI GES OUTRO MMAR Ca taey 163 SOSATS PRIPAASAVPASGVGS PAD ea GGGARRLRTAYTNTOLLELEKEFHFNKY| 167 1 eS ILCRPRRVE TAALLDLTERQVKVWFOQNRRMKHKROTO: JONSIGGKF KN . ISDKVE 222 Wee SS SD SSSR BoP G0 UUT RDP ed LON Ora els se sey) Od cee KO. PPDGIRAC PGI IICDP. . 226 EEEKSLF AL ESV | LLEMEGY TEQONA LSOQOAENGHNGRSOTF AS PLYSNEKNLKH 282 rs A PY SAS . -WEACCEPPEVVIQCAL SAMPRPL vane ee eee RO 266 FQHOSPTY rr STMGONCGAGLNNEBPZEATEVPS NGF s#2DSCLOLSI 344 LEGAGAS SIG@ALRGAGGLEPGPLPEDVFSGROMSY. . 2. FE FigAAehfemeass 320 AVEDSD E38 Proton pels Bu) T SF apoimeil Ny* LSPSLEGSLDSPViggSiaome ST Le T DLO Fic. 3. The amino acid sequence in single-letter code of murine Hox-1.11 protein is compared with that of human HOX2H protein (24). The amino acid residues of the conserved hexapeptide and the homeodomain are underlined. White amino acid residues on a black background correspond to identical amino acid residues; black amino acid residues on a grey background represent groups of conservative amino acid replacements, which are as follows: S$, T, G, A, P/L,M. I, V/ E, D, Q, N/R, K, H/ F, Y. W/ and C.   72 56 Www therefore, 211 nucleotide residues are missing from the 5‘ end of ATc2 Hox-1.11 cDNA. The untranslated regions of the Hox-1.// gene contain possible sites for proteins that are known to regulate gene expression, such as AP-1, AP-2, AP-4, SP-1, and HiNF-A (seven sequential ATTT direct repeats constitute two HiNF-A sites). The 3’ untranslated region also contains 30 sequential T residues; two additional polypyrimidine regions; a (CA)2) repeat, which is expected to assume the conforma- tion of Z-DNA under appropriate conditions; and a region rich in G residues. The amino acid sequence of the murine Hox-1.11 homeo- domain is identical to that of the Hox-2.8 (21), which suggests that both homeobox proteins may bind to the same or similar nucleotide sequences in DNA. Hox-1.3 (22) and Hox-2.1 (23) also have homeodomains with identical amino acid se- quences. A comparison of the amino acid sequences of murine Hox-1.11 protein and human HOX2H (24) protein (the equivalent of murine Hox-2.8) is shown in Fig. 3. The sequence of the human, rather than the murine homeobox protein is shown because only the sequence of the homeobox region of murine Hox-2.8 has been reported thus far. The homeodomain of Hox-1.11 and HOX2H is the most highly conserved region of each protein; however, many identical amino acid residues or conservative amino acid replacements are present in the N-terminal region, near the conserved hexapeptide, and in the C-terminal region of each protein. Approximately 50% of the amino acid residues of the murine Hox-1.11 and human HOX2H proteins are identical, and an additional 18.6% are conservative amino acid replacements. These results suggest that the Hox-/.// and Hox-2.8 genes originated by duplication of the same ancestral gene and then gradually diverged by mutation. Murine homeobox gene clusters Hox-/, Hox-2, Hox-3, and Hox-4 are located on chromosomes 6, 11, 15, and 2, respec- tively. The chromosome that contains the Hox-/.// gene was identified by Southern analysis of DNA preparations ob- tained from 17 Chinese hamster x mouse somatic hybrid cell lines that contain different sets of identified mouse chromo- somes (Table 1). Genomic DNA from each hybrid cell line was incubated with HindIII, subjected to gel electrophoresis, transferred to a nylon membrane, and hybridized to a 32P- labeled Hox-1.11 cDNA fragment. The Hox-1.11 cDNA probe hybridized to a 4 kb murine HindIIL DNA fragment and a 3-kb hamster DNA fragment. DNA from 10 of the 17 hybrid cell lines examined contained the murine 4-kb fragment of the Hox-1!./1 gene. Correlation of the murine chromosome con- tent of each hybrid ceil line with the results of Southern Proc. Natl. Acad. Sci. USA 89 (1992) 6283 Table 1. Analysis of concordance between Hox-/.1] DNA hybridization to mouse DNA from 17 Chinese hamster x mouse somatic hybrid cell lines and the mouse chromosomes present in each cell line   No. of hybrids*   Mouse % chromosome +/+ -/- +/- f+ discordance 1 8 5 2 2 24 2 9 2 1 5 35 3 4 4 4 2 43 4 7 5 3 2 29 5 2 6 8 1 53 6 10 7 0 0 0 7 8 1 2 6 47 8 8 5 2 2 24 9 5 5 4 2 38 10 1 6 9 1 59 ll 0 7 10 0 59 12 6 1 3 5 53 13 7 5 2 2 25 14 1 6 8 1 56 15 10 0 0 6 38 16 6 6 2 1 20 17 9 1 1 6 42 18 9 4 1 1 13 19 8 4 2 3 29 x 7 3 3 4 41   *Symbols represent the presence (+/—) or absence (—/ ) of Hox- 1.11 DNA probe hybridization to a 4-kb mouse DNA AindIll restriction fragment determined by Southern analysis and the pres- ence (/+) or absence ( /—) of the mouse chromosome that contains the Hox-]./] gene. The number of discordant observations is the sum of the +/— and —/+ observations. The percent discordance is the number of discordant observations, divided by the total number of observations, multiplied by 100. The results show that the Hox-/.// gene resides in mouse chromosome 6. analysis showed that the Hox-/./] gene resides on murine chromosome 6, which suggests that the Hox-/.// gene is a member of the Hox-/ cluster of homeobox genes. The amino acid sequence similarity of Hox-1.11 and Hox-2.8 suggests that Hox-/.1/ is the second gene from the 3’ end of the Hox-/ cluster of genes located between Hox-/.5 and Hox-1.6, and that Hox-1.11, like Hox-2.8, is a member of the Drosophila proboscipedia (pb) subfamily of homeobox genes. DAYS MOUSE EMBRYO 10 12 14 16 18   Fic. 4. Northern analysis of Hox-1.11 poly(A)* RNA at different stages of mouse embryo development. Each lane contains 10 ug of poly(A)* RNA from mouse embryos 10, 12, 14, 16, or 18 days after fertilization as indicated. Poly(A) RNA was subjected to electro- phoresis, transferred to a Nytran membrane, and hybridized to the 5’ portion of Hox-1.11 3?P-labeled cDNA (without the homeobox). The positions of 28S and 18S ribosomal RNA (4718 and 1874 nucleotide residues, respectively), a 1.6-kb RNA standard, and a diffuse band of Hox-1.11 poly(A)+ RNA, approximately 1700 resi- dues long are shown. 6284 Biochemistry: Tan et al.     Bae Fic.5. In situ hybridization of a 416-nucleotide-residue Hox-1.11 358-labeled RNA probe (without the homeobox) to Hox-1.11 poly(A)* RNA ina parasagittal section of a mouse embryo 12.5 days after fertilization (A) and a transverse section of a 14-day-old mouse embryo (B), exposed to x-ray film for 5 and 12 days, respectively. MY. mid and posterior mylencephalon; SC, spinal cord; M, mesen- chyme near the larynx; T, thymus; S, sternum; PV, prevertebrae;: TE, telencephalon; and DI, diencephalon. The expression of Hox-1.11 poly(A)t RNA during mouse embryo development was determined by Northern analysis of poly(A)* RNA from embryos at different stages of devel- opment (Fig. 4). The **P-labeled Hox-1.11 ATc2 cDNA probe contained nucleotide residues 1-510 shown in Fig. 1 but not the homeobox. Only one diffuse band of Hox-1.11 poly(A)* RNA, approximately 1.7 kb long, was detected. The abun- dance of Hox-1.11 poly(A)* RNA is low in 10-day-old embryos, maximum in 12-day-old embryos, and then pro- gressively decreases in abundance in 14-, 16-, and 18-day-old mouse embryos. The size of Hox-1.11 poly(A)+ RNA found by Northern analysis (1.7 kb) agrees well with the size of Hox-1.11 mRNA determined by nucleotide sequence analy- sis [1454 nucleotide residues without a poly(A) tail, and 1704 nucleotide residues assuming a poly(A)* tail of 250 residues]. Tissues that contain Hox-1.11 poly(A)* RNA in 12.5- and 14-day-old mouse embryos were identified by in situ hybrid- ization (Fig. 5). A Hox-1.11 S-labeled RNA probe without the homeobox was hybridized to Hox-1.11 poly(A)+ RNA in sagittal or transverse serial sections of mouse embryos, and sections were subjected to autoradiography. Hox-1.11 poly(A)* RNA is expressed prominently in mid and posterior myelencephalon (but not in the pons), spinal cord, larynx, thymus, sternum, and vertebrae. In other sections not shown here, Hox-1.11 poly(A)” RNA also was detected in the VII and VIII cranial ganglia, spinal ganglia, lungs, ribs, and intestine. These results agree with and extend a previous report that Hox-1.11 poly(A)+ RNA in mouse embryos is expressed in posterior hindbrain, cranial ganglia VII and VIII, and mesenchyme of the second and third branchial arches (8). Nothing is known about the functions of Hox-1.11 protein. However, homologous recombination in embryonic stem cells and transgenic mouse technology have been used to obtain strains of mice with Hox-1.6 (25, 26) or Hox-/.5 (27) mutations. Homozygous loss of function mutants of the Hox-1.6 or Hox-1.5 genes have different phenotypes, but each phenotype consists of the absence of some tissues and anatomical defects in other tissues in the head and thorax. Proc. Natl. Acad. Sci. USA 89 (1992) Therefore, Hox-1.6 and Hox-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p2q3_53vf~8npt", "00000000-0000-0000-2242-5D6592A09CD8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Hox-1.11 and Hox-4.9 Homeobox Genes", "101584910X111", null, "1992", "April 1992", "Since the late 1980s, Nirenberg has studied the homeobox genes that code for proteins that bind to special nucleotide sequences in DNA and either activate or inhibit the expression of corresponding genes.  This article describes the nucleotide sequences of two homeobox genes.", "Articles", "Amino Acid Sequence,Base Sequence,Genes, Homeobox ; Molecular Sequence Data", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of the National Academy of Sciences (U.S.).", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2883-2887, April 1992 Biochemistry Hox-1.11 and Hox-4.9 homeobox genes (Hox-4.3 / Hox-4.2 /homeobox nucleotide sequences) ADIL NAZARALI, YONGSOK KIM, AND MARSHALL NIRENBERG* Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Marshall Nirenberg, December 17, 199] ABSTRACT Mouse Hox-1.11 and Hox-4.9 genes were cloned, and the nucleotide sequences of the homeobox regions were determined. In addition, nucleotide sequence analysis of the homeobox regions of cloned Hox-4.3 and Hox-4.2 genomic DNA revealed some differences in nucleotide sequences and in the deduced homeodomain amino acid sequences compared with the sequences that have been reported.   Homeobox genes code for proteins that bind to specific nucleotide sequences in DNA and either activate or inhibit the expression of the corresponding genes (for reviews, see refs. 1-5). Homeobox proteins are related to one another primarily in the sequence of the 60-amino acid residue DNA-binding-site portion of the protein, the homeodomain. The homeobox family of genes is large; more than 50 mouse homeobox genes or species of cDNA have been reported thus far, and additional homeobox genes undoubtedly will be found in the future. Many homeobox genes reside at neigh- boring sites in the chromosome in clusters of homeobox genes (5, 6). Whereas the Drosophila genome contains only one copy of the Antennapedia (Antp) and Ultrabithorax (Ubx) clusters of homeobox genes, mammalian genomes contain four copies of the combined Antp—Ubx cluster of homeobox genes, which presumably originated by succes- sive duplications of an ancestral cluster of genes (7). The amino acid sequences of the homeodomains encoded by genes that originated as copies of the same ancestral gene, which are located in different clusters of genes, are more closely related to one another than the homeodomains en- coded by other genes within the same cluster. Both the amino acid sequence of the homeodomain encoded by each gene and the order of the genes within the four mammalian Antp-Ubx clusters of genes have been highly conserved during evolution. Why the organization of genes within each cluster has been maintained during evolution is not known, but several clues have been found. There is considerable overlap in the expression of many of the homeobox genes in the Antp—Ubx clusters of genes along the anterior—posterior axis of the embryo, but the anterior border of gene expression is successively displaced towards the posterior, starting with the second gene from the 3’ end of the cluster and progressing toward the gene at the 5’ end of the cluster (8, 9). Thus, different combinations of homeobox genes are expressed in different regions along the anterior—posterior axis of the embryo (10, 11). In addition, treatment of cultured human embryonal carcinoma cells with retinoic acid results in the gradual, sequential activation of many homeobox genes in each cluster over a period of days, starting with the gene at the 3’ end of the cluster and proceeding towards the 5’ end of the cluster (6). These results suggest that the order of homeobox genes within each cluster may be involved in determining the topographic position and/or the develop-   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement”’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2883 mental time of initiation of expression of these homeobox genes in the embryo. In this report, the nucleotide sequences of the homeobox regions of Hox-/.1] and Hox-4.9 genes are described.* METHODS AND MATERIALS Clones of PCR-Amplified Mouse Genomic DNA. The ho- meobox regions of many mouse homeobox genes were am- plified by PCR. Multiple species of oligodeoxynucleotides that correspond to highly conserved sequences in the ho- meoboxes of many mouse homeobox genes were synthesized with the aid of an Applied Biosystems DNA synthesizer model 380B and purified by OPC (Applied Biosystems) column chromatography. The (+)-oligodeoxynucleotide primers consisted of 64 species of oligodeoxynucleotides, each 28 nucleotide residues long, with a Sac I site near the 5’ terminus; the (—)-oligonucleotide PCR primers consisted of 48 species of oligodeoxynucleotides, 28 nucleotide residues long, with an EcoRI site near the 5’ terminus. (See Fig. 2 for the nucleotide sequences of the primers.) A programmable DNA thermal cycler (Perkin-Elmer/ Cetus) was used for the amplification of DNA. A typical 25-1 reaction mixture contained 1 yg of BALB/c mouse liver genomic DNA; 50 mM KCI; 10 mM Tris-HCI (pH 8.3); 1.5 mM MgCl; 0.01% gelatin; 15.6 nM of each species of (+)-oligodeoxynucleotide primer and 20.8 nM of each species of (—)-oligodeoxynucleotide primer; 1.0 mM each of dATP, dCTP, dGTP, and dTTP; and 2.5 units of Tag polymerase. Reaction mixtures were covered with 50 yl of mineral oil and were incubated for 35 PCR cycles; each cycle consisted of incubation for 1 min at 94°C, 2 min at 37°C, and 3 min at 65°C. After the last cycle, the reaction mixtures were incubated for an additional 10 min at 65°C. The DNA was precipitated with ethanol, incubated with EcoRI and Sac I, and subcloned in pBluescript II KS(+) (Stratagene). RNA Probes. **P-labeled (+)-RNA probes were prepared by using a modification of the Stratagene RNA transcription protocol. A typical 10-yl reaction mixture contained 40 mM Tris-HCI (pH 8.0), 8 mM MgCl,, 2 mM spermidine, 50 mM NaCl, 10 mM dithiothreitol, 10 uM [a-**PJUTP (800 Ci/ mmol; 1 Ci = 37 GBq), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 194 fmol of linear proteinase K-treated DNA, 10 units of RNase inhibitor, and 4 units of phage T7 RNA polymerase. Reaction mixtures were incubated at 37°C for 30 min; then RNA was precipitated with sodium acetate and ethanol. Clones of Unamplified Homeobox Genomic DNA. A mouse genomic DNA library in AGEM-11 (Promega) was screened for some of the homeobox genes that had been found with PCR-amplified DNA. E. coli KW251 cells (2 x 10°) infected   *To whom reprint requests should be addressed at: Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Building 36, Room 1C-06, 9000 Rockville Pike, Bethesda, MD 20892. *The sequences for Hox-/./1, Hox-4.9, Hox-4.3, and Hox-4.2 have been deposited in the GenBank data base (accession nos. M87801— M87804, respectively). 2884 Biochemistry: Nazarali et al. Proc. Natl. Acad. Sci. USA 89 (1992)                  MOUSE 13 12 11 10 9 8 7 6 5 4 3 2 1 CHROMOSOME m7 ji-t0} 2 |] Hox-1 6 ij P101 Pil P8B- P1160 439 (12) 433 (2) 23} |} fe} ee ee} ee} ee Ha] toe P155 P31 (69) yoeeny : ; Hox-3 15 3G 3F P8A (3) Hox X P30 a4 (3) rg} Hox-4 2 41 P9IB P24 P167 P125 (2) 49 (4) 240 26 (6) rat Fic. 1. Mouse homeobox gene clusters. Genes that code for proteins with similar homeodomain amino acid sequences that are thought to be copies of the same ancestral gene are aligned vertically. The numbers 1 through 13 (from right to left) at the top of the figure represent the vertical sets of related genes in different clusters. Clones of homeobox genes described in this report are shown with shaded backgrounds. Homeobox nucleotide sequences shown in this report are indicated by boxes drawn with thick solid or dashed lines. The chromosomal location of Hox-X is uncertain. The boxes drawn with thin dotted lines indicate that no mouse homeobox sequence has been reported; the names of the human HOX genes (6, 13) are shown beneath these boxes. Some DNA clones described in this report also are shown beneath the appropriate box; the number of DNA clones found is enclosed within parentheses. DNA clones that begin with P are clones of PCR-amplified mouse genomic DNA; clones prefaced by A are clones of mouse genomic DNA that were not amplified prior to cloning. with 25,000 recombinant phages were plated on each 150-mm States Biochemical) with universal phage M13 primers or Petri dish. Phage DNA adsorbed to replica nytran filters specific primers by the dideoxynucleotide chain-termination (GeneScreenPlus, DuPont) was hybridized overnight at 60°C method (12), and also by using an automated DNA sequencer with **P-labeled RNA (35 fmol/ml, 2 x 10° cpm/ml) synthe- (Applied Biosystems Model 373A) with Tag DNA polymer- sized from cloned PCR-amplified DNA. The hybridization ase at 70°C, dITP instead of dGTP, dideoxynucleotides or buffer contained 1 M NaCl, 50 mM Tris-HCI (pH 7.6), 1% primers labeled with fluorescent dyes, double- or single- SDS, and 100 ug of yeast tRNA per ml. Filters were washed stranded DNA preparations, and other components accord- twice with 2x SSC (300 mM sodium chloride/30 mM sodium ing to the manufacturer’s instructions. citrate, pH 7.0) at room temperature for 15 min (each wash), followed by two washes in 2x SSC/1% SDS at 60°C for 60 min (each wash) and finally by one wash in 0.1 SSC at 24°C RESULTS AND DISCUSSION     for 30 min. Filters then were exposed to x-ray film in Clones of PCR-Amplified Homeobox DNA. Small DNA cassettes at —70°C. Recombinant phage with matching pos- fragments that correspond to part of the homeobox of many itive signals on autoradiograms of replica filters were cloned. mouse homeobox genes were amplified from mouse genomic DNA inserts were excised with Sac I and cleaved with DNA with the use of sets of primers, each set consisting of various restriction enzymes; some DNA fragments were multiple species of oligodeoxynucleotides that correspond to subcloned into pBluescript II SK(+). conserved nucleotide sequences within the homeobox (nu- DNA Sequencing. Both strands of cloned DNA fragments cleotide residues 43-68 and 142-162). The amplified DNA were sequenced manually by using Sequenase 2.0 (United was subcloned, and the chain lengths of the DNA inserts from (+) PRIMERS - 7 | (-) PRIMERS (+)5' TGGAG CTC GPA AAA SAR TTT CAT TTT AAl ACC PAA GTT TTA GCC FCT TAC TTARGCT S*¢(-) G G G c c c G ct G ce PERCENT REF. G HOMOLOGY NOS. 9 69 bs 75 39 195 120 135 141 189 162 (69-1416) Hox ‘.11 P&B SAG CTC GAG ARG GAA TTT CAC TIC ARC FAG TAC CIT TGC AGA CCC CGC FOG GTG GAA ATC GCC GCG CTG CIG GAT TTG ACC GAG AGA CAR GiG AAA GIG: TGG TTC CAG AAC COG ACG FIG 100 Hox 2.8 SAG CT GAG ARG GAG] ITE cae TIC AAT AAG TAC CT) ToC Coe] ccG) cst (Cel) ork) caGi atc ocl cere cre GA (Cre) acc CAR AGG) cA] GiT) AAA GIG} GG Tre cAG AAC COAIMCE ATG §=671 [16] Hox 4.9 PI25 GAG CTC GAG AAG GAG TTT CAT TTT AAT AAG TAC CTA ACT AGA GCC CGA CGC ATC GAG ATR GCC ARC TGT TTA CAG CTG AAT GAC ACC CAG GTC AAR ATC) 1GG TTC CAG AAC COG COC ATG 100 Hox 1.6 GAG CTGi GAG AAG GAG TT) CAR TTC) AAC] AAG TAC CTfT] ACiA|/GcA seb) coc, {rt Gifs: GAG ATH! Gcc TEC] CTA CAG CTC} AAT GAG] ACC CAG GTS) ARG) RTC) TGS TTC CAC ART] COR) COC ATO 7E [24,25] Hox 2,9 GAG cid GAG AAG GAR) TIT CAT TIC) AAC fl TAC CT i fl bec cs Fi GIG! GAG ATich ccc C| cr CTC} RAT GAA! ACK] CAG GTG RAG) ATC) TGS TTC CAG ARC COG CGC ATG 64 [16,17] Hox 2.9 GAG CTG) GAG AAG GAR) TTT CAT TTC] ARK) A TAC CTO} AL i] GEC CGS Gl | 9G Arie} Gci) i G Ci¢) RAT GAIA) ACH] CAG GTG! AAG ATC} TGG TTC CAG AAC COG CEC ATE 63 f18] Hox X P30 GAG CTC GAG AAG GAG TTT CAT TIC AAC CGC TAC CTA TGC COG CCG CGC CGO STG GAG ATG GCC AAC CTS CTG AAC CTC AGC GAG CGC CAG ATC AAG ATCHTGG TTC CAR AAC CGG COG ATG 100 Hox 1.9 GAG CT] GAG AAG GAG TTI) CAC TIC AAC CGC TAC CTA Bill coc cco CGC CGC GIG GAG AIG GCC AAC CTS CTG AAC CTC GAG CGC CAG ATC AAG ATCITGG TT cA] AAC coc Cc ATC 95 [24-26] Hox 4.1 AG cTs| GAG AAG GAG TT oR TIC AAC CGC TAR) CT] ToC CGG CCG CGC CGE GTG GAG ATG GCC AAC CTS CTG AAC CTC A GRA CGC CAG ATC AAG ATC| To te ca Ape Coll ¢ RIG 93 [27] Hox 2.7 CAG eT] GAG AAG GAG Tri] cAG TIc AAC cof) TAL MMT) Toc coG ceo CoC CGC GTK] GAG PTG Gee AA CTS CTG AAC CTC AGC GAG CGC CAG ATC AAG ATC) TOG TTC CAG) AAC Col ATG 92 [28} Hox 4,3 P24 GAG CTC GAG RAG GAA TTT CAT TTC AAK CCT TAT CTG ACC AGG AAG AGG AGA ATC GAG GIC TCC CAF ACT CTG GCC CTC ACG GAS ASA CAG GTA AAA ATC! T66 TiC CAR ARC CGG CGC ATG 100 Hox 4,3 Gac (r@ cA aac cAa Trl ct 11] AAC CCT TAT CTG ACC AGG AAG AGS AGA ATC GAG GTC Tee CAT AGT CTG GCC CTC ACG GAS AGA CAG GTA AAA ATC! TGG Tre caG aac [Acc Rog atc 99 [t4] Hox 4.2 P16? GAG CTC GAA AAG GAG TFT CAC TIT AAC ASG TAT CTG ACC AGG CGC CGT C&G ATT GAA ATC GCF CAC ACC CTE TOT CTG TCT GAS CSC CAG ATC ARG are] TG TTC CAG AAS CGG CGC ATG 100 Hox 4,2 GAR CTK] GAA AAG GAA TIT cap) TIT RAC AGG TAT CTG ACC AGG COC CGT CSG ATT GRA ATC GCF CAC ACC CTG TOT CTG (QCT GAG eSc CAG ATC ARG ATC) 166 TFC CAG ARC CG [ok] ATG 99 (15) Hox 2.9 P31 GAG CIC GAR AAG GRG TIT CAT TTT SAIC AAA TAC CTG AGC COT GCC COG ASG GIG GAG ATC GCC GCC ACC CTO GAG CTC : 100 kox 2.9 GAG CTK GA AAG GAA: TTT CAT TTR) AAC AAA TAC CTG AGC COT GCC CGG ASG GIG GAG ATC GCC GCC ACC CTG GAG CIC tog (16,17) kox 2.9 GRG CTIG) GAG RAG GAA! TIT cpT TT] AAC AAA TAC CTG AGC CGT GCC SGG AGG CTG GAG ATC Gc (cc ace CiG CAG CTC | 96 (18)     Fic. 2. The nucleotide sequences of six homeobox DNA clones, obtained by PCR amplification of mouse genomic DNA, are shown and are compared with the sequences of the most closely related mouse homeobox genes. The numbers at the top correspond to homeobox nucleotide residues. The sequences of oligodeoxynucleotide primers for PCR amplification of DNA are shown at the top. The asterisks above the primers indicate that only one nucleotide residue is present at the position indicated; thus oligodeoxynucleotides will base-pair correctly if the codon sequence in DNA is complimentary to that of the oligodeoxynucleotide but not if the DNA contains other synonym codons for the same amino acid. The symbol t indicates that (—)-oligodeoxynucleotide primers do not contain A at this position; hence, correct base pairs can form if the DNA contains five of the six arginine codons but not if the DNA contains the arginine codon CGT. Biochemistry: Nazarali et al.                  —_- —__——_ (+) PRIMERS (-)PRIMERS PEACENT CO=HECTX TI CO=HETTE 2) (O-HELTX 3} HOMOLOGY 15 21 24 28 38 42. 47 52 54 AA BASE Hox 1.11 P8B ELEKEFH FNKYLC RPRRUEIAALL OLT ERQUKUWFONR RM 100 100 Hox 2.8 ELEKEFH FNKYLC APARVEIAALL OLT ERQUKUWFQNR AN 100 7 Hox 4,9 P12S ELEKEFH FNKYLT RARRIEJANCL OLN OTQUKIFQNR RN 109 1900 Hox 1.6 ELEKEFH FNKYLT RARRMEIARGL OLN EITQUKIFONR Bn 83 7 Hox 2.9 ELEKEFH FNKYCS) RARRME | AAITIL Ee TQUK IWFQNR RM 75 64 Hox 2.9 ELEKEFH FNKYLIS) RARRMEAPIIL [ELN EJTQUKIIWFONR AN 75 63 Hox % P30 ELEKEFH FNRYLC APRRUEMANLL NLS ERQIKIMFQNA BN 100 100 Hox 2.7 ELEKEFH FNRYLC RPRAVENANLL NLS ERQIKHWFOQNA RN 100 92 Hox 1.5 ELEKEFH FNRYL(T] RPRAYENANLL ne ERQIKIRFQNA RM 92 95 Hox 4.1 ELEKEFH FNAL] APRAVEMANLL NLIT] EAQIKIMFQNA AN 8393 Hox 4,3-P24 ELEKEFH FMPYLT RKARIEUSHTL ALT ERQUKIIWFQNR RM 400 100 Hox 4,3 ELEKEFR] FNPYLT RKARIEUSHEL ALT ERQUK!WFONR Rt 96 99 Hox 4.2-P167 ELEKEFH FNRVLT ARAATETAHTL CLS ERQIKINFQNA AN 100 160 Hox 4.2 ELEKEFH FNAYLT RARRIEIAHTL CLP] ERQIKIWEONR AN 96 99 Hox 2,9-P31 ELEKEFH FNKYLS RARRVEIAATL EL 100 100 Hox 2.9 ELEKEFH FNKYLS RARRVEIRATL ELN ETQUKIMFQNR AN log 100 Hox 2.9 ELEKEFH FNKYLS RAARUEIAPITL ELN ETOUKIMFONR AN 96 96   Fic. 3, The amino acid sequences deduced from the nucleotide sequences of PCR-amplified cloned mouse genomic DNA (Fig. 2) are shown and are compared with the most closely related mouse homeodomain amino acid sequences. The percent homology be- tween related amino acid sequences for the central region between the PCR primers (amino acid residues 24-47) and the percent homology between nucleotide sequences shown in Fig. 2 (nucleotide residues 69-141) are shown. The numbers at the top refer to homeodomain amino acid residues. The positions of a-helices 1-3 in the Antennapedia homeodomain (1) of Drosophila also are shown. 93 clones were determined. The nucleotide sequences of some of the DNA inserts also were determined. Of 93 clones examined, 85 were identified as homeobox genes. Two clones, P8 and P91 with different DNA inserts, consist of two homeobox DNA fragments amplified from separate genes that were joined by ligation and cloned. The DNA clones found correspond to 13 mouse homeobox genes as shown in Fig. 1. The nucleotide sequences of six amplified homeobox ge- nomic DNA clones are shown in Fig. 2 and are compared with the sequences of the most closely related mouse homeobox genes. The percent homology also is shown between the central region of each cloned DNA insert without the primer sequences (homeobox residues 69-141) and the correspond- ing sequences of the most closely related mouse homeobox genes. The percent homology was calculated for only the central region of the cloned DNA between the primers because oligodeoxynucleotides that hybridize to DNA with some incorrectly paired bases can serve as PCR primers. Proc. Natl. Acad. Sci. USA 89 (1992) 2885 Clone Hox-/.1] P8B corresponds to a novel mouse ho- meobox gene of the Drosophila proboscipedia homeobox class. The nucleotide sequence of clone Hox-/.// P8B is most closely related to that of the mouse Hox-2.8 gene, but only 71% of the nucleotide residues compared are identical. Clone Hox-4.9 P125 is a member of the labial class of homeobox genes, probably the first gene in the Hox-4 cluster of homeobox genes (see Fig. 1). The amino acid sequence of the Hox-4.9 homeodomain was reported recently (11), but the nucleotide sequence has not been described. The nucleotide sequence of Hox-4.9 P125 differs considerably from the sequences of other labial class mouse homeobox genes, Hox-1.6 (71% homology) and Hox-2.9 (63-64% homology). Clone Hox-X P30 may correspond to an unreported murine homeobox gene, probably the third gene in the Hox-3 cluster of homeobox genes (Fig. 1). The nucleotide sequence of the Hox-X P30 homeobox clone is most closely related to that of Hox-1.5 (95% homology). The nucleotide sequence of clone Hox-4.3 P24 differs from that of Hox-4.3 (14) by only 1 of the 73 residues in the central region between the (+)- and (—)-oligonucleotide homeobox primers, which suggests that clone Hox-4.3 P24 corresponds to the Hox-4.3 gene. The nucleotide sequence of clone Hox-4.2 P167 differs from the sequence reported for Hox-4.2 (15) by only 1 nucleotide residue. Of the 85 homeobox clones obtained, 69 were found to be only 78 nucleotide residues long because of the presence of a Sac I site within the homeobox. Sequence analysis of 9 of the 69 clones revealed one kind of DNA insert with Sac I sites at both the S’ and 3’ termini. The nucleotide sequence of a representative clone, Hox-2.9 P31, shown in Fig. 2, is iden- tical to the sequence of the Hox-2.9 gene reported by Rubock et al. (16) and Frohman ef al. (17) and differs by only 2 nucleotide residues from the Hox-2.9 sequence reported by Murphy and Hill (18). The amino acid sequences deduced from the nucleotide sequences of PCR-amplified and cloned DNA are shown in Fig. 3 and are compared with the most closely related mouse homeodomain amino acid sequences. The amino acid se- quence of clone Hox-/./] P8B is the same as that of the Hox-2.8 gene, but the nucleotide sequences differ markedly (71% homology). Clone Hox-4.9 P125 is a labial class ho- meobox gene with an amino acid sequence identical to the sequence recently reported for the Hox-4.9 gene (11). Hox- 4.9 P125 differs from the other labial class homeobox genes, Hox-1.6 and Hox-2.9, in both nucleotide and amino acid sequences. The amino acid sequence of the homeodomain of clone Hox-4.3 P24 (residues 24-47) differs from that of the Hox-4,3 gene by 1 amino acid residue. Since the correspond- ing nucleotide sequences differ by only 1 residue, Hox-4.3 Hox 1,11 433 ~89  GACCOTTCCACCTTCARCTGTATGIGIGICICTIGTIGGITICCCTTTCTGCAGAR -34   -tt -! Fl Hox 1 Hox 1 Hox 2.8 2       W433 S LE | A OG $ 6 6G ERRLRTA Y Vt 433 TCCCTGGARATAGC TGATGGCAGCGGCGGGGGATCCAGGCGTCTGAGARCCGCGTACACCARCACTCAGCTT              TQe 13 39   GG--CC-G-T-GC-A--AT -~G-----A-C--C)---C -CA-A---C-C--G--C-----------G--A--6 Hox 2.8 6 PGLPECG-sg§-/,f------ - ~- - LoEL eK —€F HPF NXKY LECRPRRUY A AL 37 Fic. 4. The nucleotide sequence and de- Hox 1.11 433. /TTGGAGCTGGAAAAGGAATTICATTTCRACARGTACCTTTGCAGACCCCECAGGGTGGARATCGCCGCGCTG | 111 duced amino acid sequence of the Hox-/.// Hox 2.8 [-~-------- G----- G--C--C----- To-+----- G---C-G--G~-TC-C--C--G-----T--CT-~ (clone A33) homeobox and flanking regions Ts 7 are shown and are compared with the se- LoLTERQUKUUFEONRANKH R QT 61 quences of the most closely related mouse Hox 1.11 233 |CTGGATTTGACCGEAGAGACAAGT GARAGTGIGGTTICAGRACCGGAGRATGRAGCATARGAGGCAARC 183 homeobox gene, Hox 2.8 (16). Dashes in the Hox 2.6 = [~---- cl-c--~--- f--G--G~-C----- C----- (-------- AC-C~----A--C---C----6-- nucleotide sequence or amino acid sequence SH - - represent a Hox-2.8 nucleotide or amino acid residue that is identical to the corresponding cK ENQNS EG KF KNLEODSOK EEDE 85 . : Hox 1.11 933. TGCAAGGAGRACCARARCAGCOARGSGARATTTARARACCTSGAGGACTCGGACAAAGTGGAGGARGACGAG 255  MuCleotide or residue shown for Hox-1 11. The homeobox sequence is enclosed within EEK § L FEQA 94 a box. The arrowhead represents an intron— Hox 1,11 433 GRAGAGAAGTCACTCTTTGAGCAAGCC 282 exon junction. 2886 Biochemistry: Nazarali et al. Proc. Natl. Acad. Sci. USA 89 (1992)   ~10 -! -5) K L S$ E ¥ GAT S P CCTTGTCTTTATGTTGCAGGCARACTGTCCGRATATGGAGCCACARGCCCT + a4) P A4l Hox Hox Hox   iP   SR s A | ICCCAGTGCCATCCGCACARRT RT N R T N       FS TK Q LT ELE K EF HFN K YLT RAR A 3) Hox 4.9 41 | TTCAGCACCAAGCARC TGACAGAGCTAGAGARAGAGTITCATTTCAATAAGTACCTARCTAGAGCCCGACEC | 93 Hox 4.9 FS TK Q LT ELE K EF HEN KYL TR ARR | ETANCLQOLNDTOUKIUN FE ONR AO kK I 55 Fic. 5. The nucleotide sequence and de- Hox 4.9 A41 | ATCGAGATAGCCAACTETTTACAGCTORATGACACCCAGGTCAARATCTGGTICCAGAACCGTAGGATGARS 1165 duced amino acid sequence of the homeobox Hox 4.9 | EtantectQbLbNoTQukKInWrFQONRAN EK region of the Hox-4.9 gene (clone A41) are shown. The amino acid sequence of Hox-4.9 Qk kK REREGLLATAAS YA STK L PRS 79 A4lis compared to the recently reported (11) Hox 4.9 241 | CAGRAGAAGAGGGARCGRGRGGSGCTICTGGCCACAGCTGCCTCTGIGGCCTCGATTARGCTICCCCGGTCA 237 : . Hox 4.9 OK KARE Hox-4.9 amino acid sequence. The dashes     ET S$ P | kK $ GRNL GS P §$ Q A Q Hox 4.9 41 P24 DNA probably corresponds to the Hox-4.3 gene. Simi- larly, the nucleotide and deduced amino acid sequences of clone Hox-4.2 P167 differ from those of the Hox-4.2 gene by only 1 nucleotide residue and 1 amino acid residue, which suggests that clone Hox-4.2 P167 corresponds to Hox4.2. The nucleotide and amino acid sequences of clone Hox-2.9 P31 are the same as those of the Hox-2.9 gene (16, 17). Clones of PCR-amplified DNA also were obtained and sequenced that correspond to Hox-!.1, Hox-1.2, Hox-!.3, Hox-!.6, Hox-2.1, Hox-3.4, and Hox-4.4 (data not shown). Homeobox Genomic DNA Clones in AGEM-11. A mouse genomic DNA library in AGEM-11 with 15-kilobase (kb) DNA inserts (average size) that were not amplified prior to cloning was screened for homeobox genes with a mixture of 32p_labeled RNA probes synthesized from PCR-amplified, cloned DNA that correspond to Hox-1.11, Hox-4.9, Hox4.3, Hox-4.2, Hox-3.4, Hox-2.9, Hox-1.2, and Hox-l.1. Two million recombinants were screened, and 29 clones of ho- meobox genomic DNA were obtained. Restriction site anal- ysis revealed seven kinds of DNA inserts that were shown by nucleotide sequence analysis to correspond to seven ho- meobox genes (Hox-!.11, Hox-4.9, Hox-4.4, Hox-4.3, Hox- 4.2, Hox-3.4, and Hox-!./). Hox-1.11. Two genomic DNA clones, A16 and A33, were found that correspond to Hox-/.//. The nucleotide sequence and deduced amino acid sequence of the Hox-1.// A33 homeobox and flanking regions are shown in Fig. 4 and are compared with homeobox sequences of the most closely related homeobox gene, Hox-2.8. The nucleotide sequence of Hox-!.11 433. DNA, which was not amplified prior to cloning, was identical to that found with Hox-/.// P8B cloned from PCR-amplified mouse genomic DNA (nucleotide residues 69-141) shown in Fig. 2. Although only 74% of the Hox-/.11 A33 and Hox-2.8 homeobox nucleotide residues are the same, the amino acid sequences of the homeodomains of Hox-1.11 and Hox-2.8 are identical. However, 9 of the 11 Hox-1.11 A33 deduced amino acid residues that precede the homeodomain and 1 amino acid residue after the homeodomain differ from those of Hox-2.8. These results show that Hox-/.//] and — P y -27 TGTIGTTTTTRATCRGCA $ GAARCAAGTCCCATCAARTCTGGCCGGAATCTAGGARGCCCTICTCAGGCTCAAGAGCETICCTGA represent Hox-4.9 amino acid residues that are identical to those of Hox-4.9 A41. The homeobox region is enclosed within a box. * 100 303 Hox-2.8 are separate genes. The intron—-exon junction shown in Fig. 4 at nucleotide residue —36 was identified by com- paring the nucleotide sequences of Hox-/.// genomic DNA and cDNA, which will be described elsewhere (D. Tan, J. Ferrante, A.N., C. Kozak, V. Guo, and M.N., unpublished data); elsewhere we also show that the Hox-/./1 gene resides in mouse chromosome 6, which suggests that the Hox-/.// gene is a member of the Hox-/ cluster of genes. The amino acid sequences of the Hox-1.11 and Hox-2.8 homeodomains are identical, which suggests that both spe- cies of homeobox proteins may bind to the same or similar nucleotide sequences in DNA. Another pair of homeobox proteins, Hox-1.3 (19, 20) and Hox-2.1 (21-23) also have identical homeodomains. Hox-4.9 41, The nucleotide sequence and deduced amino acid sequence of the homeobox and flanking regions of clone Hox-4.9 441 mouse genomic DNA are shown in Fig. 5. The nucleotide sequence of Hox-4.9 has not been reported pre- viously; however, the deduced amino acid sequence (A41) is the same as the recently reported Hox-4.9 homeodomain amino acid sequence (11), which suggests that A41 DNA is a Hox-4.9 genomic DNA clone. The nucleotide sequence of Hox-4.9 441 mouse genomic DNA (cloned from DNA that was not amplified) is identical to that found with PCR- amplified mouse genomic DNA (clone Hox-4.9 P125 ho- meobox nucleotide residues 69-141 shown in Fig. 2). Hox-4.3 440. The nucleotide sequence and deduced amino acid sequence of the homeobox and surrounding regions of Hox-4.3 440 DNA are shown in Fig. 6 and are compared with the nucleotide and amino acid sequences of the most closely related homeobox gene, Hox-4.3 (14). The nucleotide se- quence of the A40 mouse genomic DNA fragment, cloned from DNA that was not amplified, was found to be identical to that of Hox-4,3 P24, derived from PCR-amplified mouse genomic DNA shown in Fig. 2 (homeobox nucleotide resi- dues 69-141). Only 4 of the 209 A40 nucleotide residues compared differ from those reported for Hox-4.3 (14); how- ever, three of the homeodomain amino acid residues differ from those reported for Hox-4.3. The high nucleotide se- Fic. 6. The nucleotide sequence and de-       Hox 4.3 A40 Hox 4.3 a -1 +t Hox 4.3440 A P GIR RR GR QT ¥ S$ R F Q Hox 4.3 440 GCTCCTGGTIAGACGGAGAGGAAGACARACCTACAGTCGCT TCCRARCCCTAGAGT TGGRARAGGAATICCTT Hox 4.30000 wanna nee qas ncaa nrc nr nnn ncn cr sc rs secscnscne Hox 4.3 - = = pe te eR FON PY LC TR K RR E VS H 440 | TTTARCCCTTATC TGACCAGGAAGAGGAGRATCGAGGTCTCCCAT} Hox Hox a     duced amino acid sequence of the homeobox and surrounding regions of Hox-4.3 A40 mouse genomic DNA are shown and are compared with the Hox-4.3 nucleotide and amino acid sequences reported (14). Only nucleotide and amino acid residues of Hox 4.3 that differ from those of clone A40 are shown; residues that are the same are indi- cated by dashes. The homeobox region is 2 63 45 135     K | WF QN RRA K RK K E NN K OD ARARTCTGGTTCCAGAACAGGAGART GAART GGRARAAGGAGAAL Hox A40 Hox       K F P enclosed within a large box. Nucleotide and amino acid residues that differ are enclosed within small boxes. The arrowhead repre- sents an intron—exon junction reported for Hox-4.3 (14). 66 198 Biochemistry: Nazarali er al. PERCENT HOMOLOGY 1 10 20 30 40 30 60 AA BASE Hox 1.11 A433 SRRLRTAYTNTQLLELEKEFHF NK YLCRPRRUE | ARLLOLTERQUKUMFONRRMKHKRQT 100 100 Hox 2.8 100 74 ROX TK HUNAN 100 90   Hox 4,9 A4t Hox 4.9 HOX 4G HUNAN Hox 1.6 PSRIATNFSTKQL TELEKEFHFNKYLTRARR IE | ANCLOLNOTOQUK I HFONRRNKQKKRE 100 100 100 oF BB of?   Hox X P30 100 160 Hox 2.0 a eee eee eee 100 92 Hox 4.3 440  AARGROTYSRFQTLELEKEFLFNPYLTRKRRIEUSHTLALTERQUKIWFQNRAMKWKKEN 100 100 Hox $3000 wee eee ene ene RU---------------------- §----------------------- 95 98 Hox 4.2 46 PKRSRTAYTROQULELEKEFHFNRVLTRARRIEIAHTLCLSERQIKIUFONRRHKUKKOR 100 100 Hox 4.2 ween eee n eee n eee -- - P- — 98 99   Fic.7. The amino acid sequences of the homeodomains encoded by five mouse homeobox genes deduced from the nucleotide se- quences of cloned DNA are shown and are compared with the amino acid sequences of the most closely related mouse or human homeo- domains. The percent homology between the amino acid sequences of related homeodomains and between the corresponding homeobox nucleotide sequences also are shown. Only differences in amino acid sequence are shown. Dashes represent identical amino acid residues. quence homology between A40 and Hox-4.3 suggests that clone 440 DNA corresponds to the Hox-4.3 gene. However, the possibility that clone A40 DNA corresponds to a novel homeobox gene, Hox-1./2, the eighth gene in the Hox-] cluster of homeobox genes shown in Fig. 1, is not ruled out. A summary of results is shown in Fig. 7. Homeodomain amino acid sequences deduced from the nucleotide se- quences of cloned mouse genomic DNA are shown and are compared with the most closely related sequences reported for mouse or human homeobox proteins. The amino acid and nucleotide sequence homologies also are shown. The amino acid sequence of the Hox-1.11 homeodomain is identical to that of Hox-2.8; however, 9 of the 11 amino acid residues before the homeodomain and 1 amino acid residue after the homeodomain differ from those of Hox-2.8. Many differ- ences also were observed in the nucleotide sequences of the Hox-1.11 and Hox-2.8 homeobox regions. The mouse Hox- 1.11 homeodomain is the equivalent of the recently reported human HOX-1K homeodomain (6). The amino acid sequence of the Hox-4.9 A41 homeodomain is identical to the recently reported Hox-4.9 homeodomain amino acid sequence (11). The mouse Hox-4.9 homeodomain is the equivalent of the human HOX-4G homeodomain (13). Six of the 73 Hox-X P30 nucleotide residues differ from the corresponding sequence of the most closely related ho- meobox gene, Hox-2.7; however, the deduced amino acid sequence of Hox-X P30 is the same as that of Hox-2.7. The cumulative error due to misincorporation of bases during DNA amplification was estimated by comparing the DNA sequences of 10 clones of mouse genomic DNA subjected to 35 cycles of DNA amplification (730 nucleotide residues compared) with mouse genomic DNA sequences that were not amplified prior to cloning, which correspond to Hox-/./1, 4.9, -1.1, -3.4, 4.2, 4.3, and -4.4. No DNA amplification errors were detected. Comparison of the nucleotide se- quences of 13 additional clones of PCR-amplified DNA that correspond to Hox-1.2, -1.3, -1.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dzbk~7296.8dcj", "00000000-0000-0000-4B81-327319D543B5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Neuronal Tumor Cells With Excitable Membranes Grown In Vitro", "101584910X112", null, "1969", "1969", "This article is representative of the shift in Marshall Nirenberg's research toward neurobiology after 1966.  Here, Nirenberg's collaborative work suggests that mouse tumor cells are capable of generating action potentials that are characteristic of healthy neurons.  The suggestion that this phenomenon might be explained by genetic characteristics further reveals Nirenberg's belief that his work in neurobiology was a logical progression.", "Articles", "Neuroblastoma,Neurons,Neoplasms,Cell Membrane,Action Potentials", "Neuroblastoma Research, 1967-1976", "7", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "NEURONAL TUMOR CELLS WITH EXCITABLE MEMBRANES GROWN IN VITRO By Puitire Netson, WINFRED RUFFNER, AND MARSHALL NIRENBERG NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated July 14, 1969 Abstract-—\\\\Mouse neuroblastoma cells grown in vitro are capable of generating action potentials in response to electrical stimulation. A wide spectrum of re- sponses was observed with different cells ranging from passive responses to action potentials. A hypothesis is proposed concerning the acquisition of electrical ex- citability as neuroblasts mature into neurons. Tissue culture methodology affords many opportunities to study molecular aspects of information processing by cells from the nervous system.?: 2 During the last few years we have investigated the properties of tumor cells and normal cells derived from the nervous system and grown in vitro. During the course of these studies, Augusti-Tocco and Sato told us that they had established clonal lines of cells from a transplantable mouse neuroblastoma? that contain choline acetylase, acetyl! cholinesterase, and tyrosine hydroxylase.* Human neuro- blastomas also have been cultured zn vitro? and have been shown to contain en- zymes related to norepinephrine synthesis and catabolism.§° We have investigated electrophysiologic properties of the mouse neuroblastoma of Augusti-Tocco and Sato and wish to report that neuroblastoma cells, grown in vitro, are capable of generating action potentials that are characteristic of neurons. Materials and M ethods.—Neuroblastoma C-1300 is a spontaneous tumor maintained since 1940 by serial transplantation in strain A/J mice. Mice bearing this tumor were obtained from the Jackson \\\\femorial Laboratory, Bar Harbor, Maine. Neuroblastoma cells were dissociated and grown tn vitro as described by Augusti-Tocco and Sato‘ except that Dulbecco’s modification of Eagle’s medium? was used in place of F-10, and cells were grown in an atmosphere of 10% CO, and 90% air. Cells have not been cloned. The average doubling time of cells is 18 hr; hence, cells have been re- peatedly trypsinized and subcultured. The medium contains inorganic ions in the following concentrations (mM): Nat, 154; Kt, 5.4; Catt, 1.8; Mgt+, 0.8; Cl-, 118; and HCO,-, 44. Plastic petri dishes, 35 or 60 mm in diameter and containing neuroblastoma cells in the medium described above, were placed in a plastic chamber on the stage of a Zeiss inverted microscope. The temperature of the medium, measured with a thermistor probe, was maintained at 35°C with a heating element below the microscope stage. A humidified mixture of 10% CO: and 90% air flowed over the surface of the culture. Glass micropipette electrodes with tip diameter <0.5 « and filled with 3 Jf KCI were used to record transmembrane potentials from the cultured cells. The resistance of the electrodes ranged from 10 to 200 megohms; most were from 20 to 80 megohms when measured in the culture medium. The microelectrode was arranged in-a bridge circuit and connected to a Bak unity gain amplifier. This circuit allows transmembrane poten- tials to be recorded while currents are passed across the cell membranes through the im- paling microelectrode.” Results -The morphologic characteristics of mouse neuroblastoma cells cultured in vitro resemble those described by Augusti-Tocco and Sato. How- 1004 VoL. 64, 1969 BIOCHEMISTRY: NELSON ET AL. 1005 ever, the cells used in the present study are not clonal cell lines, so a heterogeneous cell population is expected. The following procedure was used to study the electrophysiologic properties of neuroblastoma cells in vitro. The cell was photographed and the tip of a micro- electrode was inserted in the interior of the cell; another electrode was immersed in the extracellular medium; within several minutes, when stable records could be obtained, the difference in voltage across the cell membrane recorded by the intracellular electrode was determined. Pulses of current 50-100 msec in dura- tion were then passed through the electrodes and the changes in voltage across the cell membrane were determined as a function of time. Steady current was then passed through the electrodes to adjust the voltage across the cell membrane to about —60 mv. Pulses of current again were passed through the electrodes and perturbations in voltage across the cell membrane were determined. This procedure allowed us to test the excitable properties of the cell membrane at the voltage level obtained after penetration of the cell by the electrode and at a standard level of —60 mv. Our objective was to determine whether neuroblastoma cells are electrically excitable im vitro and whether they generate neuron-like action potentials in response to stimuli. Of 259 cells that were examined, 149 were studied in detail. Most of the cells were found to have “active”? membranes ; however, the degree of activity varied widely. Excitable cells were found that were capable of generat- ing action potentials, as well as cells with less active membranes. An example of an excitable cell generating action potential is shown in Figure 1A and B. A resting potential of —40 mv was obtained upon penetration of the cell by the electrode. A steady current of about 2 namp was passed through the electrode to adjust the voltage across the cells membrane to —65 mv. Then a series of five pulses of stimulating current approximately 70 msec in duration, but differing in intensity, were passed across the membrane (the upper traces of Fig. 1B), evoking the changes in membrane voltage corresponding to the lower traces of Figure 1B. The weakest stimulus, labeled a in the F igure, evoked a small, smoothly increasing perturbation in voltage. The most intense stimulating pulses elicited action potentials. Partial responses were elicited by stimuli of intermediate intensity. Nine per cent of the cells examined were capable of generating action potentials. Stimulation of such cells for five msec, a pulse of current briefer than the duration of the action potential, elicited an action potential. Action potentials were elicited two days after cells were dissociated with trypsin and then subcultured. A cell exhibiting only a partial response is shown in Figure 1C and D. A resting potential of —20 mv was found on penetration of this cell, but even when the voltage across the membrane was adjusted to —60 mv with steady current, a pulse of stimulating current clicited only a partial response. A fully developed action potential could not be evoked in this cell. The observed response to a pulse of current is dependent upon the level of transmembrane voltage at the time of stimulation (Fig. 2). When the voltage across the membrane of the cell shown in Figure 2A was adjusted to —60 mv, a current pulse of one namp evoked a partial response (Fig. 2B). A current pulse Pia. 1—-(A, B). The large cell near the center of the photograph was studied elec- trophysiologically. Cells had been sub- cultured 7 days earlier and were past. the logarithmic phase of growth. Total time in vitro was 103 days. The 100 2 bar applies to both (A) and (C). Intracellular record- ings from the cell are shown in (B). Five oscilloscope traces are superimposed. The lines shown in the upper part of this and subsequent figures correspond to the cur- rents that, were passed through the elec- trodes across the cell membrane (right ordinate); the lower curves indicate the changes in voltage across the cell mem- brane (left. ordinate) evoked by these cur- rents. After the resting potential was de- termined, 2 namp of steady current were passed through the electrodes to adjust. the voltage across the cell membrane to —65 mv. Then the cell membrane was stimulated with pulses of current as in- dicated by ON and OFF. The weakest stimulating pulse, labeled a, elicited a small passive voltage response, also labeled a. The most intense stimulating pulses (¢) elicited action potentials. (C, D). The large cell near the center was examined electrophysiologically. The cell is from the same culture described: in (A). The pulse of stimulating current is indicated by the upper lines, the resaltant change in membrane voltage by the lower curve, The inflection on the rising phase of the voltage curve corresponds to 8 partial response of the cell membrane. MILLIVOLTS         {4 L l j 1 0 20 40 60 80 100 MILLISECONDS   NANOAMPERES   MILLIVOLTS     1 1 1 L 40 60 80 100 MILLISECONDS   NANOAMPERES                       Vou. 64, 1969 BIOCHEMISTRY: NELSON ET AL. 1007 w“” T a B ON : 7 “_D orf ° i poe 18 7 UPS -40F d -6O0F $s NO -80F T T mht T t + + + +—J “ 1 1 x ‘ ON D OFF 4io # ‘ | 4 1 = \" 0 H 3 i+) 4 _ | eg es! a $ ° H +2 4 = -20 \\\\ z 3 d $ S$ -40 + h -60] 4 i 1 1 1 4 1 i j 1 1 0 20 40 60 80 100 0 20 40 60 80 100 MILLISECONDS MILLISECONOS Fic. 2.—(A). The cell is from a culture that had been treated with trypsin to dissociate the cells 5 days earlier. The cell was examined electrophysiologically soon after the culture had passed the logarithmic phase of growth. Total time in vitro was 108 days. (B, C, D). An active response is shown in (B); examples of rectification in (C, D). The lines labeled D and H indicate pulses of current that evoke changes in voltage across the cell membrane labeled ct and A, respectively. S designates the steady current used to adjust the voltage across the cell membrane, s. of the same intensity but of opposite direction elicited a different wave form, 2 smoothly changing, uninflected, voltage transient. In Figure 2C, the steady voltage across the cell membrane was adjusted to —25 mv. Then, current pulses that increased or decreased transmembrane voltage did not evoke partial re- sponses; however, the decrease in transmembrane voltage was smaller than the increase. When the steady voltage was adjusted to —35 mv (Fig. 2D), asym- metric responses (rectification) were obtained, but the direction of asymmetry was reversed. However, at a steady membrane voltage of —15 mv, symmetric responses to pulses of current were obtained (not shown). The cell illustrated in Figure 3A generated repetitive action potentials in the ubsence of 2 stimulating current (Fig. 3B) and also in response to a pulse of stimulating current (Fig. 3C). The action potential shown in Figure 3D was elicited by turning off a pulse of current that increased the voltage across the cell membrane. The response appears to be identical to the “‘off-excitation” phenomenon exhibited by many neurons. Action potentials, partial responses, and rectification are characteristic of 1008 BIOCHEMISTRY: NELSON ET AL. Proc. N. A.S.                       “ Fe 2 ° > a 2 = +20k¢ T T T T g t a | | a Fd rm) b i = | a 5 0 on | , aig 9 ; s 8 Yl yar doz 3297 ON OFF | 4o 4-20 t tt z > ae : 4] z = nH ‘ = 407 -40 / 7 \\\\ Lt 1 iL a 1 1 - 60; 1 J 1 1 0 40 80 120 160 200 0 40 80 120 160 200 MILLISECONDS MILLISECONDS Fic. 3.—(A). The large round cell shown in the upper left part of the photograph and in- dicated by the microelectrode was studied. The culture was in the stationary phase of growth; total time in vitro was 66 days. (B). Repetitive action potentials recorded soon after the cell was penetrated by the micro- electrode. The action potentials occurred in the absence of stimulating current. The dashes in this and the following figure represent portions of the records that were filled in during re- production of the records. (C). Two action potentials elicited by a pulse of current. (D). An example of “off-excitation” is shown. An action potential was evoked by turning off the pulse of current. The top of the action potential is not shown. “active” cell membranes and are due to voltage-dependent changes in the perme- ability of cell membranes.\" “Active’ membranes could not be demonstrated in 36 per cent of the cells studied. Such membranes are termed “‘passive’’ be- cause even intense pulses of current produce changes in membrane voltage that have the simple form of voltage trace a in Figure 1C or trace hin Figure 2C and are symmetrical. The relative frequencies of membrane responses are summarized in Table 1. “Active” responses were observed with 65 per cent of the cells examined. Neu- ron-like action potentials were observed with 9 per cent of the cells, partial Taste 1. Summary of cell membrane responses. Resting Number Total potential Membrane properties of cells (%) (mvolts) Active membranes Action potential 13 9 40 Partial response 25 17 24 Rectification 58 39 20 Passive membranes 53 35 17 Total 149 100 22 Vou. 64, 1969 BIOCHEMISTRY: NELSON ET AL. 1009 responses with 17 per cent, and rectification with 39 per cent of the cells. Thirty- five per cent of the cells examined had ‘‘passive’” membranes. Discussion.—The results show that mouse neuroblastoma cells grown in vitro are electrically excitable and are capable of generating action potentials. Neu- rons with action potentials have been found in explant}?: 1° and dissociated cell!4 cultures. However, normal mature neurons usually do not divide, whereas at least some neuroblastoma tumor cells retain the ability to divide in vitro and also exhibit properties expected of neurons. Augusti-Tocco and Sato‘ have cloned these cells and have shown that they contain choline acetylase, acetyl cholin- esterase, and tyrosine hydroxylase. We have confirmed these observations and have also shown that the cells synthesize catechols and catechol derivatives in vetro.36 . Sixty-five per cent of the cells examined electrophysiologically had “active” membranes. However, a wide spectrum of responses was observed with different cells, even when membrane properties were studied at the same transmembrane voltage and under relatively stable recording conditions. Although cell injury may have contributed to the variability of results, it is unlikely that such a wide spectrum of responses would be observed with mature neurons. We propose as a working hypothesis that the range of membrane properties ob- served is expressed in the following sequence as neuroblasts mature into neurons: passive responses, delayed rectification, partial responses, and action potentials. The molecular events responsible for these phenomenon are not fully understood. It is possible that some neuroblastoma cells differentiate in vitro and one or more genetic programs corresponding to functional action potentials are expressed, or that events related to cell division affect the capacity of cells to generate action potentials. Alternatively, cultures may contain many neuroblastoma cell lines that differ genetically and are fixed at different stages of differentiation. Experi- ments are in progress to resolve these questions and also to determine whether neuroblastoma cells are capable of forming synapses in vitro. The mouse neuro- blastoma cell system of Augusti-Tocco and Sato and other cell lines derived from the nervous system provide many opportunities to explore various aspects of neurobiology. Note added in proof: Similar results have now been obtained with clonal neuroblastoma lines. We would like to express our appreciation to Gabriella Augusti-Tocco and Gordon Sato for telling us about their experiments with neuroblastoma cells prior to publication. + Murray, M. R., in Cells and Tissues in Culture, vol. 2, ed. E. N.Willmer (New York: Academic Press, 1965), p. 373. * Geiger, R. 8., in International Review of Neurobiology, vol. 5, ed. C. C. Pfeiffer and J. R. Smythies (New York: Academic Press, 1963), p. 1. * Augusti-Tocco, C., and G. Sato, Seminar at the Neurosciences Research Program meeting, Feb. 5, 1969, Brookline, Mass. * Augusti-Tocco, G., and G. Sato, these Proceepines, in press. * Murray, M. R., and A. P. Stout, Amer. J. Path., 23, 429 (1947). * Bohuon, C., E. H. LaBrosse, M. Assicot, and A. Amar-Costesec, in Recent Results in Cancer Research, vol. 2 Neuroblastoma, Biochemical Studies, ed. C. Bohuon (New York: Springer-Verlag, 1966), p. 16. 7 Goldstein, M., B. Anagnoste, and M. N. Goldstein, Science, 160, 767 (1968). 1010 BIOCHEMISTRY: NELSON ET AL. Proc, N. A. 5. 8 yon Studnitz, W., Pharm. Rev., 18, 645 (1966). * Dulbecco, R., and G. Freeman, Virology, 8, 396 (1959). 1” Araki, T., and T. Otani, J. Neurophysiol., 18, 472 (1955). 11 Grundfest, H., Federation Proc., 26, 1613 (1967). 12 Crain, S. M., J. Compt. Neurol., 104, 285 (1956). 18 Hild, W., and I. Tasaki, J. Neurophystol., 25, 277 (1962). M4 Scott, B. S., V. E. Engelbert, and K. C. Fisher, Exp. Neurol., 23, 230 (1969). 4 Wilson, S., J. Farber, R. Rosenberg, T. Amano, P. Nirenberg, N. Seeds, and M. Nirenberg, unpublished data.", "Nirenberg, Marshall W. ; Nelson, Phillip G. ; Ruffner, Winfred", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4ssv_x7mj-4d57", "00000000-0000-0000-190E-9AC7380CE2DF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes taken in graduate school", "101584910X113", null, "1957", "[ca. June 1957]", "This is a handwritten list of things to do, including contacting Gordon Tomkins, lab chief at NIH and an important collaborator for Nirenberg.", "Notes", null, "Biographical Information", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "v. aX op f \\\\ prt Ba re _ wh 5 eh Pato fh ORKNTH ae ve. a Th Ht Peo NTH | ——J W) EE ee BM I", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qeru.pygg~g9ai", "00000000-0000-0000-EC75-9F251DC736DB", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes taken in graduate school", "101584910X114", null, "1956", "29 May 1956", "These handwritten notes include experimental objectives, data, and conclusions in the study of glycolosis.", "Notes", "Glycolysis", "Biographical Information", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Ie.ce [¥ 3° OGh2% |a.30 OQ 3! 304.20 [4 2815; S34 00 ese 193ep [soy @ Fhe [00 | O [40 fz} so fr aro Jaa m3 PCI ZC. 1O | BFOiO [Hae uber deo Pd] sy [67 Try rex ]so 1140 [oy [1eclisa pest se | 204bs0 20) LH LO PMU TAL Sd PST frre dere |G fae []SS] S383 [PPV ise ia f~ Jing (3f US Nezjavo}ce feo furo fz [itt | reo fivepise} 93 159] so7 i sp i s3 VHGA AIG ST PSST breed rey y3sT sSpi Tp a i7e gs FS (sd                                 “4 S°Aleo fiod | 8 fo |iaoley LA fs) 135] 1-7 B71) ef | 3y           GK t > «MRR ory (3. gar phn, . a. oom Dah SR, rer ot *- fo a CGP rab 2D aid BR eee AL Baa +0 = MOR Go YO aa Yo, % 2 (24 3h ZIG. Be arth 13,8 7 DFS Sy, — . ; ON . Seerey ~ 2 hia 6.24 (633 4.0 MS 0 PYyoOT oO fy 0 O 4 [sy five — Ply —] se] - u5 ~ faults) [2S q} vel 2a 4 > J (e971 — 1155] — j 21 z> p- [SEF —]y] ~ [wit - vy | ~ tot — [ott ~ [oe -     ENG, sh al @ cy” r =Th_ J. jn y x Th fel caetNa ryfo tO a - Tel | be aely 4 We. 99) US JOR BIG~ GUT Mx VE We vor [yy |-39 fs 105. $5. IF (16) 1051163 1023.-4 a laa MIYAZ AI CRY van 98 nes 6 | SB | 19 \\\\auroe, MF + SF lacy: no 19 O.21G YNVEDGG | Io Le 4% i i. EPO. 72 | Zd¥ 239) 1] aso 1ST + ys 309 | 4 | 64: 24% Be, adU s¢ 160 F119 2649 | U9 TRS 193 BOLLS VG VOT loz 4Erl sro nSolinay 2sVqI6 92% |-44 UV ty ys 2€% j VIN | SE | : : po 3o0 [wigs S4Cl gS |r ol reins] PE 360N39) es 47114 \\\\ 013 ZS] S05 11930, Sub ats, as a MSO; 1096 |- 10 BIY 119 Lgb LID | SSUYG 73042994 ure | 134 ge (630| MAL OE IWG Yom | ys, 397 3h n Ste US Ui43. Gy 195] GY, 19% y200] YH THol sng SV IUD Ye: 219 N40 Mow YdS 136 | S¥ 1763) 25.634 aig Br40 | S25} 131 | 9d | 0 T4664 cneso, YE IS 1) ea, ssslasl $B 4SANGAGIENS [raee] 4S YP Hoc| 1134 Ana. Bus #1140 apeop GAA A Saye Gos tm t-te toe azlurx, te BOLT; BY ez tray | YeB|NT I 40 ie FD wa GO UW ADTT Ss INVES UMA VSCLVIN, UHL 200 O92, Ve (206) 203] 58 WT] ye (99) 790, 19 2Gliy 24 NOb 23 940 | ny «41 64 | | ee 13¢ [34f BL) gz (T1394 [BSD 420! 433: 329 US. (73 AW PO. fas oD. ‘ : 3 Le 596143,3173 [reo bse €] B64 ISSR B3C DOI US 4ST, Le 4 BD Boo why ) bv ony . . 3eef gs | wel SOiy pe dig, 350] YD? | BIOL 4S 12 BTY eS PTS OW | HS 3961314 22g ns WR pie éb 73 341 oe 1, Why byt STOLL! Red. es aL wS“| Yu] Yor| a7 Pir jyey [rusblv fal you Yo} Danes a ‘$13 5951470 | @ 4 SU 1576 j2064 | WhO SP Ve] al SS [6A] Cha 199 YS 1170 [rsto 1590) 99g YS) Yy3}334, and a 53S/[G2U 9371 63 OSHIGTS| 548d KNAISVG © Is S499 407; SB: 1940", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ke2y.xu5u~5zt2", "00000000-0000-0000-4E1E-3340B9450027", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francois Jacob to Marshall W. Nirenberg", "101584910X115", null, "1961", "20 December 1961", "This letter congratulates Nirenberg for the two manuscripts on protein synthesis, which he sent to the Institut Pasteur in late 1961.  Jacob refers to Nirenberg's work as a \"wonderful story.\"", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of Francois Jacob.", "Copyright may apply", null, null, "20 decembre 1961 Dear Dr. Nirenberg, Many thanks for your two manuscripts. It is a wonderful story. All my congratulations. Sincerely yours, F. Jacob", "Institut Pasteur (Paris, France) ; Jacob, Francois, 1920-2013", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p9cs~mw4u_wadk", "00000000-0000-0000-3BAD-534300A827F0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Genetic Memory", "101584910X116", null, "1968", "25 November 1968", "A reprint from the Journal of the American Medical Association article, presented as a 1968 Albert Lasker Basic Research Award Lecture at the New York University Medical Center. Nirenberg recounts developments in deciphering the genetic language, formulation of codons and the meaning of their punctuation, mechanisms of codon recognition, and the issue of universality.  As the translation apparatus of the cell accepts and follows instructions written in the appropriate molecular language, Nirenberg suggests our ability to decipher and manipulate that language means synthetic messages will probably eventually be used to \"program cells and their descendents.\"", "Articles", "DNA Replication,Codon,RNA, Messenger ; Amino Acid Sequence,Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Reprinted From The Journal of The American Medical Association November 25, 1968, Vol. 206, pp. 1973-1977 Copyright 1968, by American Medical Association Lasker Award Genetic Memory Marshall Nivenberg, PhD enetic memory resides in specific molecules of deoxyribonucleic acid. The DNA alphabet consists of four letters, the bases, A, T, G, and C. The sequence of letters in a nucleic acid message corresponds to a sequence of the 20 amino acid species in protein. Two molecules of DNA interact with one another by hydrogen bonding between bases on opposite chains. As proposed by Watson and Crick,’ adenine pairs with thymine, and guanine with cytosine. Information is retrieved by tran- scribing the DNA message in the form of ribonu- cleic acid and then translating the RNA message into protein. Triplets are translated sequentially, from left to right. The information encoded in a nucleic acid tem- plate enables the reading mechanism to select one from many species of molecules, to define the posi- tion of the molecule relative to the previous mole- cule selected, and to define the approximate time of the event relative to previous events. Hence the nucleic acid functions both as a template for other molecules and as a biological clock. Although the genetic information is encoded in the form of a one-dimensional string, the polypep- tide products fold in a specific manner predeter- mined by the amino acid sequence. In effect, a complex, three-dimensional object is created by first fabricating a linear string of letters that folds upon itself, in a fairly specific manner. Probably the principle of unidimensional sculpturing could be used by man for the construction of certain kinds of objects. Often, one molecule of messenger RNA (mRNA) contains the information for many molecules of protein, so the RNA message must also contain in- formation for the initiation and termination of the polypeptide chain. The translation must be ini- tiated properly since selection of the first word also phases the translation of subsequent words. At least three enzymes are required for the initiation process, three additional enzymes for the forma- tion of the peptide bond and movement of the ribo- some along the message, and one or more enzymes for the termination of protein synthesis. In addi- From the National Heart Institute, Bethesda, Md. Presented as a 1968 Albert Lasker Basic Research Award Lecture at the New York University Medical Center. New York, Nov 26, 1968. Reprint requests to National Heart Institute, Bethesda, Md 20014.   JAMA, Nov 25, 1968 © Vol 206, No 9 tion, specific enzymes are required for the synthesis and repair of DNA, for the synthesis of mRNA, aminoacyl-transfer RNA (AA-tRNA), and for the modification of tRNA and ribosomal RNA. The process of protein synthesis, illustrated diagram- matically and in highly abbreviated form, is shown in Fig 1. The chromosome of a relatively primative or- ganism, such as Escherichia coli, consists of approximately 3 million base pairs. Sufficient infor- mation is present to determine the sequence of 1 mil- lion amino acids in protein which is approximately the amount required for 3,000 species of protein. The human genome is 1,000 to 2,000 times larger than that of E coli, ie, information for the synthesis of 3 to 6 X 10° species of protein could be present. However, multiple copies of essentially the same gene frequently are stored; hence much information probably is redundant. The precise number of enzymes required for the storage, retrieval, and transmission of genetic in- formation has not been determined. Perhaps 200 species of protein would suffice. However, as much as 25% of the total protein synthesized by rapidly growing FE coli is utilized for the construction of new ribosomes. The Rate of Reading.—The average E coli ribo- some can read approximately 1,000 mRNA triplets per minute. The reading rate therefore is quite slow compared to a man-made computer, However, pro- tein is synthesized simultaneously at many sites within the cell. Escherichia coli with a generation time of 25 minutes contains approximately 15,000 ribosomes per chromosome. Hence, 15 million amino acids may be incorporated into protein per minute per chromosome. The RNA message usually is cov- ered by a train of ribosomes; hence one molecule of mRNA is translated simultaneously at different sites. A single molecule of mRNA may serve there- fore as a template for the synthesis of many mole- cules of protein. It seems likely though, that some species of mRNA are destroyed earlier than others. Some genes are transcribed more frequently than others. It is clear that retrieval of genetic informa- tion often is regulated selectively. Some regions of E coli DNA may be transcribed 1,000 times per generation; others may be transcribed only one or two times per generation. Deciphering the Genetic Language.—The experi- mental approaches that eventually led to the de- ciphering of the genetic code came from the study of in vitro synthesis of protein. The demonstration that mRNA is required for the in vitro synthesis of protein and that synthetic polynucleotides such as poly U serve as templates for the synthesis of polyphenylalanine provided a means of exploring many aspects of the code and the translation process.” Polynucleotides composed of different combinations of bases in random sequence were syn- thesized with the aid of polynucleotide phosphory- lase, discovered by Grunberg-Manago and Ochoa.° Synthetic mRNA preparations then were used to Genetic Memory—Nirenberg 1973 SATGCGAATGATCGAATGTCTGTTGTGCGCT...3 a a a a a a. JS TACGCTTACTAGCTTACAGACAACACGCGA,..5 2 tOR Fahy aaa asm (WETISULE ) ais an Panay nn StS 5 a} im eRs Dan io aie o a: AA ACTIVATION 1. The retrieval of genetic information is illustrated in a condensed and diagrammatic form. direct cell-free protein synthesis. The base content of the mRNA can be correlated with the amino acid content of newly synthesized protein. In this man- ner the base compositions of 53 RNA codons were assigned to amino acids.*” It was also found that three sequential bases in mRNA correspond to one amino acid in protein, that AA-tRNA is required for the translation of mRNA,* and that codons for the same amino acid sometimes require different species of AA-tRNA for their translation.” Analysis of the coat protein of mutant strains of tobacco mosaic virus provided evidence that triplets in mRNA are translated in a nonoverlapping fashion, because the replacement of one base by another in mRNA usually results in only one amino acid re- placement in protein.* Alternate codons for the same amino acid were shown in most cases to contain two bases in com- mon. Common bases were assumed to occupy the same base position of synonym triplets. Base Sequence of Codons.—Codon base se- quences were established in several ways: by di- recting in vitro protein synthesis with polyribonu- cleotides containing repeating doublets, triplets, or tetramers of known sequence as described by Kho- rana in the accompanying communication, and by stimulating the binding of aminoacyl-tRNA to rib- osomes with trinucleotides of known sequence.’ Since aminoacyl-tRNA binds to ribosomes prior to peptide bond formation, the process of codon recog- nition can be studied without peptide bond synthesis. A simple method for determining '*C-aminoacy]l- tRNA bound to ribosomes was devised that depends upon the selective retention of '*C-aminoacyl-tRNA bound to ribosomes by disks of cellulose nitrate; unbound '*C-aminoacyl-tRNA is removed by wash- ing. At the time that the trinucleotide template ap- proach to codon sequence was devised, most of the 64 trinucleotides had not been prepared. Elegant 1974 JAMA, Nov 25, 1968 ® Vol 206, No 9     Table 1.—The Genetic Code*   UUU UCU UAU UGU PHE TYR cYsS uuc ucc UAC uGcc SER UUA UCA UAA TERM UGA TERM LEU UUG UCG UAG TERM UGG TRP cuu ccu CAU CGU HIS cuc ccc CAC cGCc LEU PRO ARG CUA CCA CAA CGA GLN CUG CCG CAG CGG AUU ACU AAU AGU ASN SER AUC ILE ACC AAC AGC THR AUA ACA AAA AGA LYS ARG MET, AUG MET ACG AAG AGG GUU GCU GAU GGU ASP GUC Gcc GAC GGc VAL ALA GLY GUA GCA GAA GGA GLU MET, GUG GCG GAG GGG   *Nucleotide sequences of RNA codons were determined by stimu- lating binding of E coli AA-tRNA to E coli ribosomes with trinucleotide templates. F-Met corresponds to N-formy!-Met-tRNA, the initiator of protein synthesis. TERM corresponds to terminator codons. chemical methods for oligoribonucleotide synthesis, devised by Khorana and his colleagues, are de- scribed in the accompanying communication. We have employed enzymatic methods for oligoribonu- cleotide synthesis. Leder and co-workers showed that primer-dependent polynucleotide phosphory- lase, in the presence of a dinucleoside monophos- phate primer and nucleoside diphosphate, catalyzes the synthesis of oligonucleotides of low chain jength’®; a similar method was also reported by Thach and Doty.’* Another enzymatic method for oligonucleotide synthesis, reported by Bernfield,'*''’ is based upon the demonstration by Heppel et al’* that RNase A catalyzes the synthesis of oligonu- cleotides from pyrimidine-2’,3’-cyclic phosphate moieties in the presence of mononucleotide or olig- onucleotide acceptors. The 64 trinucleotides were synthesized and as- sayed for template specificity in stimulating bind- ing of EF coli aminoacyl-tRNA to ribosomes.’°’* A summary of the code is shown in Table 1. Almost all triplets were found to correspond to amino acids. In most cases, synonym codons differ only in the base occupying the third position of the tyriplet. Thus synonym codons are systematically related to one another. Only four unique patterns of degenera- cy were found, each pattern determined by the bases that occupy the third positions of synonym triplets. Patterns of alternate third bases are as fol- lows: () G @) U=C (3) A=G (4) U=CrHA Genetic Memory—Nirenberg A fifth pattern, U = C = A =G, was found also, but may be formed by combining two or more simpler patterns such as [(U =C) + (A =G)] or [((U =C=A) + (G)}. Codons specifying the initiation of protein syn- thesis differ in that alternate bases occupy the first rather than the third position of the codons. For example, N-formyl-Met-tRNA responds to AUG and GUG. Three triplets, UAA, UAG, and UGA, serve as terminator codons. Hence the degeneracy pattern again is unusual (discussed under Punctua- tion). One consequence of logical degeneracy is that mutations resulting from the replacement of one base pair in DNA by another often do not result in the replacement of one amino acid by another in protein. Hence, many mutations are “silent.’? The code appears to be arranged so that the effects of error often are minimized. Amino acid replace- ments in protein that result from an alteration of one base per triplet can be derived from Table 1 by moving horizontally or vertically from the amino acid in question, but not diagonally. Punctuation Codon Positinn.—Each triplet can occur in three structural forms: as a 5’-terminal-, 3'-terminal-, or internal-codon. Substituents attached to terminal or internal ribose hydroxyl groups can influence the template properties of codons profoundly. Relative template activities of oligo U preparations, at limit- ing oligonucleotide concentrations, are as follows: p-5'-UpUpU > UpUpU > CH;0-p-5'UpUp > UpUpU-3’-p > UpUpU-3’-p-OCH; > UpUpU-2’, -3’-cyclic phosphate. Trimers with (2’-5’) phospho- diester linkages, (2’-5')-UpUpU and = (2’-5’)- ApApA, do not serve as templates for phenylala- nine- or lysine-tRNA, respectively. The relative template efficiencies of oligo A preparations are as follows: p-5'-ApApA > ApApA > ApApA-3’-p > ApApA-2’-p.”” Many enzymes have been described that catalyze the transfer of molecules to or from terminal hy- droxyl groups of nucleic acids. It is possible there- fore that modifications of terminal hydroxyl groups sometimes regulate the reading of RNA or DNA. Initiation.—-Two species of methionine-tRNA are found in E coli; one species, Met-tRNA, is convert- ed enzymatically to N-formyl-Met-tRNA,,’® and functions as an initiator of protein synthesis in ex- tracts of EF coli in response to the codons AUG or GUG; the other species, Met-tRNA,,, does not ac- cept formyl groups and responds only to AUG.'*:° Translation of mRNA is initiated near the 5’- terminus of the RNA and proceeds three bases at a time toward the 3’-terminus. The first amino acid to be incorporated into protein is the N-terminal amino acid; the C-terminal amino acid is the last to be incorporated. At least three nondialyzable factors are required for the initiation of protein synthesis.”**° However the reactions have not been clarified fully. It seems JAMA, Nov 25, 1968 ® Vol 206, No 9 probable that one factor, the C protein, is required for the attachment of the 30S ribosomal subunit to the 5’-terminus of the nascent chain of mRNA prior to the detachment of the mRNA from the DNA template.”? Another factor is required for bind- ing of N-formyl-methionyl-tRNA to the 30S ribo- somal subunit in response to AUG or GUG. Termination.—Results obtained by Stretton and co-workers” and by Garen®* demonstrate that UAA, UAG, and UGA are terminator-codons. Capecchi has reported that a protein, termed the release fac- tor, is required for terminator-codon dependent release of polypeptides from ribosomes.”° Terminal events in protein synthesis have recent- ly been studied with trinucleotide codons.” Initiator and terminator trinucleotides sequentially stimu- late N-formyl-methiony]-tRNA binding to ribo- somes and the release of free N-formyl-methionine from the ribosomal-intermediate. The release factor and a terminator trinucleotide is required for this reaction. The release factor has been fractionated into two components; R1, which corresponds to the terminator codons UAA and UAG; and R2, which corresponds to UAA and UGA.” The specificity of R therefore is related to the codon. These results suggest that terminator codons may be recognized by release factors. However, the mechanism of ter- mination remains to be clarified, and it is certainly possible that terminator-codons are recognized by components that have not been detected thus far. Mechanism of Codon Recognition Cells often contain multiple species of tRNA for the same amino acid. Soon after the code was found to be degenerate, the specificity of separate species of tRNA'™ for codons was examined. Randomly ordered poly UG and poly UC preparations are templates for different species of Leu-tRNA.” Thus alternate codons for the same amino acid some- times are recognized by different species of tRNA. When base sequences of synonym codons were es- tablished, it became abundantly clear that synonym codons are logically related to one another. Since only a few general degeneracy patterns were found, each pattern was thought to represent a general mechanism for codon recognition. Evidence that one molecule of AA-tRNA can re- spond to two kinds of codons was obtained by show- ing that >99% of the available molecules of ‘*C- Phe-tRNA bind to ribosomes in response to poly U, and >65% of the molecules also bind in re- sponse to UUC.*® Hence >65% of the Phe-tRNA molecules respond both to UUU and UUC. Addi- tional evidence was obtained by fractionating AA- tRNA and determining the responses of the sep- arated fractions of **C-AA-tRNA to trinucleotide codons. Results obtained thus far in our laboratory with purified fractions of AA-tRNA from E coli, yeast, and guinea pig liver are summarized in Fig 2. It is clear that one species of tRNA may recog- Genetic Memory—Nirenberg 1975   Table 2.—Alternate Base Pairing™   tRNA mRNA Anticodon Codon U A G c G A U G c U I U c A   Alternate base pairing. The base in a tRNA anticodon shown in the left-hand column forms antiparallel hydrogen bonds with the base(s) shown in the right hand column, which usually occupy the third position of alternate mRNA codons. Relationships are “wobble’’ hydrogen bonds suggested by Crick,30 nize 1, 2 or 3 synonym cod- ons that differ only in the base occupying the third position of the codon. Five unique patterns of degeneracy were found, each pattern determined by alternate third bases of synonym triplets recognized by a tRNA species. Patterns of alternate third bases of synonym codon sets are as fol- lows: (1) (2) (3) (4) (5) A Gran Cc G Cc G Crick proposed a mechanism that would enable a base in the tRNA anticodon to pair with alternate bases occupying the third position of synonym mRNA codons.*° By changing positions slightly, that is, by wobbling, bases in the appropriate posi- tion of the tRNA anticodon form alternate pairs with bases occupying the third position of synonym mRNA codons. Antiparallel Watson-Crick hydro- gen bonds form between the first and second bases of the mRNA codon and corresponding bases in the tRNA anticodon and wobble hydrogen bonds form between bases occupying the third positions of synonym mRNA codons and a corresponding base in the tRNA anticodon as shown in Table 2. Hence, U in a tRNA anticodon pairs with A or G in the third position of synonym mRNA codons; C pairs with G; G pairs with C or U; and I pairs with U, C, or A. Additional evidence supporting this mech- anism of codon recognition stems from the elucida- tion of base sequences of tRNA anticodons. The data are fully consistent with wobble base pairing. In summary, degeneracy patterns for amino acids observed with unfractionated AA-tRNA often re- sult from recognition of several codons by a single Species of tRNA and from the presence of multiple species of tRNA for the same amino acid that respond to different sets of codons. Universality Although the results of many studies indicate that the genetic code is largely universal, the fidelity A U 1976 JAMA, Nov 25, 1968 @ Vol 206, No 9 corresponds to Met Release factors 1 and UGA, respecti   2. Responses of purified AA-tRNA fractions to trinucleotide codons. Joined sym- bols adjacent to codons represent synonym codons recognized by a purified AA-tRNA fraction from @ E coli, A yeast, and JJ guinea pig liver. The number between sym- bols represents the number of redundant peaks of AA-tRNA found responding to that set of codons. The open symbols represent ambiguous AA-tRNA responses; MET; corresponds to N-formyl-Met-tRNA, the initiator of protein synthesis; MET,, “tRNA; TERM corresponds to termination of protein synthesis. and 2, rather than RNA, correspond to UAA and UAG, or UAA vely (a signifies uncertain). of translation can be altered in vivo and in vitro by altering components or conditions required for protein synthesis. The extent of such alterations was examined by studying the fine structure of the code with tRNA from different organisms. Almost identi- cal translations of nucleotide sequences of amino acids were found with bacterial, amphibian, and mammalian aminoacyl-tRNA. However, E coli tRNA did not respond detectably to certain codons. Therefore aminoacyl-tRNA preparations were frac- tionated by column chromatography and responses of tRNA fractions to trinucleotide codons were determined.** A summary of the results is shown in Fig 2. Many “universal” species of aminoacyl-tRNA were found, however seven species of mammalian tRNA were not detected with E coli preparations; conversely, five species of tRNA from E coli were not found with mammalian preparations. The re- sults also suggest that some organisms contain little or no aminoacyl-tRNA for certain codons (AUA, AGA, or AGG). The remarkable similarity in codon base se- quences recognized by bacterial, amphibian, and mammalian AA-tRNA suggests that most, perhaps all, forms of life on this planet use essentially the same genetic language. The code probably evolved more than 5 X 10° years ago. It is possible that some species-dependent dif- ferences in the codon recognition apparatus serve as regulators of protein synthesis. The possibility that embryonic differentiation may be dependent upon changes in codon recognition remains to be ex- plored. At the present time, the biological conse- Genetic Memory—Nirenberg quences of a modifiable translation apparatus are largely unknown. Fidelity.—Since multiple species of tRNA for the same amino acid often recognize separate sets of codons, the synthesis of two proteins with similar amino acid compositions may require different spe- cies of tRNA. Some codons probably occur more frequently in mRNA than others for the same amino acid. Most codons probably are translated with little error (0.1% to 0.01%). However, with some codons the level of error may be as high as 50%. Therefore, the accuracy of codon translation can vary at least 5,000-fold. Errors usually are specific ones, be- cause two out of three bases per codon often are translated correctly. The code seems to be ar- ranged so that the consequences of error often are minimized. The biological significance of a flexible, easily modified codon translation apparatus is not known. One intriguing possibility is that the codon recog- nition apparatus is modified in an orderly, predicta- ble way at certain times during cell growth and differentiation and that such modifications selec- tively regulate the kinds and amounts of proteins synthesized. In accord with this hypothesis, many factors have been found that influence the rate and the accuracy of protein synthesis in vitro. In addi- tion, one may also consider the structural hetero- geneity of components required for protein synthe- sis. For example, it seems probable that tRNA may be extensively modified by enzymes after the tRNA polynucleotide chain has been synthesized. Since tRNA contains many trace bases, a spectrum of intermediates probably exists for each species of tRNA. Whether such reactions play a role in regu- lating gene expression remains to be determined. One intriguing possibility is that infection of a cell by a virus may result in the production of a factor that modifies tRNA and, in consequence, alters the rate of synthesis of mRNA or protein. It is clear that the translation apparatus of the cell will accept and follow in robot-like fashion any instructions written in the appropriate molecular language. Since the language has been deciphered, the informational properties of the genetic message can be defined in terms of molecular structure. It seems probable that synthetic messages will even- tually be used to program cells and their descen- dants. References 1. Watson, J.D., and Crick, F.H.C.: Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid. Nature 171:737-738 (April 25) 1953. 2. Nirenberg, M.W., and Matthaei, J.H.: The Dependence of Cell-Free Protein Synthesis in E coli Upon Naturally Occurring or Synthetic Polyribonucleotides, Proc Nat Acad Sci USA 47: 1588-1602 (Oct 15) 1961. 3. Grunberg-Manago, M.: Ortiz, P.J.; and Ochoa, S.: Enzymic Synthesis of Polynucleoctides, Biochim Biophys Acta 20:269-285, 1956. 4. Speyer, J.F.. et al: Synthetic Polynucleotides and Amino Acid Code, Cold Spring Harbor Symp Quant Biol 28:559-567, 1963. 5. Nirenberg, M.W., et al: On Coding of Genetic Informatien, Cold Spring Harbor Symp Quant Biol 28:549-557, 1963. 6. Nirenberg, M.W.: Matthaei, J.H.; and Jones, O.W.: An In- termediate in the Biosynthesis of Polyphenylalanine Directed by Synthetic Template RNA, Proc Nat Acad Sci USA 48:104- 109 (Jan 15) 1962. 7. Weisblum, B.; Benzer, S.; and Holley, R.W.: A Physical Basis for Degeneracy in the Amino Acid Code, Proc Nat Acad Set USA 48:1449-1454 (Aug) 1962. 8. Wittmann, H.G., and Wittmann-Liebold, B.: Tobacco Mosaic Virus Mutants and the Genetic Coding Preblem, Cold Spring Harbor Symp Quant Biol 28:589-595, 1963. 9. Nirenberg, M.W., and Leder, P.: RNA Codewords and Pro- tein Synthesis: The Effect of Trinucleotides Upon the Binding of sRNA to Ribosomes, Science 145:1399-1407 (Sept 25) 1964. 10. Leder, P.; Singer, M.F.; and Brimacombe, R.L.C.: Syn- thesis of Trinucleoside Diphosphates With Polynucleotide Phos- phorylase, Biochemistry 4:1561-1567 (Aug) 1965. ll. Thach, R.E., and Doty, P.: Enzymatic Synthesis of Tri- and Tetranucleotides of Defined Sequence, Science 148:632-634 (April 30) 1965. 12. Bernfield, M.R.: Ribonuclease and Oligoribonucleotide Synthesis: I. Synthetic Activity of Bovine Pancreatic Ribo- nuclease Derivatives, J Biol Chem 240:4753-4762 (Dec) 1965. 13. Bernfield, M.: Ribonuclease and Oligoribonucleotide Syn- thesis: II. Synthesis of Oligonucleotides of Specific Sequence, J Biol Chem 241:2014-2023 (May 10) 1966. 14. Heppel, L.A.; Whitfield, P.R.; and Markham, R.: Biochem J 60:8, 1955. 15. Nirenberg, M. et al: RNA Codewords and Protein Syn- thesis: VII. On the General Nature of the RNA Code, Proce Nat Acad Sct USA 53:1161-1168 (May) 1965. 16. Sél, D., et al: Studies on Polynucleotides: XLIX. Stimula- tion of the Binding of Aminoacyl-sRNA’s to Ribosomes by Ribotrinucleotides and a Survey of Codon Assignments for 20 JAMA, Nov 25, 1968 © Vol 206, No 9 Amino Acids, Proc Nat Acad Sci USA 54:1378-1385 (Nov) 1965. 17. Rottman, F., and Nirenberg, M.: RNA Codons and Pro- tein Synthesis: XI. Template Activity of Modified RNA Codons J Molec Biol 21:555-570 (Nov) 1966. 18. Marcker, K., and Sanger, F-.: N-Formyl-methiony]-S- RNA, J Molec Biol 8:835-840 (June) 1964. 19. Clark, B.F.C., and Marcker, K.A.: The Role of N-Formyl- methionyl-sRNA in Protein Biosynthesis, J Molec Biol 17:394- 406 (June) 1966. 20. Kellogg, D.A., et al: RNA Codons and Protein Synthesis: IX. Synonym Codon Recognition by Multiple Species of Valine-, Alanine-, and Methionine-SRNA, Proc Nat Acad Sci USA 55: 912-919 (April) 1966. 21. Anderson, J.S., et al: GTP-Stimulated Binding of Initia- tor-tRNA to Ribosomes Directed by /2 Bacteriophage RNA, Nature 216:1072-1076 (Dec 16) 1967. 22. Nomura, M., and Lowry, C.V.: Phage F2 RNA-Directed Binding of Formylmethionyl-TRNA to Ribosomes and the Role of 30S Ribosomal Subunits in Initiation of Protein Syn- thesis, Proc Nat Acad Sci USA 58:946-953 (Sept) 1967. 23. Iwasaki, K., et al: Translation of the Genetic Message: VII. Role of Initiation Factors in Formation of the Chain Ini- tiation Complex With Escherichia coli Ribosomes, Arch Bio- chem 125:542-547 (May) 1968. 24. Stretton, A.O.W.; Kaplan, S.; and Brenner, S.: Nonsense Codons, Cold Spring Harbor Symp Quant Biol 31:173-179, 1966. 25. Garen, A.: Sense and Nonsense in Genetic Code, Science 160:149-159 (April 12) 1968. 26. Capecchi, M.R.: Polypeptide Chain Determination in Vi- tro: Isolation of a Release Factor, Proce Nat Acad Sci USA 58: 1144-1159 (Sept) 1967. 27. Caskey, C.T., et al: Sequential Translation of Trinucleo- tide Codons for the Initiation and Termination of Protein Syn- thesis, Science 162:135-138 (Oct 4) 1968. 28. Scolnick, E., et al: Release Factors Differing in Specificity for Terminator Codons, Proce Nat Acad Sci USA, to be published. 29. Bernfield, M.R., and Nirenberg, M.W.: RNA Codewords and Protein Synthesis: The Nucleotide Sequences of Multiple Codewords for Phenylalanine, Serine, Leucine, and Proline, Science 147:479-484 (Jan 29) 1965. 80. Crick, F.H.C.: Codon—Anticodon Pairing: The Wobble Hypothesis, J Molec Biol 19:548-555 (Aug) 1966. 31. Caskey, C.; Beaudet, A.; and Nirenberg, M.: RNA Codons and Protein Synthesis: 15. Dissimilar Responses of Mammalian and Bacterial Transfer RNA Fractions to mRNA Codons, J Molec Biol, to be published. Genetic Memory—Nirenberg 1977", "Nirenberg, Marshall W.", null, "Journal of the American Medical Association", "American Medical Association", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3z7m.e6x7.9xis", "00000000-0000-0000-C78F-CE0F20B800E4", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Current Status of the RNA Code", "101584910X117", "101584910X51", "1963", "1963", "This article discusses the state of the field approximately a year after Nirenberg's groundbreaking poly-U experiments were discussed in Moscow and the Proceedings of the National Academy of Science.  The article addresses the efficiency and specificity with which synthetic polynucleotides have been found to direct amino acids into proteins, the process by which coding amino acids takes place, and the problem of degeneracy and its relationship to the general nature of the code.  This article covers the same subject in \"Qualitative Survey of RNA Codewords,\" which is also available on this site.", "Monographs, Excerpts", "Nucleotides,Amino Acids,RNA, Viral ; RNA", "Translating the Code of Life and the Nobel Prize, 1962-1968", "15", "pages", "Text", "English", "Reproduced with permission of the Academic Press.", "Copyright may apply", null, null, "SyMPosiy REPRINTED FRO M ON INFORMATIONAL McronoLecy ACADEMIC PRESS one ” The Current Status of the RNA Code’ MARSHALL W. NIRENBERG AND OLIVER W. JONEs, Jr. National Heart Institute, National Institutes of Health, Bethesda, Maryland Rather than review all of our work concerning the genetic coding problem, only one aspect which we have been investigating (up to September, 1962) will be presented; that is, the extent of degeneracy and its relationship to the general nature of the code. A degenerate genetic code was suggested a number of years ago by Gamow (10) and by Crick (4). In such a code, an amino acid may be directed into protein by two or more codewords. Previous work demonstrated that C!4-amino acids were directed into protein by synthetic polynucleotides in cell- free Escherichia coli extracts (19) and that leucine incorporation was stimulated by either poly UG,? UC, or UA (16, 17, 29). Thus the code was shown to be degenerate with respect to leucine (16, 17, 29). Initially, all of the codewords found contained U. However, assuming a triplet code, the proportion of U compared with other nucleotides in codewords seemed unusually high, for natural template RNA, such as viral RNA, did not contain such a preponderance of U. To resolve this paradox, a more degenerate code was proposed with both non-U and U containing codewords (17). An alternative hypothesis was advanced by Roberts, who suggested a doublet code; for in such a code the pro- 1 This report is limited to the data which were presented at the Symposium on Informational Macromolecules in September, 1962. Data obtained after this date are not included. * The following abbreviations are used: poly U, polyuridylic acid; poly A, poly- adenylic acid; poly C, polycytidylic acid; poly G, polyguanylic acid; poly UGAC, polyuridylic-guanylic-adenylic-cytidylic acid; poly ACG,  polyadenylic-cytidylic- guanylic acid; poly AC, polyadenylic-cytidylic acid; poly CG, polycytidylic-guanylic acid; poly UG, polyuridylic-guanylic acid; poly UC, polyuridylic-cytidylic acid; poly UA, polyuridylic-adenylic acid; poly UCG, polyuridylic-cytidylic-guanylic acid; poly UAG, polyuridylic-adenylic-guanylic acid; G-G, guanylic-guanylic; A, adenylic acid; G, guanylic acid; C, eytidylic acid; U, uridylic acid. 451 452 MARSHALL W. NIRENBERG AND OLIVER W. JONES, JR. TABLE I Base Ratio* (Moles Per Cent)     Input ratio Base ratio Designation Polymer of nucleotides of nucleotides UGAC UGAC Ap231 UGAC 40:20:20:20 55:32: 5: 8 Ap232 UGAC 58:14:14:14 56:25; 5:13 Ap233 UGAC 20:20:20:40 3:45: 9:43 Ap234 UGAC 12:12:12:64 23:21: 4:52 Ju 258 UGAC 29:13:29:29 27 22:92:29 Ju 2510 UGAC 29:29:29:13 27:43:21: 9 ACG ACG J 251 ACG 60:20:20 46:32:22 M 76 ACG 7:86: 7 2:89: 9 M 75 ACG 10:80:10 4:77:19 M 74 ACG 30:60:10 16:72:12 AC AC J 104 AC 9:91 3:97 J 103 AC 12:88 6:94 J 102 AC 20:80 12:88 J 101 AC 33:67 30:70 ‘J 109 AC 75:25 67:33 J 108 AC 83:17 80:20 CG CG M 141 CG 88:12 90:10 M71 CG 92: 8 87:13 F 120 CG 88:12 82:18 F 135 CG 50:50 9:91 AG AG J 106 AG 80:20 73:27 J 107 AG 66:33 48:52   ® Polyribonucleotides were synthesized, as described previously, with the aid of polynucleotide phosphorylase partially purified from Micrococcus lysodeikticus according to the method of Singer and Guss (27). The base-ratio of each polynucleotide preparation was determined by analysis. Polynucleotides were hydrolyzed by incubation in 0.4 N KOH at 25° for 18 hours. Under these conditions, little deamination occurred.2 Such mild conditions were not sufficient to hydrolyze certain polymers; however, in such cases, incubation in 0.3 N KOH at 37° for 18 hours resulted in complete hydrolysis (5). Mono- nucleotide products were separated either by paper electrophoresis (Whatman No. 3 MM paper, 0.05 M ammonium formate, pH 3.7) or by descending paper chroma- tography (Whatman No. 3 MM paper and a solvent system containing 0.1 M sodium phosphate, pH 7.0 and 3 M ammonium sulfate), Two % or greater contamination of polynucleotides by U would have been detected. No contamination by U was found. Mononucleotides and appropriate blanks were eluted by shaking small paper 3 We thank Dr. M. Grunberg-Manago for this protocol. THE CURRENT STATUS OF THE RNA CODE 453 portions of nucleotides would be within the range found in viral RNA (25, 26). The existence of non-U codewords was suggested when poly AC was found to direct small amounts of proline and threonine into protein (13, 21). Recently, in a careful study, Bretscher and Grunberg-Manago clearly demonstrated coding by non-U words (2). Several poly AC preparations were reported to code well for proline, threonine, histidine and, to a lesser extent, for glutamine. This work indicated that other non-U polynucleotides might have template activities. In this communication, further qualitative analysis of coding by such poly- nucleotides will be reported. RESULTS Base-Ratio Analysis The synthetic polynucleotides used in this study are listed in Table I. The base-ratio analysis of each polymer is compared with the ratio of nucleoside diphosphates present during the synthesis of each poly- nucleotide. In many cases, the base-ratio of the polymer product dif- fered slightly from the input ratio of the substrates. In polymers con- taining two or three different nucleotides, preferential incorporation into polynucleotide of either G or C relative to A was observed. Bret- scher and Grunberg-Manago have reported that Azotobacter polynu- cleotide phosphorylase also catalyzes a preferential incorporation of C and G into poly UC and UG (2). Stimulation of Amino Acid Incorporation by Polynucleotides Containing Four Bases The data of Table II demonstrate that synthetic polynucleotides containing four bases stimulate the incorporation of a large number of amino acids into protein. In the last column is given the basal level of C'4-amino acid incorporation obtained in the absence of polynucleotide; other figures refer to the net increase above basal incorporation due to addition of polynucleotide. The base-ratios of the polynucleotides vary widely. The fifth polynucleotide (Ju-258) contains approximately equal proportions of U, G, A, and C, whereas the other polynucleotides con- tain predominant amounts of two or three nucleotides. All of the poly- nucleotides were active in directing amino acid incorporation, except polynucleotide Ju-2510. Although 10 yg of polynucleotide were added to each reaction mixture, the total amount of C*4-amino acid directed into protein by each polynucleotide varied more than 50-fold. As we have shown previously, the template activity of polynucleotides is de- pendent upon factors other than nucleotide sequence. For example,   strips immersed in 0.1 N or 0.01 N HCl and determining UV absorption at appro- priate wavelengths in a Beckman DU spectrophotometer.                 TABLE II STIMULATION OF AMINO Act INCORPORATION BY Poty UGAC Polynucleotide: UGAC UGAC UGAC UGAC UGAC UGAC U 55 U 56 U 3 U 23 U 2 U QT Base ratio G 32 G 25 G 45 G 21 G 22 G 43 Minus (moles per cent) A 5 A 5 A 9 A A A 22 A 21 polynucleotide Cc 8 Cc 13 Cc 43 C 52 Cc 29 c 9 control Designation: Ap231 Ap232 Ap233 Ap234 Ju258 Ju2510 Incorporation above control C4-Amino acid A uuMoles¢ Alanine 110 127 62 152 31 5 10 Arginine 69 270 68 212 99 57 11 Aspartic acid (—NH,?) 10 40 9 10 25 12 12 Glutamic acid (—NH,?) 16 52 12 9 14 — 23 Glycine 62 663 25 40 12 6 13 Histidine 20 8 11 24 13 0 4 Isoleucine 68 301 60 90 0 0 22, Leucine 168 1,243 125 418 12 0 41 Lysine 10 21 0 3 25 — 4 Methionine 9 64 0 9 15 4 12 Phenylalanine 152 606 86 64 14 0 10 Proline 50 140 125 1,007 121 10 7 Serine 179 807 181 445 37 0 47 Threonine 15 54 19 78 44 0 7 Tryptophan 23 8 16 8 1 — 45 Tyrosine 14 80 11 12 6 0 17 Valine 100 602 57 70 43 13 7 Total 1,075 5,086 867 2,651 512 107 292   Var ‘uf “SANO[ ‘A WOATIO ONV OYRANAYIN ‘A TIVHSUVIL TABLE II (Continued)   _uMoles represents the difference between C14-amino acid incorporation into protein in the presence and absence of poly- ides. Basal incorporations obtained when polynucleotides were omitted are presented in the last colum (minus poly- ide). iction mixtures used to determine Cl4-1-amino acid incorporation into protein contained the following components: Tris (hydroxymethylaminoethane) pH 7.8; 0.01 M magnesium acetate; 0.05 M KCl; 6 x 10-3M mercaptoethanol; 1 x 4 ATP; 5 x 10-8M potassium phosphoenolpyruvate; 5 wg of crystalline phosphoenolpyruvate kinase (California Bio- al Corporation); 0.8 x 10-4M C14-amino acid (approximately 30,000-150,000 counts/minute/reaction mixture); 3.2 xX 4 each of 19 C12-L-amino acids minus the C14-amino acid; 10 ug of polynucleotide/reaction mixture, when specified; and ; preincubated $-30 extracts (1-2 mg protein/reaction mixture). Total volume of each reaction mixture was 0.3 ml. Re- mixtures were incubated at 37° for 30 minutes; thus, total amino acid incorporation rather than rate of incorporation was ed. A Nuclear-Chicago thin-window, gas flow counter was used, aqdOO VNY AHL JO SALVLIS INSYWHOO FHL ocr 456 MARSHALL W. NIRENBERG AND OLIVER W. JONES, JR. large polymers of chain length greater than 100 units are considerably more active than shorter ones (17). Single-stranded polynucleotides are active, whereas double- or triple-stranded polymers are not (19). In addition, randomly-mixed copolymers which have a high degree of sec- ondary structure are inactive in coding (28). In particular, polymers containing much G have little activity, possibly because of G-G inter- actions. Thus the relative inactivity of the last poly UGAC preparation (Ju-2510) should not be ascribed necessarily to the presence of a high proportion of nonsense nucleotide sequences. Such considerations make it difficult to compare with validity the relative abilities of different polynucleotides to code for the same amino acid; thus, such comparisons should be made with caution. The fact that polynucleotides containing four bases coded so well for so many amino acids strongly suggested that most nucleotide sequences could be read. In addition, a high pro- portion of U clearly was not required for messenger RNA activity. Stimulation of Amino Acid Incorporation by Poly ACG The coding activities of polymers which did not contain U are given in Table III. Base-ratio analyses of each poly ACG preparation failed to detect contamination by U. Poly ACG preparations stimulated the incorporation of many amino acids tested, including alanine, arginine, glutamic acid, lysine, proline, and threonine. Such high incorporations of glutamic acid, lysine, and threonine were not observed previously. A number of amino acids did not appear to be coded by any ACG preparations, which suggested that U may be an absolute requirement in coding for some amino acids. Since the template activities of some poly ACG preparations equaled those of our best synthetic template RNA preparations, U clearly was not required for coding other amino acids. Stimulation of Amino Acid Incorporation by Polynucleotides Containing Two Bases The data of Table IV demonstrate stimulation of amino acid incor- poration by poly AC preparations. The polynucleotides are listed in order of decreasing C content. In accord with the findings of Bretscher and Grunberg-Manago (2), poly AC stimulated incorporation of proline, threonine, and histidine. In addition, poly AC was found to direct aspartic acid, glutamic acid, and lysine into protein. Bretscher and Grunberg-Manago (2) report that glutamine is coded by such polymers. We have not been able to obtain C'*-asparagine or C*-glutamine and, thus, have not been able to study this point.* Although the addition of 4 Recently, we have confirmed the finding of Bretscher and Grunberg-Manago (2) that glutamine rather than glutamic acid is directed into protein by poly CA. In addition, we find that poly CA codes for asparagine rather than aspartic acid. THE CURRENT STATUS OF THE RNA CODE 457 C™-aspartic acid and C'°-glutamic acid to reaction mixtures completely diluted the incorporation of C*+-aspartic and C14-glutamic acids, re- spectively, the possibility of conversion of the free acid to the amide during incubation of reaction mixtures does not allow us to distinguish between the acid and amide forms. Many of the polynucleotides were found to have template activities equal to the most active poly U prep-         TABLE III STIMULATION OF Amino Aci INCORPORATION BY Poty ACG Polynucleotide: ACG ACG ACG ACG Minus . A 46 A 2 A 4 A 16 poly- Base ratio Cc 32 Cc 89 Cc 77 C 72 nucleotide (moles per cent) G2 G 9 Gi19 G 12 control Designation: J251 M 76 M 75 M 74 Incorporation above control Cl4.Amino acid A puMoless Alanine 123 45 56 85 8 Arginine 128 30 40 74 9 Aspartic acid (—-NH.?) 167 0 0 24 13 Clutamic acid (—NH,?) 326 0 0 33 21 Glycine 5 0 0 0 13 Histidine 71 6 9 95 5 Isoleucine 0 0 0 0 20 Leucine 0 10 0 0 40 Lysine 820 5 0 23 6 Methionine 1 4 0 0 10 Phenylalanine 0 0 1 6 9 Proline 147 320 185 41 8 Serine 182 24 30 55 45 Threonine 250 11 13 11 8 Tryptophan 1 0 0 0 43 Tyrosine 0 4 0 0 18 Valine ] 5 5 7 6 Total 2,222 464 339 454 282   @ ApwuMoles represents the difference between C1l4-amino acid incorporation into protein in the presence and absence of polynucleotides. Assay procedures are described in the footnote of Table II. arations tested. Poly AC (J-104) contained 97% C, yet actively directed proline into protein. Thus, it appears probable that one codeword for proline may contain only C. Relatively large amounts of lysine were directed into protein by AC (J-109) and (J-108), which contained 67 and 80% A, respectively. These data suggest that a codeword for lysine may contain only A. The data of Table V demonstrate the effects of poly CG and AG preparations in directing amino acids into protein. The first three CG 458 MARSHALL W. NIRENBERG AND OLIVER W. JONES, JR. polymers contain high proportions of C and directed alanine, arginine, and proline into protein. The last poly CG preparation (F-135) con- tains 91% G and was inactive as template RNA. Poly AG directed in- corporation of glutamic acid and lysine into protein.           TABLE IV STIMULATION OF AMINO AciD INCORPORATION BY PoLy AC Minus Polynucleotide: AC AC AC AC AC AC poly- (moles percent) f{ A 3A 6A LWA SOA 67 A 80 nucleotide Base ratio 1c 97 C 94C 88 C 70 C 33C 20. control Designation: ylo4 J103 =—-id2—s f101 yjio9 j108 Incorporation above control C14-Amino acid A puMoles¢ Alanine 0 1 0 0 0 0 Ml Arginine 0 4 1 1 0 0 12 Aspartic acid (—NH,?) 0 0 9 51 157 53 24 Glutamic acid (—NH,?) 4 19 24 53 135 53 15 Glycine 5 16 0 2 0 0 6 Histidine 0 0 5 198 85 17 23 Isoleucine 6 0 0 0 0 0 42 Leucine 0 0 — — — 1 3 Lysine 5 10 14 47 909 44] 5 Methionine 0 10 0 0 0 0 10 Phenylalanine 0 0 1 0 4 0 1l Proline 625 1,132 643 1,102 140 20 9 Serine 11 19 18 16 9 8 46 Threonine 30 65 75 170 176 105 9 Tryptophan 23 0 1 1 1 9 44 Tyrosine 14 2 2 0 0 0 19 Valine 0 0 0 0 0 0 5 Total 723 «1,278 793 1,641 1,616 707 294   @ A puMoles represents the difference between C1!4-amino acid incorporation into protein in the presence and absence of polynucleotides. Assay procedures are described in the footnote of Table IT. Quantitative Aspects of Data A comparative study of polynucleotides of varying base-ratios is helpful in evaluating amino acid incorporation data, for relative amino acid incorporations easily can be correlated with changes in base-ratio. Occasional inconsistencies and the significance of minor incorporations become apparent. Isotope dilution experiments were performed routinely to detect the possible presence of radioactive impurities in C14-amino acids. The THE CURRENT STATUS OF THE RNA CODE 459 presence of C'4-impurities seemed unlikely, for incorporation of a Cl amino acid was lowered sharply if the reaction mixture contained both a C'4-amino acid (0.05 pmoles) and the same C'-amino acid (1.0 umole). The purity of each C!4-amino acid also was determined by           TABLE V STIMULATION OF AMINO ACID INCORPORATION BY Poty CG anp AG Minus Polynucleotide: CG CG CG CG AG AG poly- Base ratio C90 C87? C8 C9 AT A 48 nucleotide (moles per cent) IG 10 G13 G18 G91 G27 G52 control Designation: MI41 M71 F120 F135 J106  J107 Incorporation above control Cl4-Amino acid A uuMoles@ Alanine 30 20 63 0 0 0 14 Arginine 39 16 86 1 10 8 13 Aspartic acid (—NH,?) 0 0 6 3 12 10 26 Glutamic acid (—-NH,?) 0 0 0 0 44 5 ll Glycine 5 0 8 0 2 0 4 Histidine 0 0 0 0 0 0 26 Isoleucine 0 0 0 0 1 0 39 Leucine 0 0 0 5 0 11 7 Lysine 2 0 0 0 110 8 3 Methionine 0 0 0 0 0 0 12 Phenylalanine 5 5 4 8 0 0 14 Proline 144 202 356 2 1 1 8 Serine 18 0 6 0 0 0 42 Threonine 0 0 1 0 1 0 5 Tryptophan 0 1 17 1 0 0 40 Tyrosine 2 6 2 0 0 0 14 Valine 1 1 0 0 0 0 4 Total 246 251 549 20 181 43 282   @ A uuMoles represents the difference between C14-amino acid incorporation into protein in the presence and absence of polynucleotides. Details of the assay procedures are described in the footnote of Table II. paper electrophoresis followed by radioautography as described pre- viously (17). Limiting amounts of polynucleotides were added to reaction mix- tures and total amino acid incorporations were measured rather than rates of amino acid incorporations. E. coli extracts contain nucleases which rapidly degrade synthetic polynucleotides and the nuclease con- tent may vary from one preincubated S-30 preparation to another. Since many different enzyme extracts were used in this study, the data are 460 MARSHALL W. NIRENBERG AND OLIVER W. JONES, JR. not useful for quantitative analyses. Comparisons between theoretical frequencies of triplets, etc. in polynucleotides and relative amino acid incorporations have not been presented because the data do not permit such calculations to be made with accuracy. The data demonstrate only qualitative aspects of the code; that is, nucleotide compositions of code- words and the degree of code degeneracy. SUMMARY OF INCORPORATION DaTa Table VI summarizes all of the coding data previously published (19, 16, 17, 29, 15, 14, 30) and obtained in this study. Only polynu-     TABLE VI Summary oF Copinc Data4 C14-Amino acid Stimulated by poly- Phenylalanine U(98) Proline C(?) CA(87) CUu(60) CG(80) Lysine A(?) AC(53) AG(60) AU(?) Threonine AC(15) Serine UC(23) UGG(23)P Valine UG(15) Leucine UG(14) UC(13) UA(?) Glycine UG(5) Cysteine UG(8-15) Glutamic acid (—-NH,?) AC(7) AG(20) Isoleucine UA(8) Tryptophan UG(6) Tyrosine UA(9) Arginine CG(15) Methionine UAG(1) Histidine AC(10 Alanine CG(11) Aspartic acid (—NH,?) AC(8)   ® Polymers used for these calculations represent optimal base-ratio directing Cl4-amino acids into protein. Ami id i orated 100 Numbers in parentheses refer to: mino acid’ Incorporated’   Sum of incorporation of 17 amino acids cleotides containing the minimum number of bases capable of stimu- lating an amino acid into protein are given in Table VI. The coding of proline by poly C and lysine by poly A was suggested by the poly AC experiments presented in Table III. The fact that poly C and poly A code so weakly may be due either to inhibitory effects of secondary structure or to difficulty in precipitating peptides. At acid pH, poly A in solution is double-stranded (9, 24), and poly C also may have or- dered structure (8). THE CURRENT STATUS OF THE RNA CODE 461 A surprising conclusion revealed by this summary is that almost every amino acid tested could be coded by a polymer containing only two bases. Methionine could be coded only by poly UGA as reported previously (17, 30), but the amount of methionine directed into protein was small; thus this codeword remains questionable. Assuming a triplet code, a summary of codewords estimated thus far is presented in Table VII. Previously, poly UCG was found to direct alanine and arginine into protein, and codewords containing U, C, and G were proposed for these amino acids (16, 17, 14, 30). The observed frequencies of incorporations (17) suggest coding of alanine and ar-     TABLE VII TENTATIVE SUMMARY OF CODEWORDS Cl4-Amino acid Codewords* Alanine CCG Arginine CGC Aspartic acid (—NH,?} ACA Asparagine UAC or UAA® Cysteine UUG or UGG¢ Glutamic acid (—NH,?) ACA AGA AGU4 Glycine UGG Histidine ACC Isoleucine UVA Leucine GUU CUU AUUb (UUU) Lysine AAA AAC AAU Methionine UGA4 Phenylalanine UUU Proline CCC CccU CCA CCG Serine UCG UCU Threonine CAC CAA Tryptophan UGG Tyrosine UAU Valine UGU   @ Nucleotide sequence in codewords is arbitrary. % Proposed by Speyer et al. (30). ¢ We cannot differentiate between these possibilities at present. 4 Jt is not entirely clear whether these codewords require U. ginine by either UCG or CCG, but not by both codewords. In addition, the data of Table V show that poly CG codes for alanine and arginine; thus, codewords corresponding to these amino acids do not appear to contain U. Since it is not possible at this time to distinguish between triplet and double codes, etc., the assignments in Table VII represent current approximations of codewords. It seems probable that additional code- words will be found. 462 MARSHALL W. NIRENBERG AND OLIVER W. JONES, JR. Discussion Codeword Specificity in Protein Synthesis The term degeneracy refers to the phenomenon whereby one amino acid is coded by two or more codewords. This term is inadequate when applied to the mechanism of coding, for it does not indicate codeword specificity. A degenerate code may have high or low specificity depend- ing upon the fidelity of protein synthesis. In most cases the fidelity of protein synthesis in vivo appears to be high, and amino acid replace- ments other than those due to mutation have not been found. However, although the amino acid sequence analyses would reveal mistakes at one site occurring with a frequency higher than 1 or 2%, they would not reveal occasional mistakes occurring at different sites. Thus, occasional coding errors of 1 or 2%, distributed at random over entire protein molecules, might not be detected. In the in vitro system, codewords direct amino acids into protein with very striking specificity (21). In Table IV for example, poly AC prepara- tions do not direct the incorporation into protein of alanine, arginine, glycine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyro- sine, or valine. The specificity of coding by poly CG and AC preparations in Table V is equally apparent. Such negative data clearly demonstrate the very high fidelity of codeword recognition during protein synthesis in this cell-free system. The codewords corresponding to both leucine and valine contain U and G (16, 17, 29). Although the nucleotide content of these code- words are identical, each word was shown to code only for the appro- priate amino acid (21). Thus, nucleotide sequence as well as chemical structure confers specificity upon codewords. However, one example of ambiguity has been found, but this occurs to a large extent in our experiments only under unusual conditions. Poly U directs about 3-5% as much leucine into protein as phenylalanine (17). Bretscher and Grunberg-Manago also have reported this phe- nomenon (2). In the absence of phenylalanine, using well-dialyzed E, coli extracts, poly U coded for leucine about 50% as well as it would code for phenylalanine (20). The molecular basis of this ambiguity is unknown. In the absence of phenylalanine, it is possible that leucine is attached to phenylalanine transfer RNA and then is coded like phenyl- alanine. On the other hand, the ambiguity may occur at the level of the coding units. It is important to note that phenomena of this type also may occur in vivo (3). THE CURRENT STATUS OF THE RNA CODE 463 Efficiency of Synthetic RNA in Coding In spite of the previously mentioned difficulties in comparing tem- plate activities of RNA preparations with different chain lengths and degrees of secondary structure, it seems clear that synthetic poly- nucleotides containing 4, 3, or 2 bases code as well in this system as natural template RNA obtained from viruses (19, 22, 32, 18). The efficiency in coding displayed by synthetic polynucleotides suggests that most nucleotide sequences direct amino acids into protein and that relatively few nonsense nucleotide sequences are present. Although alternative explanations of coding efficiency, such as non- random polynucleotides or nonsequential reading of template RNA, may be considered, such efficiency cannot be ascribed simply to random error in directing amino acids into protein, for amino acids are coded with marked specificity. Considerations such as these may be used to approximate the coding ratio. In a doublet code, only 16 base permutations are possible; thus, the information content would be insufficient to code specifically for all amino acids. Triplet and quadruplet codes would contain 64 and 256 codewords, respectively. Since almost every amino acid tested was found to be coded by polynucleotides containing only two bases, specific and efficient coding by quadruplet words would not seem likely. The data suggest either coding of all amino acids by triplet words, or coding of some by triplets and others by doublets (mixed doublet-triplet code). Recently, Weisblum, Benzer, and Holley (33) have established a molecular basis of degeneracy by demonstrating that multiple species of transfer RNA recognize different codewords with specificity. Multiple peaks of transfer RNA corresponding to at least four amino acids have been found independently by Holley et al. (11), Sueoka et al. (31), and Doctor et al. (6). If a triplet code is assumed, each cell would require almost 64 transfer RNA species. Alternatives which do not require so many transfer RNA species deserve consideration. For example, Donohue and others have described many models other than Watson-Crick pairing (7). The demonstrated interaction between poly A and poly I (23), and the type of base-pairing suggested by Hoogsteen (12) also might be cited. Theories which require recognition of either the 2- or 6-substituents of bases (34) are not supported by the demonstration that hypoxanthine functions in codewords like G (28, 1). The 2-amino group of G does not appear to be required for coding. A triplet code may be constructed wherein correct hydrogen bonding between two out of three nucleotide pairs may, in some cases, suffice for 464 MARSHALL W. NIRENBERG AND OLIVER W. JONES, JR. coding. Correct pairing of a base at one position in the triplet sometimes may be optional. It should be noted that a triplet code of this type in some respects would bear a superficial resemblance to a doublet code and would be in accord with all of the data available. Any theory concerning the physical basis of the code must attempt to explain the following experimentally obtained data: (a) High coding efficiency by synthetic polynucleotides. (b) Marked codeword specificity. (c) Degenerate codewords. (d) The 2-amino group of G is not essential for proper coding. (e) RNA with a high degree of secondary structure has little ability to code. (f) Almost all amino acids tested can be coded by polynucleotides containing only two bases. SUMMARY Synthetic polynucleotides containing 4, 3, or 2 bases have been found to direct amino acids into protein with high efficiency and specificity. Many additional RNA codewords which do not contain uridylic acid have been determined. Almost all amino acids could be coded by polynucleotides containing only 2 bases. These results have been discussed in terms of the general nature of the code. REFERENCES J. Basirio, C., Wansa, A. J., Lencyen, P., Speyer, J. F., anp Ocuoa, S., Proc. Natl. Acad. Sci. U.S., 48, 613 (1962). BRETSCHER, M. S., AND GRUNBERG-Manaco, M., Nature, 195, 283 (1962). Couen, G. N., Ann. Inst. Pasteur, 94, 15 (1958). Crick, F. H. C., in “Structure and Function of Genetic Elements, Brookhaven Symposia in Biology, No. 12,” 1959, p. 35. Davinson, J. N., AND SMELLIE, R. M. S., Biochem. J., 52, 594 (1952). Docror, B. P., Apcar, J., AND Hotiey, R. W., J. Biol. Chem., 236, 1117 (1962). Donouvus, J., Proc. Natl. Acad. Sci. U.S., 42, 60 (1956). Fresco, J. R., Trans. N.Y. Acad. Sci., Series II, 21, 653 (1959). Fresco, J. R., anp Dory, P., J. Am. Chem. Soc., 79, 3928 (1957). Gamow, G., Nature, 173, 318 (1954). Hottey, R. W., Doctor, B. P., Mennity, S. H., anp Saap, F. M., Biochim. et Biophys, Acta, 35, 272 (1959). 12, HoocsTEen, K., Acta Cryst., 12, 822 (1959). 13. Jonzs, O. W., anp Martin, R. G., Federation Proc., 21, 414 (1962). 14. Lencyet, P., Speyer, J. F., Basttio, C., anp Ocuoa, §S., Proc. Natl. Acad. Sci. U.S., 48, 282 (1962). ho bo oo _ HSoaN 15. 16. 17. 18. 19. 20. 21, 22, 23. 24, 25. 26. 27. 28. 29, 30. 31, 32. 33. 34, THE CURRENT STATUS OF THE RNA CODE 465 LencyeL, P., Speyer, J. F.. anp Ocuoa, S., Proc. Natl. Acad. Sci. U.S., 47, 1936 (1961). Martin, R. G., Matruar, J. H., Jones, O. W., anp NirENBERG, M. W., Biochem. Biophys. Research Communs., 6, 410 (1962). Marruagl, J. H., Jones, O. W., Martin, R. G., aND NIRENBERG, M. W., Proc. Natl. Acad. Sci. U.S., 48, 666 (1962). Natuans, D., Norani, G., Scuwartz, J. H., anp ZINpER, N. D., Proc. Nail. Acad. Sci. U.S., 48, 1424 (1962). NinENBERG, M. W., AND MATTHAEI, J. H., Proc. Natl. Acad. Sci. U.S., 47, 1588 (1961). Nimenserc, M. W., Matruart, J. H., anp Jones, O. W., unpublished observations. NiIRENBERG, M. W., Matruacl, J. H., Jones, O. W., Martin, R. G., And Baronpes, S. H., Federation Proc., 22, 55 (1963). OFENGAND, J., AnD Hasetxonn, R., Biochem. Biophys. Research Communs., 6, 469 (1962). Ricu, A., Nature, 181, 521 (1958). Ricu, A., Davies, D. R., Crick, F. H. C., anp Watson, J. D., J. Mol. Biol., 3, 71 (1961). Rozents, R. B., Proc. Natl. Acad. Sci. U.S., 48, 897 (1962). Roserts, R. B., Proc. Natl. Acad. Sci. U.S., 48, 1245 (1962). SincER, M. F., anp Guss, J. K., J. Biol. Chem., 237, 182 (1962). Sincer, M. F., Jones, O. W., Matruaes, J. H., AND NigENBERG, M. W., unpublished observations. Speyer, J. F., Lencyer, P., Basmio, C., anp Ocnoa, S., Proc. Natl. Acad. Sci. U.S., 48, 63 (1962). Speyer, J. F., Lencyet, P., Bastuio, C., anp Ocnoa, S., Proc. Natl. Acad. Sci. U.S., 48, 441 (1962). Suroxa, N., AnD YAMANE, T., Proc. Natl. Acad. Sci. U.S., 48, 1454 (1962). Tsucira, A., FRAENKEL-ConraT, H., Ninenserc, M. W., AND MATTHAEI, J. H., Proc. Natl. Acad. Sci. U.S., 48, 846 (1962). WEIsBLUM, B., BENZER, S., AnD Hotiey, R. W., Proc. Natl. Acad. Sci. U.S.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-sfn2~agcz-tmgf", "00000000-0000-0000-D4C6-4F097A78EDFF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Cell-Free Protein Synthesis Directed by Messenger RNA", "101584910X118", null, "1964", "1964", "Nirenberg's work with synthetic RNA and E. coli provided the basis for his genetic research in the early 1960s.  This article describes the preparation of reagents, procedures for reaction mixtures, methods for growth of E. coli, and preparation of extracts.  Their experiments ultimately proved that messenger RNA, which transcribes genetic information from DNA, directs protein synthesis. That is, messenger RNA transmits the DNA messages that prescribe the assembly of amino acids into the complex proteins that drive living processes.", "Articles", "Amino Acids,RNA, Messenger ; Enzymes", "Translating the Code of Life and the Nobel Prize, 1962-1968", "7", "pages", "Text", "English", "Reproduced with permission of the Academic Press.", "Copyright may apply", null, null, "{3] CELL-FREE PROTEIN SYNTHESIS BY MESSENGER RNA 17   [3] Cell-Free Protein Synthesis Directed by Messenger RNA By MarsHaLL W. NIRENBERG Assay Method Principle. Cell-free extracts of Escherichia coli incorporate C**-amino acids into protein. Under certain conditions this amino acid incorporation is dependent on the addition of synthetic’ or natural\"* messenger RNA, thus providing a sensitive assay for such RNA fractions. In the absence of added messenger RNA, amino acid incorporation presumably is directed by preformed and newly synthesized endogenous messenger RNA. Preincubation of extracts with DNase prevents new messenger RNA synthesis and depletes endogenous messenger RNA. C**-Amino acid incorporation by these extracts is therefore dependent on added messenger RNA and over a certain range is proportional to the amount added. E. coli extracts (“preincubated” S-30 fractions) are incubated with an ATP-generating system, a C1*-amino acid, nineteen C’?-amino acids, and messenger RNA. After incubation, proteins are precipitated and treated to remove C™-amino acyl transfer RNA and free C**-amino acid. The C*! in the washed protein precipitates then is determined. Reagents 1. Mix I contains: 10.0 ml. of 2M Tris buffer, pH 7.8; 2.0 ml. of 1.4.M magnesium acetate; 5.0 ml. of 2 KCI; 3.0 ml. of 6.66 x 2M. W. Nirenberg and J. H. Matthaei, Proc. Natl, Acad. Sci. US. 47, 1588 (1961). ?J. H. Matthaei, O. W. Jones, R. G. Martin, and M. W. Nirenberg, Proc. Nail. Acad. Sci. U.S. 48, 666 (1962). *J, F. Speyer, P. Lengyel, C. Basilio, and S. Ochoa, Proc. Natl. Acad. Sei. US. 48, 63 (1962). *A. Tsugita, H. Fraenkel-Conrat, M. W. Nirenberg, and J. H. Matthaei, Prac. Natl. Acad. Sci. US. 48, 846 (1962). 18 ENZYMES OF NUCLEIC ACID METABOLISM {3]   10? M ATP, Na salt, and 2.0 10°? M GTP, Na salt. Store in 5-ml. aliquots at —20°. 2. Phosphoenolpyruvate kinase, crystalline, 10 mg./ml., obtained as a suspension in (NH,)}.SO, solution (California Biochemical Corp.). 3. 7.5 10°? M phosphoenolpyruvate, K or Na salt, crystalline (California Biochemical Corp.) Store in small aliquots at —20°. The tricyclohexylammonium salt of phosphoenolpyruvate inhibits C'4-amino acid incorporation and should be converted to the potassium salt as follows: 5 g. of phosphoenolpyruvate, tricyclo- hexylammonium salt, are dissolved in minimal quantities of H,O at 3°, and the solution is added to a washed Dowex 50 (H*) column of approximately 2.5 15 cm. Phosphoenolpyruvic acid is eluted with H.O, and the pH of the eluate is determined rapidly with pH paper. When the pH of the eluate drops to 2, collection of the solution is begun, after the pH rises to 4, collection is discontinued. The phosphoenolpyruvic acid solution is converted to the potassium salt by the addition of M KOH with vigorous stirring. The pH of the final solution should be 5.5 to 6.0. 4, 2-mercaptoethanol. . C%-L-Amino acid. 10°? M, 4 me./millimole. 6. Mixture of nineteen C?*-L-amino acids, minus the appropriate C**-amino acid, 2 < 10°? M (each amino acid). 7. Poly U (Miles Chemical Co., Clifton, New Jersey), 30 O.D. units/ ml. at 260 mu, or other messenger RNA preparation. Poly U solutions should be stored at —20° in small aliquots. It is im- portant to note that poly U undergoes a conformational change at 6°, so that solutions which have been frozen and thawed may have lower absorbancies at 260 mu. 8. “Preincubated” S-30 or S-100 and W-RIB enzyme fractions, de- scribed below. nr Procedure. (a) For ten reaction mixtures, prepare mix II each day as follows: 250 yl. of mix I; 1 ul. of 2-mercaptoethanol; 250 yl. of phospho- enolpyruvate, 4 pl. of phosphoenolpyruvate kinase suspension; 250 ul. of C?-amino acid mixture, and 50 yl. of C**-amino acid solution. (6) Add components of each fraction mixture in the following se- quence: 80 ul. of mix II; appropriate amount of H.O to make final volume 0.25 ml.; 5 to 20 ul. of poly U solution (15 to 20 ug. of poly U or 100 vg. of natural messenger RNA usually saturates a reaction mixture) ; and E. coli extracts (0.5 to 2 mg. of protein). Reaction mixtures are incubated at 37° for 15 minutes. C!*-Amino acid incorporation should be {3] CELL-FREE PROTEIN SYNTHESIS BY MESSENGER RNA 19   dependent on the addition of messenger RNA, and, when limiting amounts of messenger RNA are added (see Remarks), incorporation should be proportional to the amount of messenger RNA added. (c) Protein is precipitated at the end of incubation by the addition of 3.0 ml. of 10% trichloroacetic acid at 3°, and precipitates are washed by the method of Siekevitz.* For exploratory work and preparation of extracts, the following fast procedure’ for washing protein precipitates may be used for all C1*-amino acids incorporations except C™4-tryptophan. Reaction mixtures depro- teinized by the addition of 10% trichloroacetic acid are placed in a water bath at 90° to 95° for 20 minutes to hydrolyze amino acyl transfer RNA. Tubes then are chilled in ice for 30 minutes. Protein precipitates are dispersed by stirring or by vigorous agitation, and each suspension is filtered under suction through a Millipore filter (HA Millipore filter, 25 mm. in diameter, 0.45-y pore size, held in an appropriate Millipore filter funnel, Millipore Co., Bedford, Massachusetts). Each precipitate then is washed rapidly with five 5-ml. aliquots of cold 5% trichloroacetic acid. If the radioactivity of the washed protein precipitate is to be counted in a thin-window gas-flow B-scaler, the Millipore filter is glued with rubber cement to a disposable planchette, dried for 5 to 10 minutes under an infrared lamp, and counted. Curled Millipore filter edges should be reglued, for they will tear windows of gas-flow counters. Growth of E. coli Escherichia coli W-3100 (other strains may be used) are grown at temperatures between 25° and 37° (30° is advantageous) in a medium containing 8 g. of nutrient broth (Difco Co., Detroit, Michigan) and 5 g. of glucose per liter. A 60% solution of glucose is autoclaved and is added to autoclaved nutrient broth solution after both have cooled. A typical preparation consists of five 19-1]. carboys, each containing 15 1. of nutrient broth-glucose solution and equipped for aeration. About 1 g. of Antifoam A (Dow-Corning Corp.) is suspended with stirring in approxi- mately 50 ml. of H,O, and the suspension is centrifuged at 3000 X g for 10 minutes to remove undissolved lumps. The supernatant solution of antifoam is autoclaved, and approximately 1 to 2 ml. are added to each carboy before inoculation with bacteria. Additional antifoam may be needed to prevent foaming during growth of bacteria; however, minimal amounts should be used. One liter of nutrient broth-glucose solution in a 6-1. Ehrlenmeyer flask is inoculated with H. coli grown on the surface 5P. Siekevitz, J. Biol. Chem. 195, 549 (1952). *M. W. Nirenberg, unpublished data. 20 ENZYMES OF NUCLEIC ACID METABOLISM [3]   of an agar slant, and the flask is aerated by shaking at 25° for 8 to 12 hours. While the culture is still in the logarithmic growth phase, 200-ml. aliquots are added to each carboy. The carboys are vigorously aerated for 1.5 to 6 hours, depending on the temperature of the medium and the degree of aeration. At 30°, 2.5 to 3.0 hours of growth will suffice. Jt is important to follow cell growth carefully so that aeration may be stopped at the proper time. Aliquots of cultures should be removed at intervals so that turbidities may be determined spectrophotometrically. Aeration should be stopped when a reading of 25 to 35 Klett spectrophotometer units at 520 mp». is obtained, compared to a sterile nutrient broth-glucose solution set at zero. The carboys then are chilled in ice water, and the cells are harvested by means of a refrigerated Sharpless centrifuge. A yield of 0.5 to 0.75 g. of packed cells, wet weight, per liter of medium is optimum. When more cells are obtained, the extracts often are less active. The packed cells are washed by rapid suspension in 3 vol. (w/v) of 0.01 M Tris, pH 7.8, 0.014 M@ magnesium acetate, and 0.06 M potassium chloride at 3°, and are centrifuged at 15,000 x g in preweighed tubes for 15 minutes at 3°. The supernatant solutions are decanted, and the pellets are drained. Centrifuge tubes again are weighed to obtain wet weights of packed cells. Washed, packed cells may be frozen quickly and stored for several months at —20° if desired. Preparation of Extracts Step 1. All operations, unless specified, are carried out in a cold room or at 3°. Cells may be disrupted either by grinding with alumina in a mortar, or with a French press (American Instrument Co.). If the alumina grinding method is to be used, about 30 g. of fresh or thawed packed cells are transferred to a large prechilled, unglazed porcelain mortar (20 cm. in diameter), and 30 g. of alumina A-301 (Aluminum Corporation of America) are added. The cells are ground vigorously with a pestle into a thick paste; cracking, popping noises should be produced during grinding. As the cells break, the paste becomes more fluid, and an additional 30 g. of alumina are slowly added to keep the paste thick and to maintain the popping noise. After about 15 minutes of grinding, 60 ml. of “standard buffer” containing 0.01 M Tris, pH 7.8, 0.014 M magnesium acetate, 0.06 M potassium chloride, and 0.006 M 2-mercaptoethanol freshly prepared are added to the mortar, and the paste is suspended evenly by gently stirring. Alumina, intact cells, and debris are removed by centrifugation at 20,000 < g for 20 minutes. The supernatant fluid is decanted, and the pellet is discarded. Cells to be broken in a French press should be evenly suspended in 1.5 to 2.0 vol. of “standard buffer” (w/v) and disrupted with 18,000 p.s.i. in a pre- (3) CELL-FREE PROTEIN SYNTHESIS BY MESSENGER RNA 21   chilled cylinder. Additional 2-mercaptoethanol (6 micromoles/ml. of extract) is added after the cells have been broken. Step 2. Extracts disrupted in either manner are treated as follows: Two micrograms of pancreatic DNase (twice crystallized, Worthington, Biochemical Co.) are added with gentle mixing to each milliliter of E. coli extract. (Caution: Some commercial DNase preparations are contaminated with pancreatic RNase, which strongly inhibits C’*-amino acid incorporation into protein.) Before the DNase is added, the extract obtained by alumina grinding should be noticeably viscous and should contain some gelatinous clots. Although the extract is maintained at 3°, 5 minutes after addition of the DNase the viscosity and clots disappear. The extract is centrifuged at 30,000 xg for 30 minutes at 3°. The supernatant solution is removed by aspiration to within 1 cm. of the pellet and again is centrifuged at 30,000 x g for 30 minutes. The upper four-fifths of the supernatant solution is removed by aspiration, and henceforth will be referred to as the S-30 fraction. At this stage the activities of 25 to 150-n1. aliquots of the 8-30 frac- tion should be assayed rapidly to determine how much C1*-phenylalanine can be incorporated into protein in the presence and in the absence of poly U. One milligram of S-30 protein added to a 0.25-ml. reaction mix- ture containing 15 to 20 wg. of poly U should incorporate 1 to 20 milli- micromoles of C*-phenylalanine into protein in 15 minutes at 37°. Hf, in the absence of poly U, 1 mg. of S-80 protein directs more than 0.1 millimicromole of C**-phenylalanine into protein, the “preincubation” procedure described in step 3 should be followed. (Step 3 usually is neces- sary.) If less than 0.1 millimicromole of C'*-phenylalanine is incorpo- rated in the absence of poly U, the S-30 fraction may be dialyzed for 8 hours as described in step 3. For routine messenger RNA assays and many amino acid incorpora- tion studies, ribosomes need not be separated from the 8-30 fraction. If the S-30 preparation at this stage can be used for assays, the dialyzed extract should be divided into 1- to 3-ml. aliquots (one tube of extract per experiment), frozen quickly, and stored until needed. Step 3. “Preincubation.” S-30 extracts carried through this step are almost completely dependent on added messenger RNA. The components of a reaction mixture appropriate for “preincuba- tion” of 100 ml. of 8-30 are: 10 ml. of M Tris, pH 7.8; 2.0 ml. of 0.14M magnesium acetate; 4.0 ml. of 0.02 M ATP (neutralized); 12 ml. of 7.5 % 10°? M phosphoenolpyruvate, K or Na salt; 1 mg. of pyruvate kinase, crystalline; 0.04 ml. of 2-mercaptoethanol; and 1 micromole each of twenty t-amino acids. After addition of 100 ml. of 8-30 fraction, the reaction mixture is incubated at 37° for 80 minutes. The reaction mix- 22 ENZYMES OF NUCLEIC ACID METABOLISM [3]   ture then is cooled to 3° and dialyzed against 120 vol. of “standard buffer” at 3° for 8 hours. The dialyzing medium should be changed once. In most cases the “preincubated” 8-30 fraction can be used after dialysis without further preparation to assay messenger RNA. The extract is divided into 1- to 3-ml. aliquots and frozen quickly for storage. One milligram of dialyzed “preincubated” S-30 protein added to a 0.25-ml. reaction mixture containing 15 to 20 yg. of poly U (see Remarks) should incorporate 1 to 40 millimicromoles of C1-phenylalanine into pro- tein during 15 minutes of incubation at 37°. Step 4. If washed ribosomes and supernatant solution are required, the dialyzed S-30 extract is centrifuged at 105,000 x g for 120 minutes in a Spinco Model L preparative ultracentrifuge at 3°. The upper four- fifths of the supernatant solution is aspirated (S-100 fraction). The lower fifth of the supernatant solution is decanted and discarded. Ribo- somal pellets are suspended in the initial volume of “standard buffer” by gentle homogenization (four to five passes) in a Potter-Elvejehm homog- enizer. The ribosomal suspension is centrifuged again at 105,000 * g for 2 hours, and the supernatant solution is decanted and discarded. The ribosomal pellets are suspended, as described previously, in one-fourth the original volume of “standard buffer,” and the suspension is centri- fuged at 10,000 x g for 5 minutes to remove aggregates. The washed ribosomal fraction will be designated W-RIB. W-RIB and S-100 frac- tions are divided into small aliquots, frozen quickly, and stored until needed. Remarks Stability of E. coli Extracts. The extracts appear to retain more activity if they are frozen rapidly in either dry ice~acetone mixture or in liquid N, than if they are frozen slowly at —20°. Little loss in activity of 8-30 fractions can be detected after storage in liquid N, refrigerators (Linde Corp.) for 6 months and longer. S-30 fractions stored at —20° lose less than 5% activity per week. S-100 and W-RIB preparations also retain more activity when stored under liquid N, than when stored at —20°. Some amino acid-activating enzymes may be more labile than others; thus, S-30 and 8-100 fractions are not frozen and thawed more than once. However, W-RIB fractions may be frozen and thawed several times without undue loss of activity. Template Activities of Poly U Preparations. Optimal amounts of poly U or natural messenger RNA required for reaction mixtures may depend on the nuclease content of Z. coli extracts.2 Another variable factor related to the template activity of poly U is the average molecular [3a] RNA POLYMERASE 23   weight of each poly U preparation. Poly U molecules composed of less than one hundred uridylic acid residues are almost completely inactive.” Therefore, poor C’*-phenylalanine incorporations need not be ascribed always to inactive E. colt extracts. Most poly U preparations contain traces of nucleases; to prevent degradation, solutions may be stored in small aliquots at —20° and kept at 3° when thawed.", "Nirenberg, Marshall W.", null, "Methods in Enzymology", "The Academic Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ynen-78sn-zb2z", "00000000-0000-0000-075A-5428F6A79098", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Genetic Code: II", "101584910X119", "101584582X281", "1963", "1963", "This article describes how combinations of bases, or code letters, provide the information used by cells to construct proteins from twenty common kinds of amino acids.  Nirenberg recounts recent developments in the field of genetics and illustrates the components and structure of DNA and RNA, and the process of protein synthesis.  The process of breaking the code and the \"dictionary\" of the genetic code are also covered.", "Articles", "DNA,RNA,Genetics,RNA, Messenger ; Genetic Code", "Public Reactions to the Genetic Code, 1961-1968", "12", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "THE GENETIC CODE: II A sequel to F. H. C. Crick’s article of last October, which discussed how the hereditary material embodies the code for the manufacture of proteins. The nature of the code has now been further elucidated ust 10 vears ago James D. Watson and Francis H. C. Crick proposed the now familiar model for the structure of DNA (deoxyribonucleic acid), for which they, together with Maurice H. F. Wilkins, received a Nobel prize last vear. DNA is the giant helical molecule that embodies the genetic code of all living organisms. In the October 1962 issue of Scientific American Crick described the general nature of this code. ee   by Marshall W. Nirenberg By ingenious experiments with bacterial viruses he and his colleagues established that the “letters” in the code are read off in simple sequence and that “words” in the code most probably consist of groups of three letters. The code letters in the DNA molecule are the four bases, or chemical subunits, adenine, guanine, cytosine and thymine, respectively de- noted A, G, C and T. This article describes how various   EXPERIMENT BEGINS when cells of the colon bacillus are ground in a mortar with finely divided aluminum oxide. “Sap” released from ruptured cells still synthesizes protein. 80 combinations of these bases, or code let- ters, provide the specific biochemica! information used by the cell in the con- struction of proteins: giant molecules as- sembled from 20 common kinds of amino acids. Each amino acid subunit is di- rected to its proper site in the protein chain by a sequence of code letters in the DNA molecule (or molecules) that each organism inherits from its ancestors. It is this DNA that is shaped by evolution. Organisms compete with each other for survival; occasional random changes in their information content, carried by DNA, are sometimes “advantageous in this competition. In this way organisms slowly become enriched with instruc- tions facilitating their survival. The exact number of proteins required for the functioning of a typical living cell is not known, but it runs to many hun- dreds. The great majority, if not all, of the proteins act as enzymes, or biolog- ical catalysts, which direct the hundreds of different chemical reactions that go on simultaneously within each cell. A typi- cal protein is a molecular chain contain- ing about 200 amino acid subunits linked together in a specific sequence. Each protein usually contains all or most of the 20 different kinds of amino acids. The code for each protein is carried by a single gene, which in turn is a par- ticular region on the linear DNA mole- cule. To describe a protein containing 200 amino acid subunits a gene must contain at least 200 code words, repre- sented by a sequence of perhaps 600 bases. No one yet knows the complete base sequence for a single gene. Viruses, the smallest structures containing the blueprints for their own replication, may contain from a few to several hundred genes. Bacteria may contain 1,000 genes; a human cell may contain a million. The human genes are not strung together in STEPS IN CODE BREAKING are shown in this sequence of photo- graphs taken in the author’s laboratory at the National Institutes of Health in Bethesda, Md. The open test tubes at upper left contain samples of the cell-free bacterial system capable of synthesizing protein when properly stimulated. The photograph shows stimulants being added. They include synthetic “messenger RNA” (ribonucleic acid) and amino acids, one of which is radioactive. The protein is Fy Fy rd 5 P| reeeees 2 2)   produced when the samples are incubated 10 to 90 minutes. At up- per right the protein is precipitated by the addition of trichloro- acetic acid (TCA). At lower left the precipitate is transferred to filter-paper disks, which will be placed in carriers called plan- chettes. At lower right the planchettes are stacked in a radiation counting unit. Radiation measurement indicates how well a given sample of messenger RNA has directed amino acids into protein. 8]   ADENINE /\\\\     THYMINE URACIL CYTOSINE CHAIN COMPONENTS        | i a pole LANI ls    =x   i Tee i <= =—o—oam   =i DEOXYRIBOSE RIBOSE PHOSPHORIC ACID COMPONENTS OF DNA (deoxyribonucleic acid) are four bases adenine, guanine, thy- mine and cytosine (symbolized A, G, T, C), which act as code letters. Other components, deoxyribose and phosphoric acid, form chains to which bases attach (see below). In closely related RNA, uraci! (U) replaces thymine and ribose replaces deoxyribose. DNA STRUCTURE         tias DNA MOLECULE resembles a chain ladder ( actually twisted into a helix) in which pairs of bases join two linear chains constructed from deoxyribose and phosphate subunits. The bases invariably pair so that A links to T and G to C. The genetic code is the sequence of bases as read down one side of the ladder. The deoxyribose-phosphate linkages in the two linear chains run in opposite directions. DNA molecules contain thousands of base pairs. 82 one long chain but must be divided among at least 46 DNA molecules. The minimum number is set by the number of human chromosomes (46). which col- lectively carry the hereditary material. In fact, each chromosome apparently carries not one or two but several copies of the same genetic message. If it were possible to assemble the DNA in a single human cell into one continuous thread, it would be about a yard Jong. This three-foot set of instructions for each in- dividual is produced by the fusion of egg and sperm at conception and must be precisely replicated billions of times as the embryo develops. The bottom illustration at left shows how the bases in DNA form the cross links connecting two helica] strands com- posed of alternating units of deoxyribose (a simple sugar) and phosphate. The bases are attached to the sugar units and always occur in complementary pairs: A joined to T, and G joined to C. Asa result one strand of the DNA molecule, with its associated bases, can serve as the template for creating a second strand that has a complementary set of bases. The faithful replication of genes during cell division evidently depends on such a copving mechanism. The coding problem centers around the question: How can a four-letter al- phabet (the bases A, G, C and T) spec- ifv a 20-word dictionary corresponding to the 20 amino acids? In 1954 the theo- retical physicist George Gamow, now at the University of Colorado, pointed out that the code words in such a dictionary would have to contain at least three bases. It is obvious that only four code words can be formed if the words are only one letter in length. With two letters 4 X 4, or 16, code words can be formed. And with three letters 4 x 4 x 4, or 64, code words become available—more than enough to handle the 20-word amino acid dictionary [see top illustration on page 90]. Subsequently many sugges- tions were made as to the nature of the genetic code, but extensive experimental knowledge of the code has heen obtained only within the past 18 months. The Genetic Messenger It was recognized soon after the for- mulation of the Watson-Crick model of DNA that DNA itself might not be di- rectly involved in the synthesis of pro- tein, and that a template of RNA (ribo- nucleic acid) might be an intermediate in the process. Protein synthesis is con- ducted by cellular particles called ribo- somes, which are about half protein and half RNA (ribosomal RNA). Several years ago Jacques Monod and Francois Jacob of the Pasteur Institute in Paris coined the term “messenger RNA” to de- scribe the template RNA that carried genetic messages from DNA to the ribo- somes. A few vears ago evidence for the en- zymatic synthesis of RNA complemen- tary to DNA was found by Jerard Hur- witz of the New York University School of Medicine, by Samuel Weiss of the University of Chicago, by Audrey Stevens of St. Louis University and their respective collaborators [see “Messenger RNA,” by Jerard Hurwitz and J. J. Furth; SciENTIFIC AMERICAN, February, 1962]. These groups, and others, showed that an enzyme, RNA polymerase, catalvzes the synthesis of strands of RNA on the pattern of strands of DNA. RNA is similar to DNA except that RNA contains the sugar ribose instead of deoxyribose and the base uracil in- stead of thymine. When RNA is being formed on a DNA template, uracil ap- pears in the RNA chain wherever adenine appears at the complementary site on the DNA chain. One fraction of the RNA formed by this process is messenger RNA, it directs the synthesis of protein. Messenger RNA leaves the nucleus of the cell and attaches to the ribosomes. The sequence of bases in the messenger RNA specifies the amino acid sequence in the protein to be synthesized. The amino acids are transported to the proper sites on the messenger RNA by still another form of RNA called transfer RNA. Each cell contains a spe- cific activating enzyme that attaches a specific amino acid to its particular trans- fer RNA. Moreover, cells evidently con- tain more than one kind of transfer RNA capable of recognizing a given amino acid. The significance of this fact will become apparent later. Although direct recognition of messenger RNA code words by transfer RNA molecules has not been demonstrated, it is clear that these molecules perform at least part of the job of placing amino acids in the proper position in the protein chain. When the amino acids arrive at the proper site in the chain, they are linked to each other by enzymic processes that are only partly understood. The linking is accomplished by the formation of a peptide bond: a chemical bond created when a molecule of water is removed from two adjacent molecules of amino acid. The process requires a transfer en- zyme, at least one other enzyme and a cofactor: guanosine triphosphate. It ap- pears that amino acid subunits are bonded into the growing protein chain one at a time, starting at the end of the chain carrying an amino group (NH,) and proceeding toward the end that terminates with a carboxyl group (COOH). The process of protein synthesis can be studied conveniently in cell-free ex- tracts of the colon bacillus (Escherichia coli). The bacteria grow rapidly in suitable nutrients and are harvested by sedimenting them out of suspension with a centrifuge. The cells are gently broken open by grinding them with finely pow- dered alumina [see illustration on page 80]; this releases the cell sap, containing DNA, messenger RNA, ribosomes, en- zymes and other components. Such ex- tracts are called cell-free systems, and when they are fortified with energy-rich substances (chiefly adenosine triphos- phate), they readily incorporate amino acids into protein. The incorporation process can be followed by using amino acids containing carbon 14, a radioactive isotope of carbon. Optimal conditions for protein syn- thesis in bacterial cell-free systems were determined by workers in many labora- tories, notably Alfred Tissiéres of Har- vard University, Marvin Lamborg and Paul C. Zamecnik of the Massachusetts General Hospital, G. David Novelli of the Oak Ridge National Laboratory and Sol Spiegelman of the University of Illi- nois. When we began our work at the National Institutes of Health, our   MESSENGER RNA is the molecular agent that transcribes the genetic code from DNA and carries it to the sites in the cell (the ribosomes) where protein synthesis takes place. The letters in mes-   senger RNA are complementary to those in one strand of the DNA molecule. In this example UUAGUCAA is complementary to AATCAGTT. The exact mechanism of transcription is not known. progress was slow because we had to prepare fresh enzyme extracts for each experiment. Later mv colleague J. Hein- rich Matthaei and I found a way to stabi- lize the extracts so that they could be stored for many weeks without apprecia- ble loss of activity. Normally the proteins produced in such extracts are those specified by the cell’s own DNA. If one could establish the base sequence in one of the cell’s genes—or part of a gene—and correlate it with the amino acid sequence in the protein coded by that gene, one would be able to translate the genetic code. Although the amino acid sequence is known for a number of proteins, no one has vet determined the base sequence of a gene, hence the correlation cannot be performed. The study of cell-free protein syn-   DNA DOUBLE HELIX UGU GUU UUG GGU ACA   MESSENGER RNA MESSENGER RNA 3 yUG   RIBOSOME 4 PROTEIN   UUA UUG UGU GUU        by thy TRANSFER RNA'S UGU GUU UUG      GGuU ACA CGC GGU         a , >< oy 4 z3 Ss Ly 2S oy Crs, es ss vw? mrt C4 bh GCG y GGU ACA CGC GGU SYNTHESIS OF PROTEIN begins with the genetic code embodied in DNA (1). The code is transcribed into messenger RNA (2). In the diagram it is assumed that the message has been derived from the DNA strand bearing dark letters. The messenger RNA finds its way to a ribosome (3), the site of protein synthesis. Amino acids, indicated by numbered rectangles, are carried to proper sites on the messenger RNA by molecules of transfer RNA (see illustration on opposite page). Bases are actually equidistant, not 84 “4   yuu thesis provided an indirect approach to the coding problem. Tissiéres, Novelli and Bernard Nisman, then at the Pasteur Institute, had reported that protein syn- thesis could be halted in cell-free ex- tracts by adding deoxyribonuclease, or DNAase, an enzyme that specifically de-   CAC yuA ccc   oy AS Ss   grouped in triplets, and mechanism of recog- nition between transfer RNA and messenger RNA is hypothetical. Linkage of amino acid subunits creates a protein molecule. stroys DNA. Matthaei and I also ob- served this effect and studied its char- acteristics. It seemed probable that protein synthesis stopped after the mes- senger RNA had been depleted. When we added crude fractions of messenger RNA to such extracts, we found that they stimulated protein synthesis. The de- velopment of this cell-free assay for messenger RNA provided the rationale for all our subsequent work. We obtained RNA fractions from vari- ous natural sources, including viruses, and found that many of them were highly active in directing protein syn- thesis in the cell-free system of the colon bacillus. The ribosomes of the colon ba- cillus were found to accept RNA “blue- prints” obtained from foreign organisms, including viruses. It should be em- phasized that only minute amounts of protein were synthesized in these ex- periments. It occurred to us that synthetic RNA containing only one or two bases might direct the synthesis of simple proteins containing only a few amino acids. Syn- thetic RNA molecules can be prepared with the aid of an enzyme, polynucleo- tide phosphorylase, found in 1955 by Marianne Grunberg-Manago and Severo Ochoa of the New York University School of Medicine. Unlike RNA poly- merase, this enzyme does not follow the pattern of DNA. Instead it forms RNA polymers by linking bases together in random order. A synthetic RNA polymer containing only uracil (called polyuridylic acid, or poly-U) was prepared and added to the active cell-free system together with mixtures of the 20 amino acids. In each mixture one of the amino acids con- tained radioactive carbon 14; the oth- er 19 amino acids were nonradioactive. In this way one could determine the particular amino acid directed into pro- tein by poly-U. It proved to be the amino acid phenyl- alanine. This provided evidence that the RNA code word for phenylalanine was a sequence of U’s contained in poly-U. The code word for another amino acid, pro- line, was found to be a sequence of C’s in polycytidylic acid, or poly-C. Thus a cell-free system capable of synthesizing protein under the direction of chemically defined preparations of RNA provided a simple means for translating the genetic code. The Code-Word Dictionary Ochoa and his collaborators and our group at the National Institutes of   AMINO ACID + ADENOSINE TRIPHOSPHATE —       AMINO ACID ACTIVATING ENZYME     AMINO ACID       TRANSFER RNA is a special helical form of RNA that transports amino acids to their proper site in the protein chain. There is at least one transfer RNA for each of the 20 common amino acids. All, however, seem to carry the bases ACC where the amino acids attach and G at the opposite end. The attachment requires a specific enzyme and energy supplied by adenosine triphosphate. Unpaired bases in transfer RNA (AAU in the example) may provide the means by which the transfer RNA “recognizes” the place to deposit its amino acid package. (MORAL   RNA STRUCTURE can take various forms. Transfer RNA (top) seems to be a fairly short double helix (probably less perfect than shown) that is closed at one end. Some RNA molecules contain a mixture of coiled and uncoiled regions (bottom). Health, working independently, have now synthesized and tested polymers containing all possible combinations of the four RNA bases A, G, C and U. In the initial experiments only RNA polv- mers containing U were assayed, but re- cently many non-U polymers with high template activity have been found by M. Bretscher and Grunberg-Manago of the University of Cambridge, and also by Oliver W. Jones and me. All the results so far are summarized in the table at the PROTEIN 4   Pre ty       MESSENGER RNA UUU yuu         RIBOSOME FIRST BREAK IN GENETIC CODE was the discovery that a syn- thetic messenger RNA containing only uracil (poly-U) directed the manufacture of a synthetic protein containing only one amino 86 \\\\ ‘ TRANSFER RNA’S Gy ae Yo + h K UUU bottom of pages YO and G1. Tt lists the RNA_ polymers containing the mini- mum number of bases capable of stimu- lating protein formation. The inclusion of another base in a polymer usually enables it to code for additional amino acids. With only two kinds of base it is pos- ’ sible to make six varieties of RNA poly- mer: poly-AC, poly-AG, poly-AU. polv- CG, poly-CU and poly-GU. If the ratio of the bases is adjusted with care, each variety can be shown to code with great specificity for different scts of amino acids. The relative amount of one amino acid directed into protein compared with another depends on the ratio of bases in the RNA. Assuming a random sequence of bases in the RNA, the theoretical probabilities of finding particular se- quences of two, three or more bases can be calculated easily if the base ratio is known. For example, if poly-UC contains 70 per cent U and 30 per cent C, the probability of the occurrence of the triplet sequence UUU is .7 X 7 X.7, or .34, That is, 34 per cent of the trip'ets in the polvmer are expected to be UUU. The probability of obtaining the se- quence UUC is .7 X .7 X .3, or .147. Thus 14.7 per cent of the triplets in such a polymer are probably UUC. This type of calculation, however, assumes ran- domness, and it is not certain that all the actual polymers are truly random. lt had been predicted by Gamow, Crick and others that for each amino acid are ye fame.   Phe }—       XXX UUU (here might be aore than one code word, since there are 64 possible triplets and only 20 amino acids. A code with multi- ple words for each object coded is termed degenerate. Our experiments show that the genetic code is indeed degenerate. Leucine, for example, is coded by RNA polymers containing U alone. or U and A,or U and C, or U and G. It must be emphasized that degen- eracy of this sort does not imply lack of specificity in the construction of proteins. It means, rather, that a specific amino acid can be directed to the proper site ina protein chain by more than one code word. Presumably this flexibility of cod- ing is advantageous to the cell in ways not vet fully understood. A molecular explanation of degenera- cy has been provided recently in a strik- ing manner. It has been known that some organisms contain more than one species of transfer RNA capable of recognizing a given amino acid. The colon bacillus, for example, contains two readily distin- guishable species that transfer leucine. Bernard Weisblum and Seymour Benzer of Purdue University and Robert W. Holley of Cornell University separated the two leucine-transfer species and tested them in cell-free systems. They found that one of the species recognizes poly-UC but not polv-UG. The other species recognizes poly-UG but not poly- UC [see top illustration on page 89]. Al- though the number of transfer RNA species per cell is unknown, it is possible a Ss s SS rod Xx J Cong] ~ SOX acid, phenylalanine (Phe). The finding was made by the author and J. Heinrich Matthaei. The X’s in transfer RNA signify that the bases that respond to code words in messenger RNA are not known. that each species corresponds to a differ- ent code word. There is, however, the possibility of real ambiguity in protein synthesis. This would occur if one code word were to di- rect two or more kinds of amino acid into protein. So far only one such ambiguity has been found. Poly-U directs small amounts of leucine as well as phenylala- nine into protein. The ratio of the two amino acids incorporated is about 20 or 30 molecules of phenylalanine to one of leucine. In the absence of phenylalanine, poly-U codes for leucine about half as well as it does for phenylalanine. The molecular basis of this ambiguity is not known. Nor is it known if the dual coding occurs in living systems as well as in cell- free systems. Base sequences that do not encode for any amino acid are termed “nonsense words.” This term may be misleading, for such sequences, if they exist, might have meaning to the cell. For example, they might indicate the beginning or end of a portion of the genetic message. An indi- rect estimate of the frequency of non- sense words can be obtained by compar- ing the efficiency of random RNA prepa- rations with that of natural messenger RNA. We have found that many of the synthetic polymers containing four, three or two kinds of base are as efficient in stimulating protein synthesis as natural polymers are. This high efficiency, to- gether with high coding specificity, sug- gests that relatively few base sequences are nonsense words. In his recent article in Scientific American Crick presented arguments for believing that the coding ratio is either three or a multiple of three. Re- cently we have determined the relative amounts of different amino acids di- rected into protein by synthetic RNA preparations of known base ratios, and the evidence suggests that some code words almost surely contain three bases. Yet, as the table at the bottom of the next two pages shows, 18 of the 20 amino acids can be coded by words containing only two different bases. The exceptions are aspartic acid and methionine, which seem to require some combination of U, G and A. (Some uncertainty still exists about the code words for these amino acids, because even poly-UGA directs very little aspartic acid or methionine into protein.) If the entire code indeed consists of triplets, it is possible that cor- rect coding is achieved, in some in- stances, when only two out of the three bases read are recognized. Such imper- fect recognition might occur more often with synthetic RNA polymers containing     Leu —             0000 ‘LEUCINE TRANSFER RNA | LEUCINE TRANSFER RNA II i\\\\e / of 1 pa XXX MESSENGER R UUG SSENGER RNA CODE WORDS a cx cx Ox TWO KINDS OF TRANSFER RNA have been found, each capable of transporting leucine (Leu). One kind (left) recognizes the code word UUG;; the other (right) recognizes UUC. @-=   CYSTEINE ALANINE H wt, le, \\\\ | ‘\\\\ ee Ny C——C——O0-—H 7 | | | | H 0 H oO ” UUG MESSENGER RNA CODE WORDS   S ~ Cys 4       >   a —   RANEY NICKEL   AY) WAY), 4 CYSTEINE TRANSFER RNA x —~ x ~~ x x<- x4     MESSENGER RNA CODE WORDS UUG INGENIOUS EXPERIMENT showed that code-word recognition depends on the specificity of transfer RNA, not on the structure of the amino acid being transported. Cysteine is coded by UUG, alanine by CCG or UCG. Cysteine was hooked to its specific transfer RNA and sulfur was removed by a catalyst (Raney nickel). With sulfur removed from the molecule, eysteine became alanine. It was still directed into protein, however, as if it were cysteine.                                                   SINGLET DOUBLET TRIPLET CODE CODE CODE (4 WORDS) ' (6 WORDS) (64 WORDS) AAR AAG AAC AAU | AGA AGG AGC AGU. ACA: ACG. ACC ACU AUA? AUG AUC AUU GAA GAG GAC GAU GGA 666 GGC GGU AA | AG | AC | AU GCA | GCG GCC . GCU GA; GG | GC . GU GUA GUG ; GUC Guu ca} C6 | cc. CU CAA | CAG CAC CAU UA | UG | UC) UU CGA | C66 CGC CCU CCA CCG) CCC CCU: CUA) CUG | CUC | CUU UAA | UAG | UAC | UAU UGA | UGG | UGC | UGU UCA | UCG | UCC | UCU | UUA | UUG UUC UUU |         CODE-LETTER COMBINATIONS increase sharply with the length of the code word. Since at least 20 code words are needed to identify the 20 common amino acids, the minimum code length is a sequence of three letters, assuming that all words are the same length. only one or two bases than it does with natural messenger RNA, which always contains a mixture of all four. The results obtained with synthetic RNA may dem- onstrate the coding potential of the cell; that is, it may reveal code words that function routinely in the living cell and potential words that would be recog- nized if appropriate mutations were to occur in the cellular DNA. The table on. page 93 summarizes the code-word dic- tionary on the assumption that all code words are triplets. The Universality of the Code Does each plant or animal species have its own genetic code, or is the same genetic language used by all species on this planet? Preliminary evidence sug- gests that the code is essentially uni- versal and that even species at opposite ends of the evolutionary scale use much the same code. For instance, a number of laboratories in the U.S. and England have recently reported that synthetic RNA polymers code the same way in mammalian cell-free systems as they do in the bacterial system. The base com- positions of mammalian code words cor- responding to about six amino acids have been determined so far. It nevertheless seems probable that some differences 90 may be found in the future. Since certain amino acids are coded by multiple words, it is not unlikely that one species may use one word and another species a different one. An indirect check on the validity of code words obtained in cell-free systems can be made by studying natural pro- teins that differ in amino acid composi- tion at only one point in the protein chain. For example, the hemoglobin of an individual suffering from “sickle cell” anemia differs from normal hemoglobin in that it has valine at one point in the chain instead of glutamic acid. Another abnormal hemoglobin hits Iwsine at the same point. One might be able to show, by examining the code-word dictionary, that these three amino acids—glutamic acid, valine and Ivsine—have similar code words. One could then infer that the two abnormal hemoglobins came into being as a result of a mutation that substituted a single base for another in the gene that controls the production of hemoglobin. As a matter of fact, the code-word dic- tionary shows that the code words are similar enough for this to have hap- pened. One of the code groups for ghi- tamic acid is AGU. Substitution of a U for A produces UGU, the code group for valine. Substitution of an A for a U vields AGA, one of the code groups for lysine. Similar analyses have been made for other proteins in which amino acid sub- stitutions are known, and in most cases the substitutions can be explained by alteration of a single base in code-word triplets. Presumably more code words will be found in the future and the cor- relation between genetic base sequences and amino acid sequences can be made with greater assurance. The Nature of Messenger RNA Does cach molecule of messenger RNA function only once or many times in di- recting the svnthesis of protein? The question has proved difficult because most of the poly-U in the experimental system is degraded before it is able to function as a messenger. We have found, nevertheless, that only about 1.5 U’s in poly-U are required to direct the in- corporation of one molecule of phenyl- alanine into protein. And George Spy- rides and Fritz A. Lipmann of the Rocke- feller Institute have reported that only about .75 U’s are required per molecule of amino acid in their studies. If the cod- ing is done by triplets, three U’s would be           AMINO ACIDS CODED U A c G PHENYLALANINE LYSINE PROLINE® LEUCINE ®     ® POLY U CODES PREFERENTIALLY FOR PHENYLALANINE @ REPORTED BY ONLY ONE LABORATORY; STILL TO BE CONFIRMED 4 REQUIRES ONLY FIRST OF TWO BASES LISTED 4 REQUIRES ONLY SECOND OF TWO BASES LISTED SPECIFICITY OF CODING is shown in this table, which lists 18 amino acids that can be coded by synthetic RNA polymers containing no more than one or two kinds of base. The only amino acids that seem to require more than two bases for coding are aspartic acid: and methionine, which need U, A and G. The relative amounts of amino acids directed into pro- required if the messenger functioned only once. Evidently each poly-U molecule directs the synthesis of more than one long-chain molecule of polyphenylala- nine. Similar results have been obtained in intact cells. Cyrus Levinthal and his associates at the Massachusetts Institute of Technology inhibited messenger RNA synthesis in living bacteria with the anti- biotic actinomycin and found that each messenger RNA molecule present at the time messenger synthesis was turned off directed the synthesis of 10 to 20 mole- cules of protein. We have observed that two factors in addition to base sequence have a pro- found effect on the activity of messenger RNA: the length of the RNA chain and its over-all structure. Poly-U molecules that contain more than 100 U’s are much more active than molecules with fewer than 50. Robert G. Martin and Bruce Ames of the National Institutes of Health have found that chains of poly-U con- taining 450 to 700 U’s are optimal for di- recting protein synthesis. There is still much to be learned about the effect of structure on RNA function. Unlike DNA, RNA molecules are usually single-stranded. Frequently, however, one part of the RNA molecule loops back and forms hydrogen bonds with another portion of the same molecule. The ex- tent of such internal pairing is influenced\\", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-pbkk-4si6~iv7x", "00000000-0000-0000-D786-AE2E37B205E3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Nirenberg, Marshall W(arren)", "101584910X120", null, "1965", "1965", "This issue of Current Biography argues Nirenberg's research on the genetic code \"opened up the gates for other scientists to vast areas of knowledge about the processes of all life on earth, its origins, evolution and future.\"  Promised benefits include disease eradication, slowing down the aging process, correction of genetic defects, and changing heredity.  A warning of possible abuse is also included.  Nirenberg is quoted for praising NIH support and is characteristically modest about his achievements.", "Articles, Biographies (documents)", null, "Biographical Information", "3", "pages", "Text", "English", "Reproduced with permission of H. W. Wilson Company.,http://www.ebscohost.com/wilson", "Copyright may apply", null, null, "CURRENT BIOGRAPHY NIRENBERG, MARSHALL W(ARREN) Apr. 10, 1927- Biochemist Address: b. National Institutes Bethesda, Md. 20014 Man’s scientific understanding of the funda- mental nature of life took a giant stride forward in 1961 when Dr. Marshall W. Nirenberg dis- covered a procedure for deciphering the “genetic code” in living cells. His findings then and his continuing research have opened the gates for other scientists to vast areas of knowledge about the processes of all life on earth, its origin, evolution, and future. Almost equally vast is the speculation over the eventual rewards and dangers to man when his mastery of the genetic code gives him the power to influence heredity. Since 1957 Nirenberg has been associated with the National Institutes of Health of the Depart- ment of Health, Education, and Welfare, and since 1962 he has been chief of the section of biochemical genetics of the National Heart Institute. Marshall Warren Nirenberg was bom on April 10, 1927 in New York City. His family moved to Orlando, Florida when he was ten years old, and he has always thought of Florida as home. As a student at the University of Florida in Gainesville, he was attracted to bio- chemistry while working with Dr. R. L. Shirley and Dr. George K. Davis of the Nutrition Lab- oratory. Biochemistry was then almost unknown to the undergraduate courses, and Nirenberg re- calls getting a good introduction at the labora- tory to radioisotope techniques that proved important to him in investigating the structures of organic compounds. He was an_assistant in the department of biology from 1945 to 1947 and a teaching assistant there the fnllowing year. He obtained his B.S. degree in 1948. His fraternity at the university was Pi Lambda Phi. While taking graduate courses at the Univer- sity of Florida, Nirenberg worked as a resear associate at the Nutrition Laboratory in 1951-52. For his M.Sc. degree in biology, which he re- ceived in 1952, he made a study of certain in- sects in Alachua County. Dr. Lewis Berner, who was directing Nirenberg’s research, recognized his deeper inclination and helped him decide to seek his Ph.D. degree in biochemistry. From 1952 to 1956 he held a teaching fellowship in the department of biological chemistry at the University of Michigan. Then as a_resear fellow in 1956-57, he completed his dissertation of Health, 1965     Ea MARSHALL W. NIRENBERG on the subject of hexose uptake in ascites tumor cells. The University of Michigan awarded him the Ph.D. degree in biochemistry in 1957. A postdoctoral fellowship of the American Cancer Society for further work in biochemistry, under the sponsorship of Dr, DeWitt Stetten, Jr., brought Nirenberg to the National Institutes of Health in Bethesda, Maryland in 1957. Dur- ing the next few years at the National Institute of Arthritis and Metabolic Diseases, Nirenberg doggedly pursued his research into the yet ille- gible hieroglyphics of life’s code, When his American Cancer Society fellowship expired in 1959, he continued at the institute under a_fel- lowship of the Public Health Service, and in 1960 he became a research biochemist in the institute’s section of metabolic enzymes. The fruits of his research were summed up in a paper on the genetic code that Nirenberg de- livered at the International Congress of Bio- chemistry in August 1961 in Moscow. The paper had an electrifying impact on his fellow-scien- tists and was followed up by two reports pub- lished in the October issue of the Proceedings of the National Academy of Sciences. In these Nirenberg described how he and Dr. J. Heinrich Matthaei, a visiting scientist from West Ger- many, performed the milestone experiment that cracked the genetic code. But although he had stirred the world of biochemistry, Nirenberg re- mained unknown to the general public until December 1961, when news of his discoveries appeared in the press. The chemical “language” used to describe Nirenberg’s findings is a metaphorical means of expressing the way that every living cell passes along its genetic message. The storehouse of genetic information, which he has helped to un- lock, is a chemical called DNA (deoxyribonucleic acid). Another chemical, RNA (ribonucleic acid), serves as a kind of messenger and trans- mits the information out of the original cell. Through the co-operation of the two acids, 305 CURRENT BIOGRAPHY specific proteins are manufactured that are basic to the development of new cells. Genetic in- formation is transmitted in the form of a code made up of precise quantities of four nucleic acids: A (adenine), T (thymine), G (guanine), and C (cytosine). From this genetic “alphabet” words and sentences are formed, and their se- quence determines each genetic trait. Dr. Nirenberg looked for clues to the code in the making of protein. He mixed a batch of chemicals containing all the amino acids, ex- cluding from them the DNA, RNA, and other components. Then he added an artificial RNA made up only of uracil, coded as UUU. “By adding radioactive carbon 14 to one amino acid at a time in each mixture,” as George Eagle explained in the Washington Post (January 3. 1965), “Nirenberg found that the amino acid directed into protein by UUU is phenylalanine.” This was the first word translated from the genetic code. A similar experiment was _per- formed later in the year by a New York Univer- sity group led by the Nobel Prize winner Dr. Severo Ochoa. In January 1962 Ochoa an- nounced that he and his colleagues had deter- mined the coding for nineteen of the twenty known amino acids and had predicted the base code for the last one. Nirenberg immediately announced substantial agreement with those find- ings for the fifteen amino acids his team had deciphered. Scientists everywhere hailed Nirenberg’s feat as the most important development in molecular biology since the two Nobel Prize winners, James Dewey Watson and Francis H. C. Crick, dem- onstrated the double-spiral structure of DNA back in 1953. Crick declared that the entire coding problem would be solved within a year —a prediction that proved to be too optimistic. Nirenberg pointed out that many other scientists in all parts of the world were adding fragments of knowledge that would help to unravel the life process. Later, when singled out for awards, he insisted to the press that at least thirteen biochemists at the National Institutes of Health share the credit with him, including Philip Leder and Merton Bernfield, who were most closely associated with him in the work on the genetic code. At a symposium on the chemistry of heredity in April 1962, in Atlantic City, a record-break- ing audience of 3,000 biologists and chemists heard Nirenberg report how he made his system turn out tobacco virus proteins. In co-operation with Dr. Heinz Fraenkel-Conrat and Dr. Akira Tsugita of the University of California, he fed tobacco virus RNA into a mixture of amino acids derived from wrung-out sewage bacteria whose code of life had been destroyed. Soon the “brainless” bacteria turned out tobacco virus proteins—something that only a tobacco leaf could do before. In a similar way, biochemists at the Rockefeller Institute have made Dr. Nirenberg’s system turn out hemoglobin, the pro- tein pigment of red blood cells of vertebrates. In 1962 Dr. Nirenberg was appointed chief of the section of biochemical genetics of the National Heart Institute. Addressing a sympo- sium at the Institute of Microbiology, Rutgers University, in September 1962, he disclosed that earlier in the year he and his associates had 1965 worked out three-letter combinations for each of the twenty amino acids. Many scientists at the symposium shared the belief that the genetic code of life is more complicated than it was thought the year before. Nirenberg supported this view by presenting evidence that in addition to the twenty three-letter words for the amino acids, there are some twenty-five other designa- tions for them in the code. In his opinion, the newly discovered complexity in the genetic code represented a step forward, since it meant that the understanding of the code was nearer reality and less of a laboratory ideal. He expressed his belief that the code would be broken in the near future. The next step in the deciphering of the cade was made public at a gathering of the world’s leading biochemists in New York on July 30, 1964. Nirenberg described how he and his col- leagues combined the chemical code symbols into a three-letter “word” and then put all the “words” into the test tubes with ribosomes, amino acids, and the proper enzymes. Each of the GUU “words,” it turned out, called for the amino acid named valine. Thus Nirenberg found out a code word that carried a specific order. George Eagle of the Washington Post reported that Nirenberg has revealed that codes can be read and written in sequence by adding radioactive material to see which component comes up first, second, and third. According to Nirenberg, his group was “just zooming” through the translation in sequence. But he said that biochemists in the molecular field still could not read long genetic messages—only fragments. The discoveries resulting from Nirenberg’s pure research are bound to have startling im- plications for the world outside his laboratory, and hardly any area of life will remain un- affected by them. His research might eventually tell doctors enough about the construction of diseased cells to help wipe out diabetes, cancer, and other illnesses. The pattern of RNA has al- ready been further clarified by Robert W. Holley and others, and its function, particularly in the process of memory, is already being investigated. Mastery of the genetic code would permit not only the improvement of the lot of individuals but also the slowing down of the aging process, the correction of genetic defects, and the chang- ing of heredity. Although most scientists look at these prospects with exhilaration, some have sounded sober warnings. Thus the Nobel Prize winner Ame Tiselius has cautioned against tam- pering with life, mind control, and heredity abuse. Against this background of awesome oppor- tunities and dangers, Nirenberg takes a cautious and realistic position. He calls his group’s work “enlightened trial and error” and prefers to speak of his immediate task in the laboratory. In his view, a long and tedious building of the foundations of basic biological knowledge is necessary before any of the medical and other implications can begin to be understood. “Ulti- mately one hopes,” he wrote in an article for Scientific American (March 1963), “that cell- free systems will shed light on genetic control mechanisms. Such mechanisms, still undis- covered, permit the selective retrieval of genetic 306 CURRENT BIOGRAPHY information.” He believes that the genetic code is essentially universal on this planet and is used even by species on opposite ends of the evolutionary scale. The National Academy of Sciences gave Dr. Nirenberg its award for distinguished research in molecular biology in 1962, including a $5,000 grant to continue his work. Other honors fol- lowed; the Paul Lewis Award in Enzyme Chem- istry of the American Chemical Society in 1963 and the Modern Medicine Award in 1964. On February 8, 1965 President Lyndon B. Johnson presented Nirenberg with one of eleven Nation- al Medals of Science. He is a member of the American Society of Biological Chemists, Amer- ican Chemical Society, Biophysical Society, Washington Academy of Sciences, and Sigma Xi, the science honor society. His articles have ap- peared in Science, Scientific American, the Pro- ceedings of the National Academy of Sciences, and other professional periodicals. Modest about his scientific achievements, Nirenberg prefers anonymity. According to one interviewer, “He shies away from talk about cancer cures, controlled heredity, and the pos- sibility of international honors.” He is six feet three inches tall, slender, and dark-haired. The scientist is said to look ten years younger than his age. His wife, the former Perola Zaltzman, met him when she was a graduate exchange student from Brazil on a fellowship at the Na- tional Institutes of Health. Mrs. Nirenberg now works in the clinical endocrinology branch of the National Heart Institute. They live in Bethesda near the institute. Dr. Nirenberg likes to return to his laboratory in the evening to pursue an interesting idea far into the night. His sense of urgency about his work has made him appreciate the way the United States has supported scientific research. He has visited laboratories in many other countries, but “nowhere else in the world,” he has said, “do the researchers get the support of anything like the National Health Institutes. Without this kind of help we would be many, many years behind where we are now.” References Sci Am 208:33 Mr 63 Washington (D.C.) Post E p2 Ja 3 °65 307 1965", "H.W. Wilson Company", null, "Current Biography", "H.W. Wilson Company", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4pgm-veav_6trm", "00000000-0000-0000-57BF-BACDF672FC76", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Will Society Be Prepared?", "101584910X121", "101584910X151,101584910X435,101584910X442,101584910X350", "1967", "11 August 1967", "Marshall Nirenberg's editorial calls for a more introspective response to advances in genetic research.  Nirenberg predicts that man will soon have the \"power to shape his own biological destiny.\"  He fears that man may be able to program his own cells before assessing the consequences and insists that we must refrain from instructing our own cells until we are \"wise enough\" to make a decision as an \"informed society.\"", "Articles", "Ethics", "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reprinted with permission from Marshall Nirenberg. \"Will Society be Prepared?\" Science 157 (11 August 1967): 633. Copyright 1967 American Association for the Advancement of Science.,http://www.sciencemag.org/", "Copyright may apply", null, null, "tt August 1967, Volume 157, Number 3739 SCIENCE Will Society Be Prepared? New information is being obtained in the field of biochemical genetics. at an extremely rapid rate. Thus far, this knowledge has had relatively little effect upon man. More information must be obtained before prac- tical application will be possible. and the technical problems that must’ be overcome are formidable. However, when these obstacles have been’ removed this knowledge will greatly influence man’s future, for man thea, will have the power to shape his own biologic destiny. Such power cag. be used wisely or unwisely, for the betterment or detriment of mankind: Salvador Luria has said: “the progress of science is so rapid that 4 creates an imbalance between the power it places in the hands of man and the social conditions in which this power is exerted. Then neither warnings of scientists, nor breadth of public information, nor wisdom of citizens may compensate for inadequacies of the institutional frame-: work to cope with the new situations.” & The public understands to some extent the recent developments in bio- chemical genetics, but has only a vague notion of what may be expected. in the future, in spite of the efforts of many scientists to inform the public about probable future developments. oS Where do we stand today? The genetic language now is known, and: it seems clear that most, if not all. forms of life on this planet use the! same language, with minor variations. Simple genetic messages now can be synthesized chemically. Genetic surgery, applied to microorganisms, is a reality. Genes can be prepared from one strain of bacterfa and. a i inserted into another, which is then changed genetically. Such changes‘ are inheritable. Thus far, it has not been possible to program mammaliaig #3 cells in this way. ~? What may be expected in the future? Short but meaningful genetic messages will be synthesized chemically. Since the instructions will be Written in the language which cells understand. the messages will be used to program cells. Cells will carry out the instructions, and the program may even be inherited. don’t know how long it will take before it will be possible to program cells with chemically synthesized messages. Certainly the experimental obstacles are formidable. However. I have little doubt that the obstacles eventually will be overcome. The only ques- tion is when. My guess is that cells will be programmed with synthetic Messages within 25 years. If efforts along those lines were intensified, bacteria might be programmed within $ years. The point which deserves special emphasis is that man may be able to program his own ceils with synthetic information long before he will be abie to assess adequately the long-term consequences of such altera- tions, long before he will be able to formulate goals, and long before he can resolve the ethical and moral problems which will be raised. When man becomes capable of instructing his own cells, he must refrain from doing so until he has sufficient wisdom to use this knowledge for the benefit of mankind. I state this problem well in advance of the need to resolve it, because decisions concerning the application of this knowledge must ultimately be made by society, and only an informed society can make such decisions wisely.—MARSHALL W. NIRENBERG, National Heart Institute   This editocial is adapted from remarks made in accepting the Research Corporation's 1966 award.", "Nirenberg, Marshall W.", null, "Science", "American Association for the Advancement of Science", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tvj9_acnt_g264", "00000000-0000-0000-4856-1733FF2A3272", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Markers for Gene Expression in Cultured Cells from the Nervous System", "101584910X122", null, "1972", "25 May 1972", "Representative of Marshall Nirenberg's early work as part of the Biochemical Genetics group at NIH, and specifically Nirenberg's experiments involving gene expression in neurons, this article assesses the activities of marker enzymes in surface cultures of new born mouse brain cells, and in glial and nonbrain cell lines.", "Articles", "Gene Expression,Neurons,Metabolism", "Neuroblastoma Research, 1967-1976", "11", "pages", "Text", "English", "Reproduced with permission of the American Society for Biochemistry and Molecular Biology.", "Copyright may apply", null, null, "THe JoURNAL oF BroLogicaL CuEMISTRY Vol. 247, No. 10, Issue of May 25, pp. 3159-3169, 1972 Printed in U.S.A. Markers for Gene Expression in Cultured Cells from the Nervous System (Received for publication, December 13, 1971) SAMUEL H. Witson,* Bruce Kk. Scunien,t Joun L. Farsper,§ Epwarp J. Toompson, Roger N. RosEen- BERG, ARTHUR J. BLUME,|| AND MARSHALL W. NIRENBERG From the Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 SUMMARY Methods are presented for preparation of extracts from cultured cells from the nervous system and for study of choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase, and catechol O-methyltransferase activities. These enzyme activities are markers that can be used for studying gene expression in neurons. The methods are sufficiently sensitive so that all assays can be performed with protein harvested from one Petri dish. Activities of the marker enzymes were assessed in surface cultures of new- born mouse brain cells, and in glial and nonbrain cell lines. Low activities of choline acetyltransferase, acetylcholines- terase, and glutamate decarboxylase were detected in all the cells tested. All of these activities, and particularly glutamate decarboxylase, were higher in cultured brain cells from newborn animals than in non-neuronal cell lines. Glutamate decarboxylase activity in glial cells and in brain cells was inhibited more than 95% by 1 mm amino-oxyacetic acid.   Techniques have been developed in this laboratory and others for culture of differentiated neurons (1-10). Activities of en- zymes important in neuronal cell metabolism are useful param- eters for following cell maturation and exploring steps in differ- entiation in such cultures. The purpose of this communication is to describe a set of methods used to explore the expression of genes that determine the metabolism of molecules involved in intercellular conmmuni- vation in the nervous system. Simple, convenient methods are presented for preparing cell-free extracts from surface cultures * Present address, National Cancer Institute, Bethesda, Md. 20014. t Present address, National Institute of Child Health and Thiman Development, Bethesda, Md. 20014. § Present address, Department of Pathology, Health Science Center, Temple University, Philadelphia, Pa. 19140. © Present address, Division of Neurosciences, University of California at La Jolla, San Diego, Calif. 92122. | Present address, Roche Institute of Molecular Biology, Nutley, N. J. 07110. and for assays of acetylcholinesterase (EC 3.1.1.7), cutechol O-methyltransferase (eC 2.1.1.1), choline acetyltransferase (EC 2.3.1.6), and glutamate decarboxylase (EC 4.1,1.15) activities. ‘The assays presented are extensive moclificutions of previously published procedures. Activities of the marker enzymes were assessed in a variety of glial and nonbrain cell lines as well as in surface cultures of new- born mouse brain cells. The results document properties and usefulness of the methods presented and also show that newborn brain cells in surface culture attained levels of glutamate de- carboxvlase activity significantly higher than in non-neuronal cell lines. A preliminary report of this work has been presented (11). METHODS Isolation and Culture of Newborn Mouse Brain Cells Whole brains from newborn Balb/c (National Institutes of Health stock) mice were placed in Solution D (137 mm NaCl, 5.4 mM KCI, 0.17 mm Na2HPO,, 0.22 mm KHPOu, 5.5 mM elu cose, and 5.9 mM sucrose) pH 7.2, 340 mosm (modified Puck's D1 solution (12)) at 0-4°, weighed, washed several times with Solution D, and minced to about 1-mm* pieces with iris scissors. The minced tissue was subjected to two 15-min treatments with 0.25% erude trypsin (Nutritional Biochemicals 1:250 or Difeo 1:300) in Solution D (100 ml per g of tissue) at 37° with eon- stant mild swirling. After each treatment, tissue pieces were allowed to sediment and the dissociated cells that remained sus- pended were collected by decantation. ‘Tryptic activity in both the cell suspension and undissociated tissue was inhibited by the addition of an equal volume of growth medium containing 106; (v/v) fetal bovine serum (Colorado Serum Co.). The undis- sociated tissue was then triturated five times by gravity flow through a 10-ml serological pipette and cells were again recovered by decantation. Cells were pooled and sedimented at 250 x 9max for 10 min at 3° and resuspended in Solution 1). Cell num- ber and viability were determined by counting in a hemocvton- eter with 0.540 (w/v) nigrosin (Allied Chemical, Morristown, N. J... Only those cells which excluded the dve were vonsidered viable. Usual cell recoveries for dissociation of newborn brain were 90 to 100 < 108 cells per g of tissue (98 ta LOOC? viable). Cells were inoculated at 107 viable cells per 150-mm Faleon polystyrene Petri dish (145 ¢ny surface area) in 20 ml of DMEM 3159 3160 (906¢ Dulbecco-Vogt modification of Eagle’s medium, 10°; fetal bovine serum with 50 units of sodium-penicillin and 10 yg of streptomycin-SO, per ml). Dishes were maintained ut 37° im an humidified atmosphere of 10 COs90¢% air. Changes of medium were performed on the 3rd day of culture, every 2nd day thereafter, and 16 to 20 hours before enzyme assay. Other Cell Lines The C-6 rat astrocytoma cell line wax obtained from the Amer- ican Type Culture Collection (No. CCL 107). HeLa cells were from Flow Laboratories, Rockville, Md. The 313 and Balb/C 373 cell lines were from Dr. George Todaro. The RG-179 line was a permunent cell line obtained after multiple passages of brain cells from 5-day-old Fisher rats. The human astrocytoma CHB and rat glioma C2; were provided by Dr. S. Pfeiffer and Dr. Hl. Schein, respectively. The C2, cell was originally de- veloped by Dr. G. Benda. Preparation of Homogenates   Surface Cultures—The procedure was designed to wash cells free of serum protein and to recover cell enzymes reproducibly in high yield. Growth medium was removed from Petri dishes and discarded and the cell monolayer was gently washed twice with 10 ml of Solution D containing 0.14 mm CaCl, and once with 10 ml of Solution D. The Petri dish was drained for 90 sata 45° angle, then the dish was scraped with a spatula 3 em wide (the end of a flexible plastic ruler) covered with disposable Teflon tape (Scientific Specialties Services, Inc., Randallstown, Md.). Cells were recovered by aspiration with a large bore micropipette and transferred to a polyallomer tube, 3 x 0.5 inch. The surface of the dish was washed twice with 0.1 to 1.5-1n] portions of Buffer A (50 mm potassium phosphate buffer, pIl 6.8; 1 mm EDTA, potassium salt) at 3°. The amount of Buffer A added was adjusted so that the final protein concentra- tion was 2 to 10 mg per ml of homogenate. Cells contained in the washes were recovered and combined with the scraped cells. The tubes were stoppered and placed in cold H.O in the chamber of an ultrasonic oscillator (Raytheon, model No. DF101); cells were lysed by sonication at 1° for 5 min. Homogenate volumes were measured; each homogenate was divided into small por- tions which were frozen quickly in a Dry Ice-acetone bath and stored in the vapor phase of a liquid nitrogen freezer. JAfouse Brain—Brain tissue from Balb/e mice was washed with Solution D, blotted, minced in approximately 7 volumes of Buffer A, and homogenized at 1° with a Potter-Elvehjem ho- mogenizer and then by sonicution as described above. The homogenate was centrifuged at 35,000 X gimax for 15 min at 1°; the supernatant fraction (4 to 12 mg of protein per ml) was divided into 0.05-ml portions, quick frozen, and stored at — 100°. Protein concentration was determined by a modification of the method of Lowry eé al. (13) with 3 to 20 wg of protein per reaction, DNA was determined by the spectrofluorophoto- metric method of Kissane and Robbins (14). Histochemical tests were kindly performed by Dr. Lloyd Guth. inzyme Assays Hfomogenates in Buffer A were thawed shortly before use and portions were diluted with appropriate modifications of Buffer A to adjust the homogenates to the specific conditions of each assay. ILomogenates were added to reactions last. Each ho- Markers for Neuronal Genes Vol. 247, No. 10 mogenate was assayed at four concentrations; values were used only if the rate of reaction was proportional to the homogenate concentration. Triplicate homogenates were prepared and assaved routinely; the average values are shown, Acetylcholinesterase Assay Acetylcholinesteruxe activity was assayed by a modification of the methods of Reed et a/. (15) and Ehrenpreis e¢ a7. (16). Neu- roblastoma clone N-18 homogenates were thawed immediately before use and adjusted to the assay conditions by mixing + volumes of homogenate with 1 volume of Buffer A containing 1.0 u NaCl and 2.66% (v/v) Triton X-100. The radioactive substrate, [2-'H}acetvicholine chloride, 250 mCi per mmole (Amersham-Searle) or [l-“Clacetylcholine iodide, 2.4 mCi per mmole (New England Nuclear) was dissolved in HO and ly- ophilized for 16 hours to remove possible volatile contaminants. The cation exchange resin AG 50W-X8 (H+ form, 100 to 200 mesh, Bio-Rad Laboratories) was converted to the Nat form with 2 x NaOH at 25° for 45 min and then washed with H.O until the pH of the effluent was 6.0. Columns (0.5 x 5.0 em) of resin were formed over small plugs of glass wool in 9-ineh disposable Pasteur pipettes and washed with H.O. Each reaction contained the following components in a final volume of 50 pl unless stated otherwise: 2.8 mm [2-°H]acetvl- choline chloride (0.15 wCi per reaction, 1.08 mCi per mmole), 200 mm NaCl, and 0.5°% Triton X-100 in Buffer A; and 0 to 40 ul of neuroblastoma homogenate, or Buffer A containing 200 mu NaCl and 0.5% Triton X-100, or both. Reactions in disposable glass tubes, 10 & 75 mm, were incubated for 10 min at 37°, then transferred to an ice-water bath and diluted rapidly by the addi- tion of 1.0 ml of HO at 1°. Each diluted reaction was immedi- ately passed over a column of the cation exchange resin; the tube was washed with two 1.0-ml portions of HO at 1°, and the washes also were passed through the column. The column effluent was collected in a glass scintillation vial, then 10 ml of Triton X-100-toluene-Liquifluor (333 ¢:666 m1:55 ml) scintilla- tion fluid were added and radioactivity was determined with a scintillation counter (82% counting efficiency). Since reactions are not deproteinized, the column step must be performed rapidly. The rate of acetylcholinesteraxe activity after dilution at 1° was approximately 1.5% that of undiluted reactions at 37°, Therefore, the assay was performed in batches of 20 tubes or less so that all reactions could be passed through columns within 3 min. In some instances distinction was made between “true” and “pseudo” cholinesterases by employing 1073 m BW 284C51 dibromide. Catechol O-Methyltransferase Assay The assay described was a modification of the method of Nikodejevic e¢ al. (17). Neuroblastoma homogenates were ad- justed to the conditions of the catechol O-methyltransferase assay immediately before use by mixing 9 volumes of homogenate with 1 volume of Buffer A containing 50 mm MgCl. The 1-S- adenosy][methyl-“C]methionine, 58 mCi per mmole (obtained from Amersham-Searle in approximately 0.001 n H.S0,), was extracted twice with 10 ml of toluene; the aqueous phase was lyophilized before use. Nonradioactive L-S-adenosylmethionine iodide and 3,4-dihydroxybenzoic acid were obtained from Cal- biochem Corp. Each reaction contained the following components in a final Issue of May 25, 1972 volume of 50 ul except where stated: 0.55 ma 1-S-adenosyl- lnethyl-4C|methionine iodide (0.318 wCi per reaction, 11.55 mCi per mmole), 2.5 mat dihydroxybenzoic acid, and 5 mm MgCl in Buffer A; and 0 to 40 yl of neuroblastoma homogenate, or Buffer A containing 5mm MgCl, or both. Each reaction was incubated ina 12-ml conical glass centrifuge tube with ground glass stopper for 20 min at 37°, then transferred to an ice-water bath and 0.2 ml of 1.0N HCl and 10 ml of toluene were added. ‘Tubes were shaken vigorously for L min, and centrifuged for 5 min at 250 x g. Then 9 ml of the toluene phase were removed and transferred to a scintillation vial containing 5 ml of toluene-Liquifluor (958 ml:42 ml) scintillation solution and radioactivity was determined (87¢¢ counting efficiency). A correction was applied so that eich value reported represented the entire toluene phase. Au- thentic 3-[4C]methoxy-4-hydroxybenzoic acid and 4-[4C]me- thoxy-3-hydroxy benzoic acid were prepared with purified rat liver cutechol O-methyltransferase obtained from Dr. C. R. Creveling. Choline Acetyliransferase Assay The assay described was a modification of the method of Schrier and Shuster (18). Mouse brain extracts were thawed immediately before use and adjusted to the choline acetyltrans- ferase assay conditions by mixing 4 volumes of brain extract with 1 volume of Buffer A containing 1.0 mM NaCl and 2.5¢% Triton X-100. The [1-\"Clacety1-CoA (50 mCi per mmole) (New Eng- land Nuclear) was lyophilized for 16 hours before use. Un- labeled acetyl-CoA (trilithium salt, trihydrate, A grade) and neostigmine methylsulfate were from Calbiochem, choline iodide was from Schwarz. The anion exchange resin AG 1-X8, (C17 form, 100 to 200 mesh, Bio-Rad Laboratories) was washed with HO until the effluent was pH 5.5, and columns of this resin for each assay were prepared as described for the acetylcholines- terase assay. Kach reaction contained the following components in a final volune of 50 wl, except where stated: 0.21 mat [1-4C]acetyl-CoA (6 mCi per mmole), 2 mM choline iodide, 200 mm NaCl, 0.L mm neostigmine methylsulfate, and 0.56 Triton X-100 in Buffer A; and 0 to 40 wl of homogenate, or Buffer A containing 200 mu NaCl and 0.46% Triton X-100, or both. Reactions were incu- bated in glass tubes, 10 x 75 mm, for 10 min at 37°, then trans- ferred to an ice-water bath and diluted by the addition of 1.0 ml of H.O at 1°. The contents of each tube were passed through an anion exchange column; the tube was washed with two 1.0-ml portions of 1,0 at 1°, and the washes were also passed through the column. The column effluent was collected in a glass scin- tillation vial, then 10 ml of Triton-toluene-Liquifluor scintillation mixture (see above) were added and radioactivity was deter- mined at a counting efficiency of 87%. Glutamate Decarboxylase Assay Two modifications of the method of Wingo and Awapara (19) were employed. Method a—Homogenates were thawed prior to use and ad- justed to the conditions of the glutamate decarboxylase assay by mixing 9 volumes of homogenate with 1 volume of Buffer A con- taining 10 mm 2-mercaptoethanol, 5% Triton X-100, and 5 mm pyridoxal phosphate. The 1-[1-\"“C]glutamic acid, obtained from Calbiochem, was neutralized with KOH and lyophilized before use. Anino-oxyacetic acid was obtained from Sigma. Each reaction contained the following components in a final Wilson et al, 3161 volune of 50 wl, except where stated: 5 mm L-{1-“C]glutamic aeid, potassium salt (1.04 wCi per reaction, 4.16 mCi per mmole), 1 mM 2-mercaptoethanol, 0.5 mm pyridoxal phosphate monohy- drate, and 0.5¢% Triton X-100 in Buffer A; and 0 to 40 ul of homogenate, or Buffer A containing 0.5% Triton X-100, 1 mat 2-mereaptoethanol, and 0.5 mx pyridoxal phosphate, or both. Reaction mixtures in glass tubes, 10 x 37 mm, were sealed with tightly fitting rubber stoppers that could be easily perforated with a needle. Reactions were incubated for 10 min at 37° and then transferred to an ice-water bath. Each tube was then placed in a glass scintillation vial containing 5 ml of Hyamine- toluene-Liquifluor scintillation solution (288 ml of 1 Mm Hyamine hydroxide in methanol to 640 ml to 42 ml). The vial was sealed with the reaction tube stopper; then 0.2 ml of a solution of 10 mM acetic acid in methanol was injected through the rubber stopper into the reaction in the tube with a hypodermic syringe. The injection needle was used to dislodge the glass reaction tube from the rubber stopper so that the tube fell to the bottom of the seintillation vial. The stoppered vial was incubated for 30 min at 24° to allow absorption of “COs by the Hyamine solution. Then the reaction tube was removed from the scintillation vial with forceps and the vial was capped with a scintillation vial top. Radioactivity was determined by scintillation counting at 80% efhciency. Vethod b—This alternate method differed only in the technique of #CO. collection. Each reaction was performed in the 6-mm diameter center well of a 10-ml Erlenmeyer flask (Kontes) equipped with a tightly fitting rubber stopper. Hydroxide of Hyamine at 37° (Packard Instruments, Downer’s Grove, TL) was placed in the outer portion of the flask and reaction compo- nents were added to the center well. The flask was sealed with the stopper and incubated at 37° for 11 min. The reaction was terminated by injection of 0.2 ml of 10 mM acetic acid in meth- TaBLe I Range and sensitivity of assays         Usual and amount Source of Incu- | . : tivity, | eat Assay homogenate ee | Radioactive product arts ' prod | or 3H | product Protein reaction min pmoles | pmoles Acetyleho- | Neuroblas- 10 H]Acetate 1,000-|75 , 000 linester- toma clone 14,000 ase N-18 [4C]Acetate 75-|75 , 000 14,000 Choline ace- | Mouse brain . 10 | [#C]Acety!- 5-| 1,500 tyltrans- (age 35 choline 700 ferase days) Glutamate Mouse brain 10 CO; 20- 150 decarbox- (newborn) 3,000 ylase Catechol O- | Neuroblas- 20 | 3-[4C]Me- 10- 7d methyl- toma clone thoxy-4-hy- | 1,200 transfer- N-18 droxybenzoic ase acid?         * Approximately 5°, of the “C-product is 4-[!4C]methoxy-3- hydroxybenzoie acid. 3162 anol through the stopper into the center well. After an addi- tional 90 min at 37° an aliquot of the Hyamine-“CQ2 solution was transferred to a scintillation vial containing 10 ml of toluene- Liquifluor scintillation mixture. Radioactivity was determined by scintillation counting at 87% efficiency. RESULTS Assays for Marker Enzymes The usual range and sensitivity observed for the four enzyme assays are shown in Table I. In evaluation of the assays neuro- blastoma clone N-18 homogenate was used for acetylcholines- terase and catechol O-methyltrausferase assays; mouse brain ex- tracts were used for evaluation of the assays for choline acetyl- transferase and glutamate decarboxylase, although these assays were also applicable to cell culture extracts. The range of linear- ity with protein for neuroblastoma choline acetyltransferase was about half that obtained with mouse brain homogenates (20). Conditions were selected so that a homogenate could be pre- pared by harvesting a single Petri dish and stored frozen for future use. About 30 mg of protein could be harvested from a                       5 1,000 A z 20.000} B - cL | o 5 » ACETYL a GLUTAMATE a CHOLINESTERASE W750 DECARBOXYLASE WJ 15,000- é ™~™ Lae E S 8 a 8 500 § 'a000} ) & x | / ol EHOLINE ACETYL- | = TRANSFERASE |r / =5) 250 7m’ 5,000 4 Q CATECHOL O-METHYL] 2 TRANSFERASE | =      Oo 10=—20.—CO30—s« 4 0 10 ©620.—30 VOLUME OF HOMOGENATE (yl) /REACTION Fic. 1. The relation between concentration of enzyme extract and reaction velocity for glutamate decarboxylase, choline acetyl - transferase, and catechol O-methyltransferase (Panel A) and for acetylcholinesterase (Panel B). Reactions contained the com- ponents described under ‘Methods,’ and enzyme extract protein at the following concentrations: 12.2, 1.06, 5.0, and 1.0 ug of pro- tein per ul of extract for glutamate decarboxylase, choline acetyl- transferase, catechol O-methyltransferase, and acetylcholinester- ase, respectively. Glutamate decarboxylase was determined by Method a.             \"GLUTAMATE C. ue oS TaMATE | & 4,000 B20, 000\" seerye- | i FE ; CHOLINESTERASE QO oO a 4 ; fe 3,000 1 ® 15,000} / ~ 5 b _o df 8 B 10,000 } 8 2,000 CATECHOL o- : Qu f we METHYL~ ao a TRANSFERASE SF 1000 owe oF a O00) TRANSFERASE | a 5,000 | ] | iW 2 A 3 g iS De i! 2 ve a ° 30 60 90 0 30 60 930 oO 30 60 90 MINUTES MINUTES hia. 2. The relation between length of incubation and amount of product. formed. Hach glitamate deearboxylase, catechol O-methylransferase, choline acetyltransferase, and acetyleholin- esterase reaction contained the eomponents described under “Methods” and 488, 200, 42.4, and 8.0 uz of protein, respectively. The dashed line in Panel B represents the theoretical linear reac- tion rate. Ghitamate decarboxylase was determined by Method a. Markers for Neuronal Genes Vol. 247, No. 10 150-mm Petri dish (145 ecru?) containing 2 confluent monolayer of neuroblastoma cells. Routinely, less than 1.0 mg of protein was used to determine the specific activity of each enzyme studied, and 40 to 100 assays were performed per day. Kinetics of Reactions As shown in Fig. 1, the rate of each reaction was proportional to concentration of homogenate protein within the range studied. Conditions were adjusted so that the proportion of radioactive substrate converted to product was <5°; in the case of choline acetyltransferase and <10% with the other three enzymes. The relation between time of incubation and reaction rate is shown in Fig. 2. Under the conditions employed, reactions ‘atalyzed by glutamate decarboxylase, catechol O-methyltrans- ferase, choline acetyltransferase, and acetylcholinesterase were linear for 60, 98, 20, and 90 min, respectively. The relation between reaction mixture pI] and enzyme activity is shown in Fig. 3. At pH 6.8 the activities of glutamate decar- boxylase and catechol O-methyltransferase were essentially maxi- mal (the latter was inhibited by Tris); however, choline acety1- transferase and acetylcholinesterase activities were 60 to 65°¢ of the observed maxima. Although these latter two activities were optimal between pl 8 and 8.5, the four enzymes were routinely assayed at pli 6.8 because acetyl-CoA and acetylcholine are more stuble at pI16.8 than at pIL8. Inaddition, thesame homogenate could be used to determine the activity of each enzyme without adjustment of pI. Enzyme Characteristics Isnzyme stability and product recovery is shown in Table I. Inzyme activity was not affected appreciably by freezing and thawing homogenates or by keeping the enzyme extracts at 1° for 2 to 3 hours prior to assay. However, each enzyme tested was completely inactivated after incubation at 100° for 10 min. Although little or no activities of the other enzymes were de- tected at 1°, acety]cholinesteraxe activity at 1° was approxi- ; 2 : VI     1200p 12,000; ; 3 Neel ACETYLCHOLINESTERASE 5 4000 “(3 4 510,000; ax i | o ° ied fra ' s ~ 800 7X 8,000, { 8 6 8 : / 8 600 caT + S 60007 sf a x a a. 7, ‘| . #4090 | eo 4,000 s n wn / 4 uw / dS 200 + 82,000 6 = = a comt | & Pee eer po OS a a pH py Fro. 3. The effect of pH upon reaction rates. Each glutamate decarboxylase, choline acetyltransferase, catechol O-methyl- transferase, and acetylcholinesterase reaction contained the com- ponents described under ‘Methods,’ the specified buffer, and 122, 26.5, 50, and 10 ug of protein, respectively. Glutamate deear- boxylase was determined by Method @. The formation of radio- active products was determined with standard 50-ul reactions; pH was determined in 0.5-ml reactions that were identical with the 50-zl reactions except that unlabeled substrates were em- ployed. Solid symbols represent reactions with 50 mat potassium phosphate buffers; open symbols represent 50 mM Tris-HCl buffers. The abbreviations used are: GAD, glutamate deearboxylase; CAT, choline acetyltransterase; COMT, catechol O-methyltransferase. Issue of May 25, 1972 Tasur II Enzyme stabtlily and product recovery Each reaction contained the components deseribed under “Methods” and 40, 15, 366, or GO wg of protein for choline acetyl- transferase, acetylcholinesterase, glutamate decarboxylase, and catechol O-methyltransferase reactions, respectively. The amount of radioactive product formed (picomoles) per complete reaction corresponding to 100°; were 610, 11,300, 630, and 114 for choline acetyltransferase, acetylehclinesterase, glutamate de- rarboxvlase, and eatechol O-methyltransferase reactions, re- spectively. Glutamate decarboxylase was determined by Method a (see “‘Methods’’). Product recovery was tested by adding the folowing eompounds to reactions in place of radioactive sub- strate: choline acetyl transferase assay, 28 nmoles of [1-!C]acetyl- choline iodide (1.58 & 10° dpm); aecetylcholinesterase, 208 1moles of sodium [4CJacetate (8.05 & 106dpm); glutamate decarboxylase, 35 umoles of NaH'CO; (3.97 « 105 dpm); catechol O-methyl- transferase, 0.133 nmole of 3-[4C]methoxy-t-hydroxybenzvie acid (3.4 * 10% dpm) and 0.015 nmole of 4-[4C]methoxy-3-hydroxy- benzoic acid (350 dpm). 1 'Catechol   Choline Acetyl- Gluta- Modification acetyl cholin- mate O-meth- Forase [P'S posettse \"feuce re Ve % % % Enzyme stability | | Complete reaction. .... ; 100 | 100 100 100 Minus enzyme. | 2 | 9 8 9 fougzvme frozen and “thawed | i | three times... 0. .0.0.0.0.0..0...... 98 | 99 | 109 108 Inzyme held at 1° for 2-3 hrs.. | 100 | 100 | 116 104 Iunzyme held at. 100° for 10 min.: 25 9 | 8 8 Reaction incubated at 19.0.0... 9 31 | 10 7 Product recovery tadioactive product added in- | stead of substrate. .......... 95 110 | OF 83     mately 20¢¢ of that found at 37°. For this reason acetylcholines- terase reactions were performed in batches (<20 tubes) so that reactions could be chilled immediately after incubation, diluted, and passed through the ion exchange columns in <3 min. Radioactive product was added to reactions instead of sub- strate to determine the percentage of product recovered. Greater than 94% of the radioactive products of the choline acetyltrans- feriuse, acety lcholinesterase, and glutamate decarboxylase reac- tions and 836% of the radioactive product of the catechol O-meth- yitransferase reaction were recovered. The effect of reaction components and other compounds on the rate of enzyme activity is shown in Table IIT. Choline acetyl transferase from mouse brain was completely dependent upon choline for activity and was stimulated by 200 mm NaCl En- zyme activity was not affected appreciably by omission of Triton X-100 or by the addition of 2mm MgCh. Neuroblastoma acetylcholinesterase activity was reduced in the absence of Triton X-100; however, omission of NaClor EDTA in the presence or absence of MgCl was without effect. Marked inhibition was observed by 107° m BW 284C41, a potent inhibitor of acetylcholinesterase (EC 3.1.1.7). The activity of glutamate decarboxylase from mouse brain was reduced slightly in the absence of pyridoxal phosphate but was not iffected appreciably by omission of EDTA or 2-mereapto- ethanol, The reaction was stimulated slightly by omission of Wilson et al. 31638 TasLE III elssay conditions Each reaction contained the components described under “Methods,’’ except as noted, and the following: 40, 15, 366, and 60 ve of homogenate protein for the choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase, and catechol O- methyltransferase reactions, respectively. Iuzyme — specific activities, in the order stated above were: 1,480, 64,400, 140, and 88 pmoles of product per min per mg of protein. Glutamate decarboxylase was measured by Method @ with {1-\"C]glutamic ac id as substrate.       : smoun : Tadioactive Modification product centae per min i pales re Choline acetyltransferase Complete. 0000 eee | G4 ; 100 Minus enzyme (mouse brain)...............43 2 3 Minus choline... .....0.0..0.0.0.000.0002 000% 2 : Minus NaCl... 00.000. ee 40 62 Minus Triton X-100..000...0.0....00....... 61 » OF Plus 2mm MgCls..0.0000000000000.0000.0... 59 91 Acetylcholinesterase Complete. 1130 * 100 Minus enzyme Cneurobl astoma). veveeeet 105 th Minus Triton X-100.....0.0..0.0...0........... - 750 » 66 Minus NaCl... 2.0.02 ee ; 1140 101 Minus EDTA.. . Le 1160 102 Minus EDTA, plus 1 mm uw MgCle. eee 1130 100 Plus 107° w BW 284C51. 0 eee. j 135 12 Glutamate decarboxylase i Complete... 0.2.0.0. | 63 » 100 Minus enzyme (mouse brain)... ........-.. | 8 Minus pyridoxal phosphate... ............... | 55 87 Minus EDTA.. ol ittttecseeeee.f 68 | 110 Minus 2-mere aptoethanol So | 60 > 96 Minus Triton X-100...000.000...............1 87 » 140 Plus l mu MgCly. o.oo eee : 76 i 120 Plus 10 mm iodoacetamide... ............... J 18 » 29 Plus 10 mm hydroxylamine.................. 8 . 2 Plus 1 mm amino-oxyacetie acid. 2.0.0.0... i 8 12 Catechol O-methyltrausferase ! Complete.....0.0.0.000.0000 000 c cee | 5.7; 100 Minus enzyme (neuroblastoma)............. 0.48 9 Minus dihydruxybenzoie acid... ............. 0.69 12 Minus dihydroxybenzoic¢ acid, plus 1 mm p- | hydroxy phenviacetic acid. .......0.0..0... 0.76 | 13 Plus 1 mm p-hydroxyphenylacetic acid...... 6.5 115 Minus MgCly.. 0000 ence 0.98 | 17 Minus EDTA... 00.000. 0c een 5.7; 100 Minus MgCl, and EDTA... 000. 3.0 } 52 Plus 0.52 (v/v) Triton N-100............... , 6.6 5 115 Plus 1 mm 2-mercaptoethanol............... 7.9 140 Plus 0.25 mm dihvdroxymethoxybenzoic acid. 1.3 j 28 Plus 1mm tropolone.. 0. 0.945 17 Triton X-100 or by the addition of 1 mm MgCl. Marked in- hibition was observed in the presence of iodoacetamide, hyvdrox- ylamine or amino-oxyacetic acid, as expected (21). The activity of neuroblastoma catechol O-methyltransferase was almost completely dependent on the substrate, dihydroxy- benzoie acid. p-Hydroxyphenylacetie acid did not serve as a substrate for the enzyme and did not inhibit methyl transfer to 3164 dihydroxybenzoic acid. Hence, the enzyme was capable of dis- tinguishing between mono- and dihydroxy substrates. Omission of MgCl, markedly reduced the rate of reaction; omission of EDTA was without effect. Enayme activity was markedly in- hibited in the presence of compounds known to inhibit catechol O-methyltransferase, such as dihydroxy methoxybenzoic acid (17) and tropolone. Product [dentification The radioactive products of the choline acetyltransferase, ace- tylcholinesterase, und catechol O-methyltransferase reactions were characterized by thin layer or paper chromatography as shown in Table TV. Virtually all of the radioactive product of the acetylcholinesterase reaction wax chromatographically indis- Markers for Neuronal Genes Vol, 247, No. 10 tinguishable from authentic sodium acetate added as carrier. Greater than 73¢¢ of the applied radioactive produet was recov- ered after chromatography. Similarly, virtually all of the radioactive product formed in the choline acetyltransferuse reactions with a mouse brain homoge- nate was chromatographically indistinguishable from authentic acetylcholine chloride. It has been shown (20), however, that with some homog", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ykds-43ik_8i8s", "00000000-0000-0000-DEE4-1E11C29E4A18", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Regulation of Adenylate Cyclase of Neuroblastoma x Glioma Hybrid Cells by a-Adrenergic Receptors. I. Inhibition of Adenylate Cyclase Mediated by Alpha Receptors", "101584910X123", null, "1979", "25 March 1979", "This article is the product of years of research on clonal hybrid cells as \"model systems\" for experimentation on receptor structure and function.  The studies show that receptors mediate both long term and short term effects on enzymatic and neural activity.", "Articles", "Neuroblastoma,Adenylyl Cyclases,Receptors, Adrenergic,Cyclic AMP,Cyclic GMP,Metabolism", "From Neuroblastoma to Homeobox Genes, 1976-1992", "8", "pages", "Text", "English", "Reproduced with permission of the American Society for Biochemistry and Molecular Biology.", "Copyright may apply", null, null, "THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol, 254, No. 6, Issue of March 25, pp. 1913-1920, 1979 Printed in U.S.A. Regulation of Adenylate Cyclase of Neuroblastoma x Glioma Hybrid Cells by a-Adrenergic Receptors I. INHIBITION OF ADENYLATE CYCLASE MEDIATED BY a RECEPTORS* Steven L. Sabol and Marshall Nirenberg (Received for publication, May 23, 1978) From the Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014 (—)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E,-stimulated adenylate cy- clase activities by 35 to 60% in homogenates of NG108- 15 neuroblastoma x glioma hybrid cells and markedly reduce adenosine 3’:5’-monophosphate levels of intact cells, but do not affect guanosine 3’:5’-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an a receptor, possibly a presynaptic a, receptor. The inhibition of adenylate cyclase by nor- epinephrine is reversed by a receptor antagonists such as dihydroergotamine or phentolamine, but not by the & receptor antagonist propranolol. The effect of nor- epinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 aM GTP. The inhibition of adenylate cyclase activity by norepi- nephrine is reduced by 10 mm NaF and is abolished by 0.05 mm guanyl-5’-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by a receptors, opiate receptors, and muscarinic acetylcholine recep- tors are not additive; this suggests that the three spe- cies of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regu- lating the enzyme.   Cellular responses to the endogenous catecholamines nor- epinephrine and epinephrine are mediated by specific receptor molecules, which have been divided into two major classes, a receptors and £ receptors, as well as into subclasses, according to the potencies of ligands for the receptors (reviewed in Ref. 1). Typical a receptor responses exhibit the specificity, in order of decreasing potency [epinephrine = norepinephrine >> isoproterenol}, and are blocked by phentolamine, phen- oxybenzamine, or dihydroergotamine. Typical £ receptor re- sponses exhibit the specificity [isoproterenol > epinephrine = norepinephrine] and are blocked by propranolol. Whereas activation of 8 receptors usually results in an increase in the activity of adenylate cyclase (EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)) and, therefore, an increase in the concentration of adenosine 3’:5’-monophosphate (2), the relationship between a receptor activation and adenylate cy- clase is less clearly defined. In certain cells, « receptor acti- vation reduces the intracellular cAMP concentration or re- duces the magnitude of responses elicited by compounds which elevate the cAMP concentration (3-9); thus, Robison et al. (2) proposed that a and @ receptors may be linked to * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. adenylate cyclase in an opposing manner. However, in cerebral cortical slices, a receptor activation is associated with an increase in cAMP (10-12). In addition, « receptor activation in certain tissues may increase permeability to Ca’* ions (13- 15) or Cl ions (16) and/or increase cellular cGMP (13, 14) independently of an alteration of cAMP levels. Clonal NG108-15 hybrid cells,’ obtained by fusion of mouse neuroblastoma N18TG-2 clone (17) with rat glioma clone C6BU-1 (18), possess adrenergic receptors which, in concert with an activating ligand, reduce the PGE,’-dependent in- crease in cellular cAMP (19). The cells also possess PGE, and adenosine receptors that are coupled to the activation of adenylate cyclase and opiate and muscarinic acetylcholine receptors coupled to the inhibition of adenylate cyclase (20- 23). In this report, we show that a receptors are coupled to the inhibition of adenylate cyclase in NG108-15 homogenates and characterize the inhibition. In the accompanying report (24), we show that prolonged « receptor-mediated inhibition of adenylate cyclase in intact NG108-15 cells results in a long lived increase in adenylate cyclase activity. Some of these results have been presented in preliminary form (25). After this work was completed, a receptor-mediated inhibition of adenylate cyclase in human platelet lysates was reported (26). EXPERIMENTAL PROCEDURES Growth of Cells and Preparation of Homogenates—NG108-15 cells (subculture 16-22) were grown in Falcon flasks (75 cm? surface area) or Petri dishes (100-mm outside diameter, 64 cm’ surface area) in 90% DME medium (Grand Island Biological Co., Catalogue No. H- 21) containing 44 mm NaHCO; and 10% fetal bovine serum (Colorado Serum Co.) supplemented with 0.1 mm hypoxanthine, 1 iM amino- pterin, and 16 um thymidine in a humidified atmosphere of 90% air, 10% CO, at 36.5°C. As cultures approached confluency, the medium was changed once or twice daily to maintain the pH between 7.2 and 7.4. Mycoplasma were not detected in the cells or culture medium. For preparation of homogenates, cells from confluent cultures (approximately 15 mg of protein/flask) were harvested 4 to 6 h after the medium was replaced with fresh medium. Cells were washed twice with D2 saline solution (0.17 mm Na:HPO,, pH 7.4, 150 mm NaCl, 5.4 mm KCl, 25 mm p-glucose, and 0.2 mm CaCl) and were dissociated with D1 saline solution (D2 solution without CaCh, pH 6.7) and washed twice with D1 by centrifugation at 250 x g for 5 min at 25°C. The final washed cell pellet was suspended in 290 mm   'T. Amano, B. Hamprecht, and M. Nirenberg, manuscript in preparation. ? The abbreviations used are: PGE,, prostaglandin E,; PGF;, pros- taglandin F,,; DME medium, Dulbecco-Vogt modification of Eagle’s minimal essential medium; Gpp(NH)p, guanyl-5’-yl imidodiphos- phate; Ro20-1724, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone; EGTA, ethylene glycol bis(8-aminoethyl ether) N,N’-tetraacetic acid; WB-4101, — 2-([2’,6’-dimethoxy ]phenoxyethylamino)methylbenzodi- oxan; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; car- bachol, carbamylcholine chloride. 1913 1914 sucrose, 25 mm Tris-HCl, pH 7.5 (4 to 6 mg of protein/ml), and 1-ml aliquots were frozen in dry ice and stored at —190°C. Immediately before use an-_aliquot was thawed and the suspension was homoge- nized at 0°C in a ground glass tube with 25 strokes of a Teflon pestle rotating at 1200 rpm. The resultant homogenate was found by phase contrast microscopy to have a ratio of intact cells to nuclei of 0.01 or less. Assays for adenylate cyclase activity were initiated within 5 min after homogenates were prepared. NG108-15 particulate fractions were prepared by centrifugation of homogenates at 30,000 x g or 130,000 x g, as indicated in the figure legends, for 15 min. The pellets were washed by dispersion and centrifugation in 290 mM sucrose, 25 mM Tris-HCl, pH 7.5. The final pellets were dispersed in the same buffer (2 mg of protein/ml) and stored at —190°C. These procedures were performed rapidly because a receptor-mediated inhibition of adenylate cyclase was found to decay significantly upon storage of homogenates at 0°C for more than 30 min: Protein was measured by the method of Lowry et al. (27) using bovine serum albumin as a standard. Assay of Adenylate Cyclase Activity—Adenylate cyclase activity was routinely determined in 100-u! reaction mixtures containing 30 or 35 mmo Tris-HCl (pH 7.5), 5 mm magnesium acetate, 58 or 116 mm sucrose, 0.25 mM Ro20-1724 (a phosphodiesterase inhibitor), 1 mm [a-\"PJATP (3 to 6 x 10° cpm), 1 mm cAMP, 0.1 mM pargyline hydrochloride to inhibit monoamine oxidase, 20 mM creatine phos- phate, 10 units (67 to 80 yg of protein) of creatine phosphokinase, 10 um PGE, where indicated, 0.48% ethanol (used as a solvent for PGE, and Ro20-1724), and unfractionated NG108-15 homogenate protein as indicated (usually 75 to 110 yg of protein/reaction mixture). Unless otherwise indicated, for assays involving catecholamines, reaction mixtures also contained 0.01 mm sodium ascorbate (see preparation of catecholamine stock solutions below). In most experiments sodium ascorbate had little or no effect on adenylate cyclase activity or on the potency of catecholamines as inhibitors of adenylate cyclase. However, for some homogenates prepared during the later stages of the work, ascorbate was found to activate adenylate cyclase. In these cases, indicated in the text, ascorbate was omitted. Reactions were started by addition of homogenate to prewarmed (2 min, 37°C) reaction mixtures. Unless otherwise stated, incubations were at 37°C for 10 min for basal activity and 5 min for PGE)- stimulated activity. Reactions were stopped by the addition of 0.8 ml of 6.25% trichloroacetic acid at 4°C and 100 pl of PH]cAMP (10,000 cpm, 39.8 Ci/mmol), and cAMP was purified according to method C of Salomon e¢ al. (28) with a recovery of [7H]cAMP of 70 to 85%. Greater than 90% of the ’P product recovered was shown previously to be cAMP (20). Duplicates usually differed by less than 3%. Less than 0.8% of the 1 mm cAMP in the reaction mixture was degraded by endogenous phosphodiesterase activity in a typical reaction mix- ture incubated for 10 min. Determination of ICs) and Apparent Hill Coefficient Values— Adenylate cyclase was determined in the presence of 8 to 12 concen- trations of each adrenergic compound tested. The data were fitted to a four-parameter logistic model (29) using the equation: A-D “Te avortP where A is the activity in the absence of ligand C is the concentration of ligand resulting in half-maximal inhibition (ICs0), D is the maxi- mally inhibited activity at saturating ligand concentration, e.g. at least 0.1 mm (—)-norepinephrine, B is the exponent which is related to the steepness of the dose-response curve and here is termed “apparent Hill coefficient,” X is the ligand concentration, and Y is the adenylate cyclase activity. The best values of B and C for each concentration curve with given A and D values were determined by iterative nonlinear least squares analysis using the MLAB program of the PDP-10 computer of the National Institutes of Health Computer Center. The ICso values obtained in this manner agreed well with those obtained by log-probit analysis as well as by inspection of the concentration curves. Assay of cAMP of Intact Cells—Culture medium was removed and 3 ml of cold 5% trichloroacetic acid were added to the cell monolayers. Cells were washed twice with 5% trichloroacetic acid (1 ml/wash). The trichloroacetic acid extracts and washes were pooled and centri- fuged at 30,000 x g for 20 min, and cAMP was purified (20) and assayed by the method of Gilman (30) in 0.2-ml reaction mixtures each containing 1 pmol of [*H]cAMP and 0.7 ug of partially purified cAMP-dependent protein kinase protein, which bound 0.3 pmol of Regulation of Adenylate Cyclase by a-Adrenergic Receptors [*H]cAMP under the conditions used. Cell protein was determined (27) from the NaOQH-solubilized trichloroacetic acid precipitates. Assay of cGMP of Intact Cells—Confluent cultures of NG108-15 cells in 100-mm Petri dishes were incubated in air for 30 min at 37°C in DME medium containing 25 mm Hepes buffer instead of NaHCOs, adjusted to pH 7.4, and adjusted to 340 mosm/kg with NaCl, and supplemented with 0.1 mm hypoxanthine, 1 um aminopterin, 16 uM thymidine, and 0.5 mM 3-isobutyl-1-methylxanthine (a phosphodies- terase inhibitor). Test compounds were then added and dishes were incubated at 37°C for 15 min. The medium was discarded and cells were suspended in 5 m! of 5% trichloroacetic acid. Intracellular cGMP was purified from the trichloroacetic acid supernatants and assayed by radioimmunoassay according to the method of Matsuzawa and Nirenberg (31). The trichloroacetic acid precipitates were assayed for protein (27). Chemicals—The following were kind donations: prostaglandins, Dr. J. E. Pike of Upjohn; Ro20-1724, Dr. H. Sheppard of Hoffmann- La Roche; methoxamine hydrochloride, Burroughs-Wellcome; oxy- metazoline hydrochloride, Schering; phentolamine hydrochloride, CIBA-GEIGY; naloxone hydrochloride, Endo; 9,10-dihydro-a-ergo- cryptine, Sandoz; fluphenazine hydrochloride, Squibb; and WB-4101, Ward-Blenkinsop Pharmaceuticals. R-(—)-Norepinephrine hydrochloride, R-(—)-epinephrine bitar- trate, (—)-phenylephrine hydrochloride, (—)-isoproterenol hydrochlo- ride, dopamine hydrochloride, (—)-dopa hydrochloride, 9,10-dihydro- ergotamine, yohimbine hydrochloride, phenoxybenzamine hydrochlo- ride, (+)-propranolol hydrochloride, atropine sulfate, carbamylcho- line chloride, adenosine 5’-triphosphate (prepared from equine muscle or by phosphorylation of adenosine), cAMP-dependent protein kinase (beef heart), and creatine phosphokinase (rabbit muscle) were from Sigma. Pargyline hydrochloride, (+)-dihydroxymandelic acid, and a- methyl-(+)-norepinephrine were from Regis. Other compounds were from the following sources: (+)-norepinephrine bitartrate, Adams; bulbocapnine, K and K; morphine sulfate, Merck; guanosine 5’-tri- phosphate, P-L Laboratories; guanyl-5’-y! imidodiphosphate, ICN; 3- isobutyl-1-methylxanthine, Aldrich; [a-\"PJATP and [G-\"H]cAMP, New England Nuclear. Other compounds were of reagent grade purity. Solutions of adrenergic compounds usually were prepared imme- diately before use and were stored at —25°C or on ice. Catecholamines were dissolved either in 0.1 mm sodium ascorbate or in 1 mm HC] at 0°C. Stock solutions of dihydroergotamine and dihydroergocryptine were prepared by titrating the free base with HC] (final pH 6.0); these compounds were less soluble at higher pH. RESULTS Effects of Norepinephrine on cAMP and cGMP Levels of Cells and on Adenylate Cyclase Activity—The effects of (—)-norepinephrine and PGE; on intracellular cAMP concen- trations of NG108-15 cells, in the presence or absence of the phosphodiesterase inhibitor Ro20-1724, are shown in Table I (Experiment 1). In the presence of Ro20-1724, 10 um norepi- nephrine reduced basal and PGE,-stimulated cAMP accu- mulation by cells to 25%. Cyclic AMP concentrations of cells were low in the absence of the phosphodiesterase inhibitor, and norepinephrine reduced basal cellular cAMP only slightly, if at all, but inhibited PGE,-dependent cAMP accumulation by 93%. These results agree well with those reported previ- ously (19, 32). Substitution of 0.2 mm EGTA for Ca?* ions in the medium did not significantly reduce the a receptor-me- diated decrease in cAMP accumulation; this suggests that the a receptor-mediated response is not dependent on extracellu- lar Ca’* ions.’ The effects of (—)-norepinephrine or PGF,, on intracellular cGMP concentrations of NG108-15 cells are shown in Table I (Experiment 2). Exposure to norepinephrine did not signifi- cantly affect the cGMP concentration of cells, but did decrease the cAMP concentration during the period examined. Expo- sure to PGF,, for 0.5 min, however, elevated the cGMP concentration 5.6-fold as previously found.‘ 3'R. McGee and M. Nirenberg, unpublished results. ‘H. Matsuzawa and M. Nirenberg, manuscript in preparation. Regulation of Adenylate Cyclase by a-Adrenergic Receptors As shown in Fig. 1, 10 um norepinephrine inhibited basal and PGE,-stimulated adenylate cyclase activities in NG108- 15 homogenates 60% and 48%, respectively. Reaction rates were linear during the 15-min period examined. In other experiments (not shown), norepinephrine inhibited adenylate cyclase for at least 60 min; thus, the effect of norepinephrine does not desensitize rapidly under the conditions used. The rates of basal and PGE,-stimulated cAMP synthesis were proportional to the amount of homogenate protein added in the range 25 to 150 ug of protein/reaction mixture (not shown). The extent of maximum inhibition of adenylate cyclase by norepinephrine varied somewhat from one batch of cells to TaBLeE I Effects of norepinephrine on intracellular cAMP and cGMP of NG108-15 cells Experiment 1: duplicate Petri dishes (60-mm diameter), each with 3 mg of cell protein, were incubated in an atmosphere of 10% COz, 90% air with 5 ml of growth medium/dish without serum, supple- mented with 0.1 mm ascorbic acid and 0.1 mm pargyline, with or without 250 um Ro20-1724, for 20 min at 37°C, Ten micromolar PGE, and/or 10 ym (—)-norepinephrine was then added where indicated, Dishes were incubated for an additional 10 min at 37°C, then intra- cellular cAMP was determined. Experiment 2: triplicate Petri dishes (100-mm diameter), each with 10 mg of cell protein, were incubated as described under “Experimental Procedures.” Then the following compounds were added where indicated: 1 um HCl (control), 10 um (—)-norepinephrine and 1 ym HCl, or 10 uM PGF,,. Cultures were incubated at 37°C for the times indicated, then intracellular cGMP and, where indicated, cAMP were determined.           . . Min- Picomoles rons Experiment No. and Additions ates p came ime pr ot ein + 1. cAMP: With Ro20-1724 None 10 160 +4 Norepinephrine 10 38 + 10 PGE, 10 4100 + 490 PGE, + norepinephrine 10 1040 + 57 cAMP: Without phosphodiesterase inhibitor None 10 12+3 Norepinephrine 10 10+ 1 PGE, 10 790 + 67 PGE, + norepinephrine 10 52 + 11 2. cGMP Control 0 0.57 + 0.12 Control 0.5 0.53 + 0.09 Control 2.0 27 +2 0.47 + 0.03 Norepinephrine 0.25 0.51 + 0.04 Norepinephrine 0.5 0.55 + 0.15 Norepinephrine 1.0 0.58 + 0.16 Norepinephrine 2.0 1641 0.36 + 0.04 PGF,, 0.5 3.0 + 0.20 ® pasar B PGE, 13500 2 250} {1 43000 2 5 PGE, 5 & 200 NONE + 12500 & a £ L 42000 & g {50 4 = 3 L 41500 3 ‘w= 100 | ef mo L PGE*NE J 1 Q008 2 3 a 50 1 { 500 = o i015 °         0 5 MINUTES Fic. 1. Effect of norepinephrine (NE) (10 um) on the rate of (°P]cAMP formation in an NG108-15 homogenate. Aliquots (50 ul) were withdrawn at the indicated times from a 600-1 reaction mixture containing 735 ug of homogenate protein. A, basal rate; B, rate in the presence of 10 um PGE). 1915 another; however, similar values were obtained with sepa- rately homogenized portions of the same batch of cells. Receptor Specificity for Adrenergic Ligands—The effects of different ligands and of ligand concentration on basal and PGE,-stimulated adenylate cyclase activities are shown in Fig. 2. Compounds known to activate a receptors, such as (—)- norepinephrine, (—)-epinephrine, and dopamine, inhibited basal and PGE,-stimulated adenylate cyclase partially; the maximum inhibitions found were approximately 50 and 35%, respectively. The concentrations of these and other ligands required for half-maximal inhibition of adenylate cyclase (ICs) are listed in Table II. Clonidine was the most potent inhibitor tested with an ICso of 0.1 uM, but the extent of inhibition of basal activity (24%) was less than that of other inhibitors. Clonidine also partially antagonized the inhibition by norepinephrine (not shown), which suggests that clonidine acts as a mixed agonist-antagonist. The ICso values for a- methyl-(+)-norepinephrine, (—)-norepinephrine, and (—)-epi- nephrine were 0.2, 0.4, and 0.5 HM, respectively, for basal activity, while dopamine and (—)-isoproterenol, a selective 8 receptor activator, were less potent inhibitors with ICso values of 5 and 60 uM, respectively. (+)-Norepinephrine was 75-fold less potent an inhibitor than (—)-norepinephrine; this indi- cates that the inhibition of adenylate cyclase is dependent on a stereospecific interaction. (—)-Phenylephrine, methoxamine, and oxymetazoline, which are potent a receptor activators in other systems, were relatively weak inhibitors of adenylate cyclase (ICso 9 to 80 um). Serotonin, an a receptor activator in some systems, did not reduce intracellular cAMP levels‘ or inhibit adenylate cyclase. A precursor of norepinephrine (dopa) and a metabolite (3,4-dihydroxymandelic acid) did not affect adenylate cyclase activity. Thus, the receptor exhibits the specificity of an a receptor. The potency order [a-meth-            HOF 100¢ 90 oOo wn Ow Oo Oo 9 wn Oo   £ oO 100¢ PERCENT OF CONTROL ACTIVITY oO wo oO oO ~ oO T a 3 x3 oF m @ wn Oo T i =z z m     Po Oo 8 i” 10° ie Gt . CONCENTRATION (M) Fic. 2. Inhibition of (A) basal and (B) PGE)-stimulated adenylate cyclase activity by adrenergic compounds. Each reaction contained 100 pg of homogenate protein and one of the following compounds at the concentrations indicated: (—)-norepinephrine ((—)-NE), (—)-epi- nephrine bitartrate (EPI, bitartrate concentration adjusted with so- dium salt to 0.1 mm in all tubes), dopamine (DA), (~)-phenylephrine (PE), (+)-norepinephrine ((+)-NE), and (—)-isoproterenol (SO). One hundred per cent corresponds to the following specific activities {(picomoles of [”P]cAMP/min/mg of protein) for the compounds listed: (A) 11.1, 10.6, 10.4, 10.3, 9.0, 10.5, respectively; and (B) 142, 167, 144, 191, 167, and 155, respectively. 1916 TABLE Il Concentrations of adrenergic compounds required for inhibition of NG108-15 adenylate cyclase activity Adenylate cyclase activity of homogenates (average 105 pg of protein/reaction mixture) was assayed with or without 10 pm PGE, in the presence of 10 to 12 concentrations of each compound in duplicate reaction mixtures. Half-maximal inhibitions (ICso + S.E.) were found from the resultant concentration curves as described under “Experi- mental Procedures.” Numbers in parentheses refer to the number of experiments averaged. Otherwise, values listed refer to the most reliable experiment. ICs Compound eo Basal PGE, pM Clonidine 0.1\" a-Methyl-(+)-norepineph- 0.2 0.1 rine (—)-Norepinephrine 0.4 + 0.1 (9) 0.6 + 0.2 (2) (—)-Epinephrine 0.5 + 0.03 (3) 0.3 Dopamine 5 20 Oxymetazoline 9 (—)-Phenylephrine 20 30 (+)-Norepinephrine 30 160 (-)-Isoproterenol 60 30 (+)-Methoxamine 80 70 (—)-Dopa >100° (—)-3,4-Dihydroxyman- >100° delic acid Serotonin >100° >100° @ Maximum inhibition less than that for catecholamines. * Little or no effect at 100 po, the highest concentration tested. ylnorepinephrine > norepinephrine > phenylephrine > meth- oxamine] suggests that NG108-15 a receptors resemble pre- synaptic a, receptors more than postsynaptic a receptors of smooth muscle (33-35). Average apparent Hill coefficients obtained from the de- pendence of inhibition of basal and PGE,-stimulated adenyl- ate cyclase on ligand concentration were as follows: (—)-nor- epinephrine, 0.71 + 0.04 (n = 9) and 0.91 + 0.16 (n = 2), respectively; and (—)-epinephrine, 0.72 + 0.11 (n = 3) and 0.67 + 0.16 (n = 2), respectively. The average apparent Hill coef- ficient for other ligands was 0.8. These results suggest either heterogeneity of receptors or adenylate cyclase or negative cooperativity in ligand-receptor interactions and/or in the functional coupling of the [ligand-receptor] complex with adenylate cyclase. Therefore, the ICso values (Table 1) do not necessarily approximate dissociation constants for the [ligand receptor] complexes. The inhibition of adenylate cyclase by norepinephrine was blocked by the reversible a receptor antagonists dihydroer- gotamine and phentolamine as well as the irreversible a receptor antagonist phenoxybenzamine, but not by the B receptor antagonist propranolol (Fig. 3). The a antagonist property of phentolamine and phenoxybenzamine could be demonstrated only in the presence of 10 to 50 pM naloxone, a - specific opiate receptor antagonist. This is because phentol- amine or phenoxybenzamine also were weak activators of the opiate receptor and thereby inhibited NG108-15 adenylate cyclase (half-maximal inhibition at 2M or 5 uM, respectively). These compounds are known to interact with rat brain opiate receptors (36). The apparent dissociation constants (Kpepp) of various re- ceptor antagonists in reversing norepinephrine-dependent in- hibition of adenylate cyclase are shown in Table III. The specificity of the receptor for antagonists is consistent with that of an a receptor, with dihydroergocryptine, dihydroer- gotamine, and yohimbine the most potent tested (Kpspp 0.005 to 0.07 pM). The Knapp of phentolamine (0.2 pM) was higher Regulation of Adenylate Cyclase by a-Adrenergic Receptors                                       BLPHENTOLAMINE + 2 D.PHENTOLAMINE SOuM NALOXONE       —NE 8 4+NE 6 > 4 4 i 12 D.PHENOXYBENZAMINE + 5 504M NALOXONE oe & 10 Dp 7 z 8 = . & 6 = 4 om AS 4 O12 F, PROPRANOLOL 3 = alo —NE a    5 4 6 5 4 0 7 6 -LOG ANTAGONIST MOLARITY Fic. 3. Effect of adrenergic receptor antagonists on the norepi- nephrine-dependent inhibition of basal NG108-15 adenylate cyclase. Reaction mixtures without (@) or with (©) 10 um (—)-norepinephrine (NE) contained 108 pg of homogenate protein and receptor antagonist at concentrations indicated. TaBLe Ill Apparent dissociation constants of receptor antagonists in blocking norepinephrine-dependent inhibition of adenylate cyclase activity Basal adenylate cyclase activity was assayed with or without 10 pM (—)-norepinephrine (NE) in the presence of different concentra- tions of antagonist. The concentrations (ECso) resulting in half-max- imal reversal of norepinephrine inhibition were used to calculate the apparent dissociation constants Kpapp of the antagonist from the equation (37) Kospp = ECso/(1 + [NE]/Ksapp), where Ksapp is the apparent dissociation constant for norepinephrine-a receptor binding, assumed here to be 0.4 uM (Table I). Compound Koapp eM 9,10-Dihydro-a-ergocryptine* 0.005 9,10-Dihydroergotamine” 0.01 Yohimbine 0.07 Phentolamine“ 0.2 Phenoxybenzamine” 0.2 WB-4101 0.2 Fluphenazine 0.2 (+)-Propranolol >100° Bulbocapnine >100° Atropine >100° Naloxone >100° * Mixed agonist-antagonist effect noted. > Mixed agonist-antagonist effect noted in some but not all experi- ments. : © Naloxone (50 4M) added to reaction mixtures to block opiate receptor activation. 4 Trreversible antagonist. © Little or no effect at 100 pM, the highest concentration tested. than that found in other systems (about 0.01 pm), but is identical with that found for the reversal of a-adrenergic inhibition of platelet adenylate cyclase (38). Fluphenazine, 4 potent antagonist of dopamine receptors in the central nerv- ous system (39), blocked NG108-15 receptors with a Knapp of 0.2 uM, which is consistent with the a antagonist property of Regulation of Adenylate Cyclase by a-Adrenergic Receptors phenothiazines. The receptor antagonists propranolol (f re- ceptors), bulbocapnine (dopamine receptors), atropine (mus- carinic acetylcholine receptors), and naloxone (opiate recep- tors) had little or no effect on norepinephrine-dependent inhibition of adenylate cyclase at concentrations of 100 um or less. Responses of Parent Cell Lines of the Hybrid Line NG108- 15—As shown in Table IV, adenylate cyclase activity in ho- mogenates of C6BU-1 rat glioma cells was stimulated by norepinephrine; however, when the f receptors of these cells were blocked by propranolol, norepinephrine had no effect on adenylate cyclase activity. However, adenylate cyclase of N18TG-2 mouse neuroblastoma cells was inhibited by nor- epinephrine, but slightly less than that of the hybrid cells. This suggests that expression of « receptors in the hybrid cells is a property derived from the neuroblastoma parent. Effect of GTP—Hormonal stimulation of adenylate cyclase has been shown to require low concentrations of GTP (see Ref. 40 for review). To determine whether a receptor-me- diated inhibition of NG108-15 basal adenylate cyclase requires GTP, a washed particulate fraction was assayed with or with- out GTP in a system in which the ATP concentration was reduced to 0.1 mm and ATP synthesized by phosphorylation of adenosine was used to reduce the level of guanine nucleotide contaminants. In the absence of added GTP (Fig. 4A), nor- epinephrine did not inhibit the initial rate of cAMP synthesis between 0 and 4 min, but norepinephrine-dependent inhibi- tion slowly appeared during further incubation. However, in the presence of 1 um GTP (Panel B), the lag in the initial rate was abolished and norepinephrine inhibited adenylate cyclase activity approximately 45% at each time tested between 2 and 15 min. The relationship between GTP concentration and the reaction rates between 0 and 4 min and between 4 and 8 min are shown in Panels C and D, respectively. Between 0 and 4 min, three effects of GTP on adenylate cyclase can be seen: (a) stimulation of the initial rate (half-maximal stimulation at 5 X 10°° m GTP); (5) enhancement of norepinephrine-depend- ent inhibition from 6% to 44% (half-maximal effect at 3 x 107 M GTP); and (c) inhibition of activity in the absence or presence of norepinephrine (>10-° m GTP). Between 4 and 8 min, GTP did not stimulate the rate of cAMP synthesis but increased norepinephrine-dependent inhibition from 17% to 46% (half-maximal effect at 3 x 10°’ m GTP) and inhibited activity above 10°\" m GTP. These results indicate that inhi- bition of adenylate cyclase by norepinephrine is dependent upon GTP. TABLE IV Effect of norepinephrine on adenylate cyclase activity of neuroblastoma X glioma hybrid NG108-15 cells and parental cell lines Adenylate cyclase reaction mixtures contained, where indicated, homogenate protein from C6BU-1 cells (subculture 24, 170 pg), N18TG-2 (subculture 12, 146 ug), or NG108-15 cells (190 pg) and 10 #M (—)-norepinephrine, 10 um PGE), or 20 um (+)-propranolol, as indicated. The activities listed are means of duplicate determinations. Parental celis     Neuroblas- iti : neu- toma x Additions Rat glio 7 nen haere glioma hy- . Ni8TG-2 brid eal pmol cAMP/min/mg protein None 43 5.5 77 Norepinephrine 113 4.1 46 PGE, 48 97 128 PGE, + norepinephrine 116 76 94 Propranolol 43 Propranolol + norepi- 43 nephrine                     1917 700 Z A. NOGTP B. 10®m GTP E 6OO} + CONTRO! & CONTROL, Fecal j & 400} + 2 NE S$ s00/ 1 2,300 Ne Ow 3 200+ ol = 100f oO 5 i0 50 5 105 MINUTES é C. 0-4.17 MIN D. 4.17- 8.17 MIN 50+ 450_ f g E F 4Qu £ 40 Z z 305 30m & f Zz 3 207 | 202 @ 10h og 110E Bi ae % INHIBITION So 0 a +s Se $ io 10” 168 mo O 16? 1” 16 6 GTA) (M) Fic. 4. Effect of GTP on inhibition of adenylate cyclase by nor- epinephrine. Reaction mixtures (400 yl) contained 0.1 mm [a-\"PJATP (unlabeled ATP prepared by phosphorylation of adenosine), 129 pg of protein of NG108-15 particulate fraction (30,000 x g, 15 min), and, as indicated, 100 um norepinephrine (NE) and 0 to 10 um GTP. A and B, adenylate cyclase activity in the absence or presence of 1 ym GTP, respectively, as a function of time. C and D, average specific activities between 0 and", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ytqe-c5vj.q4ji", "00000000-0000-0000-A558-C5E2F05F2DB0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Modulation of Synapse Formation by Cyclic Adenosine Monophosphate", "101584910X124", null, "1983", "18 November 1983", "As part of the continued study of the developing nervous system and mechanisms of gene expression conducted by Nirenberg, this article discusses studies with retinal cells, reviews studies on the plasticity of synapses formed by clonal neuroblastoma-hybrid cells with striated muscle cells, and uses monoclonal antibodies and cultured cell systems in new studies of synapse formation and plasticity.", "Articles", "Synapses,Cyclic AMP,Neuromuscular Junction,Neuronal Plasticity", "From Neuroblastoma to Homeobox Genes, 1976-1992", "6", "pages", "Text", "English", "Reprinted with permission from M. Nirenberg, S. Wilson, H. Higashida, A. Rotter, K. Krueger, N. Busis, R. Ray, J. G. Kenimer, M. Adler. \"Modulation of Synapse Formation by Cyclic Adenosine Monophosphate.\" Science 222 (18 November 1983): 794-799. Copyright 1983 American Association for the Advancement of Science.,http://www.sciencemag.org/", "Copyright may apply", null, null, "Modulation of Synapse Formation by Cyclic Adenosine Monophosphate M. Nirenberg, S. Wilson, H. Higashida, A. Rotter K. Krueger, N. Busis, R. Ray, J. G. Kenimer, M. Adler How neurons in the developing ner- vous system form synapses and distin- guish appropriate from inappropriate synapses remains one of the central, unsolved problems in neurobiology. In 1963, Sperry (1) proposed the chemoaf- finity hypothesis; namely, that neurons bear positional labels (that is, molecular addresses) that are recognized by com- plementary molecules on the synaptic target cells and thereby determine the specificity of neuronal connections. He also suggested that two gradients of mol- ecules on retina neurons at right angles to one another, which interact with com- plementary molecules on the target neu- rons in the tectum, might be a mecha- nism for matching synaptic connections and reproducing a point-to-point map of the retina in the tectum. If synapse rec- ognition molecules exist, monoclonal antibody technology should be a power- ful tool for their detection. Many investi- gators are now using this approach. Other mechanisms such as regulation of gene expression by environmental fac- tors such as hormones, neuromodula- tors, transynaptic communication, or molecules secreted by neighboring or other cells surely play important roles in the assembly of synaptic circuits. For example, Le Douarin (2) and Patterson (3) and their colleagues have shown that during development neurons from the neural crest can express either the gene for tyrosine hydroxylase, which cata- lyzes the first step in the pathway for norepinephrine synthesis, or the gene for choline acetyltransferase, which cata- lyzes the synthesis of acetylcholine, de- pending on the presence of an extracellu- lar macromolecule, purified by Weber guidance of neurites by glia (//). (4), which is secreted by other cells, or the extent of depolarization of the neu- ron. In addition, Mudge (5) has shown that the expression of somatostatin. a peptide transmitter or neuromodulator. by dorsal root ganglia sensory neurons is dependent on molecules secreted by nonneural cells. Raff et al. (6) also have shown that fetal calf serum markedly influences the differentiation pathway expressed by glial cells in the central nervous system. Edelman and his colleagues (7) discov ered a neuronal glycoprotein rich in sial- ic acid residues, termed N-CAM (neural cell adhesion molecule), that mediates intercellular adhesion in the absence of Ca2* and probably plays an important role in the development of the nervous system by conserving the topographic relationships between individual neurons or axons (or both) in a set of neurons even though axons may migrate long distances before synapsing. Molecules that mediate Ca?*-dependent intercell” lar adhesion (8) and factors that promol* retina cell adhesion, such as cognin (7). and ligand and agglutinin (/0), also have been described, but little is known about their function in the nervous syst€™ Other mechanisms such as contact £0” ance, chemotaxis, cell survival facie of th The authors are or have been member on? Laboratory of Biochemical Genetics: qa Heart, Lung. and Blood Institute, Nation? ots tutes of Health, Building 36, Room 1C-06. Be Maryland 20205. lection for synchronous or sequential yansmission across two or more synap- 8 that innervate a neuron may also play portant roles in synaptogenesis. \"ye have used monoclonal antibodies jd cultured cell systems to study syn- se formation and plasticity. Some “udies with retina cells are discussed gest, and then studies on the plasticity of ynapses formed by clonal neuroblasto- gatybrid cells with striated muscle lls are reviewed. Adorsal-ventral gradient of protein in ina. Trisler et al. (12) obtained a monoclonal antibody that recognizes a ll membrane protein distributed in a yrge dorsal-ventral topographic gradient in chick retina (Fig. 1). The concentra- tion of antigen detected at the dorsal margin was at least 35-fold higher than that found at the ventral margin of the retina, and the concentration of antigen detected varied continuously arid loga- fithmically with the logarithm of distance along the circumference of the retina fom ventral to dorsal poles of the gradi- ent. Thus, the protein defines a bilateral- ly symmetrical, dorsal-ventral axis of the retina and can be used as a marker of cell position in the retina with respect to the dorsal-ventral axis. The antigen, termed TOP (toponimic), was detected on all cells examined in dorsal and middle reti- na, but more TOP was detected on cells from dorsal retina than on cells from middle retina. The TOP antigen was solubilized and purified by antibody-agarose column chromatography and sodium dodecyl sulfate (SDS}-polyacrylamide gel elec- trophoresis. A single band of protein was obtained with a molecular weight (M,) of approximately 47,000 (/3). TOP was de- lected in optic cups of 48-hour chick embryos (/4), and evidence for a gradi- ent of TOP was found in 4-day embryo retinas. A gradient therefore is generated a neurons are generated in the retina and the gradient is maintained through- out embryonic development and in the adult. Neurons first appear in the central portion of retina and then are added in concentric, ever widening rings. Thus, central retina is the oldest portion of the tetina and peripheral retina is the young- st. How a dorsal-ventral gradient is Senerated as the retina forms and is perpetuated is not known. TOP was detected, in order of de- ‘reasing concentration, in retina, cere- brum, and thalamus; little or no antigen Was found in other parts of the nervous System or in other tissues. Gradients of TOP were found in chicken, turkey, duck, and quail retina, but the antigen Was not detected in rat, Xenopus laevis, Rana pipiens, or goldfish retina. The antigenicity of TOP is destroyed by trypsin; however, cells dissociated with trypsin from dorsal, middle, or ven- tral retina, cultured separately or com- bined in various proportions, continue to synthesize the antigen and accumulate the amount of TOP that would be expect- ed with cells from the corresponding monoclonal antibodies are specific for a single class of cells in retina such as photoreceptors, horizontal neurons, Miller cells, or ganglion neurons, or for a family of cells such as those in the inner nuclear layer of retina. Another monoclonal antibody, A,B; (/8), recog- nizes unidentified gangliosides with sial-   Summary. Synapses between neuroblastoma-hybrid cells and myotubes. exhibit a high degree of plasticity. Increase of cyclic adenosine monophosphate (AMP) levels of the hybrid cells for several days results in the appearance of functional voltage- sensitive Ca?* channels, which are required for evoked secretion of acetylcholine. The results show that cyclic AMP regulates. synaptogenesis by regulating the expression of voltage-sensitive Ca2* channels, and suggest that cyclic AMP affects posttranslational modifications of some glycoproteins and cellular levels of certain proteins.   region in the intact retina in ovo. Thus, the number of antigen molecules detect- ed on retina cells after 10 days in culture depends on the prior position of the cells in the intact retina. These results suggest that the retina is composed of a gradient of cells that express different amounts of TOP, de- pending on the position of the cells in retina along the dorsal-ventral axis of the retina. The function of TOP is not known. Monoclonal antibodies that rec- ognize an anterior-posterior gradient of molecules in retina were looked for, but were not found (/5). However, the dem- onstration that TOP is a cell membrane protein and is expressed on the basis of cell position in the retina, rather than cell type, suggests that TOP miay play a role in the specification of positional informa- tion in the retina. We are trying to clone complementary DNA (cDNA) corre- sponding to TOP messenger RNA (mRNA) to use to define the amino acid sequence of TOP and to explore the mechanism of regulating TOP expres- sion. Other monoclonal antibodies to reti- na. Grunwald et al. (16) showed that antibody 13H9 recognizes cell mem- brane protein detected on most or all cells in retina; however, antigen was not detected on neurons or glia in other parts of the nervous system. It is of interest to determine whether the protein specifies a compartment of cells; that is, functions as a cell adhesion molecule that enables retina cells to adhere preferentially to one another rather than to other cells. Three monoclonal antibodies recognize antigens that are restricted to the outer synaptic layer of retina (113F4, 92A2, and 18B8); another antibody (16G6) rec- ognizes antigen in both the inner and outer synaptic layers of retina. Antibody [8B8 binds to glycoproteins and uniden- tified species of gangliosides (/7). Other ic acid residues and glycoproteins (/7) that are markers of neurons and some glia (6, 18). Cultured retina cells, Chick retina contains abundant nicotinic and musca- rinic acetyicholine receptors that mostly are distributed in layers within the inner synaptic layer of retina (/9). Cultured neurons dissociated from chick embryo retina also express choline acetyltrans- ferase and acetylcholine receptors, and the neurons form approximately as many synapses in vitro (1.5 x 10° synapses per milligram of protein) as they do in ovo, as judged by electron microscopy (20). The specificity of synapse formation by retina neurons was examined by co- culturing dissociated chick embryo or rat retina neurons with inappropriate synap- tic partner cells such as striated muscle cells that possess many nicotinic acetyl- choline receptors. Retina neurons form functional synapses with most striated muscle cells in 90 minutes, but these synapses are transient and slowly disap- pear over a period of 5 to 10 days (2/- 23). Cholinergic neurons that are able to synapse with myotubes first appear in chick retina on day 6 of embryonic de- velopment, are most abundant on day 8 and comprise approximately 8 percent of the retina cell population, and lose the ability to form synapses with myotubes by day 16 of embryonic development (23). However, synapses between retina neurons increase during the culture peri- od and remain abundant after all synap- ses between retina neurons and muscle cells terminate. Two processes contribute to the turn- over of retina neuron synapses with myotubes. First, retina neurons are able to form synapses with striated muscle cells only for a short time during devel- opment (23); and second, synapses be- tween retina neurons and myotubes ter- minate because retina neurons preferen- tially adhere to other retina cells rather than to myotubes (2/). Preparations of neurons from chick embryo spinal cord, which presumably contain motor neurons that normally in- nervate striated muscle cells, also form synapses with cultured muscle cells, but the number of synapses remains con- stant during subsequent culture (22). Therefore, spinal cord neurons either form stable, long-lived synapses with muscle cells or attain a steady sf&te wherein the rate of synapse formation is equal to the rate of synapse termination. These results show that inappropriate synapses between retina neurons and myotubes form rapidly and are terminat- ed slowly, that synapses formed by cho- linergic neurons from retina and spinal cord turn over at different rates, and that differences in synapse turnover rates of two populations of synapses can result in the selective retention of one population and the loss of the other. Clonal Neuroblastoma Cell Lines Adult neurons do not divide; however, the establishment of clonal lines of neu- roblastoma cells from.a transplantable mouse neuroblastoma tumor (C-1300) of spontaneous origin provided a source of relatively homogeneous populations of dividing cells of neural origin (24). Char- acterization of these (24) and other (25) clonal lines of C-1300 neuroblastoma ‘showed that the cells have excitable membranes (26) and other neural proper- ties, and that the expression of genes for neural properties is inherited and thus can be perpetuated. Clonally inherited differences in phenotype also were found; for example, some neuroblastoma cell lines synthesize acetylcholine (25), others catecholamines; but most do not synthesize these compounds. Cells from neuroblastoma lines that synthesize acetylcholine were cocul- tured with striated muscle cells, which possess abundant nicotinic acetylcholine Table 1. Cell line phenotypes [see (32, 34, 35)].     K*t-Dependent Vesicles Cell ACh* receptor lines forma- Ca?* PHJACh = Small Large aggregation Synapse (No.)} tion dense . uptake release clear core protein 5 + +++ +++ + + + +++ 3 + + + + + + + 2 + - - + + + - 5 + ++ - + + + -ort+ 3 + ++ + + ~ - -ort 9 - - -   * Acetylcholine (ACh).   18 F T T T Outer segments = N T        Central segments > ['25)]F(ab’). (pmole/mg-protein) co T                 20.0 10.0 1.0 0.1           2 4 6 8 Retina section   § 10 50 Maximal distance (%) Fig. 1. Geometry of the TOP gradient in 14-day chicken embryo retina (/2). (A) Specifically bound '25J-labeled F(ab’), (pmole per milligram of protein) is shown on the ordinates in (A) and (B) and within the appropriate segment of retina tested. (B) The circumference of the retina is 14.5 mm which corresponds to 100 percent on the abscissa. (A) Strips of retina from ventroanterior (0 percent) to dorsoposterior (100 percent) retina margins were removed, and each was cut into nine segments and assayed for TOP. (1) Strips of retina from anterior (0 percent) to posterior (100 percent) margins of the retina perpendicular to the choroid fissure were prepared and assayed as above; (O) data from panel A. 79% receptors, or with cardiac muscle cells that have muscarinic acetylcholine re. ceptors. However, for several years we, and others, failed to detect synapses. We thought that these cell lines might not express all genes for proteins that might be required for synaptic communication, and therefore we fused. neuroblastoma cells with other cells and generated many somatic hybrid cell lines (26). Hybrid cell lines were found that express new neural properties not detected with parental cells (27, 28); with other hybrid cell lines some neural properties were extin- guished. Eventually five cell lines were found that synthesize acetylcholine and form many synapses with cultured myo- tubes (32, 33). The early attempts to form synapses with neuroblastoma cells failed for two reasons. (i) The extent of neural maturation and ability of cells to form synapses are regulated and are highly sensitive to environmental condi- tions, making it necessary to find condi- tions that yield populations of ‘‘differen- tiated’ cells. (ii) Most, but not all, of the cholinergic neuroblastoma cell lines that were tested do, indeed, lack reactions that are required for synapse formation. Empirically, we found that populations of neuroblastoma or hybrid cells can be shifted from a poorly differentiated, syn- apse incompetent state, to a well-differ- entiated, synapse competent state. by increasing intracellular levels of cyclic adenosine monophosphate (AMP) for days. Selection for nondividing cells also yields well-differentiated populations of cells. In Fig. 2 are shown photomicrographs of cells from four of the five cel! lines that form many synapses with striated muscle cells. The NBr10-A and NBr20-A cells originated by fusion of mouse neu: roblastoma NI8TG-2 (26) with clonal BRL30-E rat liver cells, NCB-20 cells (29) resulted from fusion of NI8TG-? cells with fetal Chinese hamster brain cells, and NG108-15 (30) resulted from fusion of N18TG-2 with C6BU-1 (28) 1 glioma cells. Few neurites or synapses were found when cells were in the log* rithmic phase of growth. However. &¥ posure of cells for 7 days to 1 mM dibur tyryl cyclic AMP, which promotes newt ite extension, and to 1 percent (rathe! than 5 percent) fetal bovine ser which reduces neurite retraction. yields cells with neurites that can be more tha\" 2 mm in length (3/). Other cell lines have high concent tions of acetylcholine, adhere well \" myotubes, but do not form synape (32). A summary of phenotypes of “ lines with or without synaptic defects shown in Table 1. The NBr10-A. NB SCIENCE. YOR” ,, NCB-20, NG108-15, and NS-26 neu- ‘splastoma cells (25) form many synap- ges with cultured myotubes (32, 33), syn- tnesize acetylcholine (32), have function- 1 voltage-sensitive Ca** channels (34), have small clear vesicles approximately ,) nm in diameter and large dense-core yesicles 180 nm in diameter (4/), and ;lease acetylcholine into the medium jad a protein that stimulates the aggrega- iun of nicotinic acetylcholine receptors on myotube plasma membranes (35). cells from three lines take up Ca’* ions slowly (34) and secrete little acetyicho- ine when depolarized by 80 mM K* ions, and form few synapses with muscle cells. Cells from two lines lack functional yoltage-sensitive Ca?* channels (34) and do not form synapses. Cells from five lines take up Ca?* when depolarized by K” ions but do not respond by secreting more acetylcholine (32), and few or no synapses were found. These cells lack a Ca’*-dependent acetylcholine secretion reaction (or reactions); however, acetyl- choline is secreted into the medium in the basal, unstimulated state. Cells from three lines have small clear vesicles but lack large dense-core vesicles and func- tional protein that induces nicotinic ace- tylcholine receptor aggregation on myo- tube membranes (35), and form few or no synapses. Nine additional cell lines have little or no choline acetylitransferase ac- tivity, and therefore they synthesize lit- tle or no acetylcholine (32) and do not form functional synapses with striated muscle cells. Regulation of synaptogenesis. Thus far, we have identified 12 species of receptors that are expressed by NG108- 1S cells, including receptors for prosta- glandin E, (PGE,) (36, 37), prostaglandin F, (PGF;) (36), adenosine (38), Met- enkephalin (36), alpha-2-adrenergic re- ceptors (39), depolarizing muscarinic acetylcholine receptors (40), serotonin and LSD receptors (29), and receptors for bradykinin, neurotensin, angiotensin Il. and somatastatin (32), and have de- fined cell responses to the ligands for these receptors. Some receptors, such as those for PGE,, mediate activation of adenylate cyclase; other receptors such as Met-enkephalin receptors, muscarinic depolarizing acetylcholine receptors, and alpha-2-adrenergic receptors medi- ate inhibition of adenylate cyclase. Increase of cyclic AMP in neuroblas- toma or hybrid cells for 5 to 7 days, obtained either by treating cells with PGE, to increase the endogenous rate of cyclic AMP synthesis or by inhibition of cyclic nucleotide phosphodiesterase with dibutyryl cyclic AMP, or theophyl- line. resulted in increases in the percent- 8 NOVEMBER 1983 Table 2. Effect of culture conditions on synap- togenesis and acetylcholine secretion by NG108-15 cells. Each value is the mean of values obtained from more than 75 myotubes. (Data from (32)]     Myo- Syn- tubes aptic : we: with re- Culture conditions syn- sponse apses fre- (%)  quency* Control 15 0.7 1 mM dibutyryl cyclic 55 14 AMP 1 mM theophylline 64 10 10 pM PGE, 63 il 10 pM PGE, + 1 mM 98 32 theophylline   *The number per minute per myotube. age of myotubes tested that were inner- vated and the rate of spontaneous secre- tion of acetylcholine from NG108-15 cells at synapses (32) (Table 2). Presum- ably, each depolarizing response of a myotube to acetylcholine is due to the spontaneous secretion of acetylcholine from a single NG108-15 vesicle. NG108- 15 cells and myotubes were cocultured and treated for 5 to 7 days with the compounds shown; then myotubes were assayed for synapses by intracellular mi- croelectrode recording. Treatment of cells with 1 mM dibutyryl cyclic AMP, 1 mM theophylline, or 10 pM PGE, in- creased the percentage of muscle cells tested that were innervated from 15 to NBri0A     NBr20A approximately 60 percent and increased 14- to 20-fold the frequency of spontane- ous synaptic responses of myotubes (the miniature end-plate potential frequency). Treatment of cells with 10 »4 PGE, and 1 mM theophylline resulted in innerva- tion of 98 percent of the myotubes tested and increased the frequency of synaptic responses of myotubes 45-fold. No im- mediate effect of these compounds on the cell membrane potential or rate of acetylcholine secretion was detected. Half-maximal increases in synapses and rate of spontaneous acetylcholine secre- tion at synapses were observed when cellular cyclic AMP levels were in- creased for 1 to 2 days; maximal in- creases were obtained when cells were treated for 3 to 5 days (32). In other experiments, NG108-15 ceils weré incubated with PGE, theophylline, dibutyryl cyclic AMP, or PGE, and theo- phylline for 5 to 7 days; then the com- pounds were withdrawn and cells were incubated for an additional 4 to 14 days to determine whether the effects on syn- apses and acetylcholine secretion were reversible. On withdrawal of the com- pounds, synapses and acetylcholine se- cretion gradually returned to control val- ues in 7 to 11 days (32). Thus, the effects of the compounds on synapses are ex- pressed slowly and are long-lived. Cyclic AMP levels of NG108-15 cells increase markedly in the presence of 10 pM PGE, and | mM theophylline and      Fig. 2. Neuroblastoma hybrid cells from lines that form many synapses with cultured myotubes were treated for 7 days with 1 mM dibutyryl cyclic AMP and the concentration of fetal bovine serum was reduced from 5 to | percent between day 5 and day 7. The bar in the lower right hand panel corresponds to 50 ym in each panel. [Data from (32)] 797 remain higher than those of control cells for seven or more days. Intracellular acetylcholine in NG108-15 cells also in- creases eight- and threefold when cells are treated for 3 days with PGE, and theophylline or dibutyryl cyclic AMP, respectively (32). NG108-15 cells treated with dibutyryl cyclic AMP (41) or PGE, and theophylline (32) for five or more days contain many large dense-core vesi- cles and small clear vesicles, whereas control cells contain few vesicles. The cyclic AMP-dependent increase in intra- cellular acetylcholine is due, at least in part, to an increase in the number of acetylcholine storage vesicles in cells. Depolarization of NG108-15 or NBr10- A cells with 80 mM K* ions, in place of 80 mM Na* ions, has no effect on the rate of acetylcholine secretion by un- treated NG108-15 or NBri0-A cells. However, cells gradually are shifted from an unresponsive to a responsive state with respect to depolarization-de- pendent secretion of acetylcholine when treated for 5 to 7 days with | mM dibu- tyryl cyclic AMP or 10 pM PGE, and 1 mM theophylline. Half-maximal and maximal increases in acetylcholine se- cretion due to cell depolarization were obtained when NG108-15 cells were treated with | mM dibutyryl cyclic AMP for 2 and 5 days, respectively (42). Depolarization of nerve terminals is known to activate voltage-sensitive Ca?* channels; Ca?* ions then flow into the cytoplasm of axon terminals and in- crease the rate of secretion of transmitter at the synapse. We therefore examined the effect of prolonged elevation of cy- clic AMP levels of NBr10-A or NG108- 15 cells on voltage-sensitive Ca** chan- nel activity. Four kinds of assays were used (34). “Ca?* flux, net uptake of Ca?* by cells was measured with a Ca’* specific electrode, Ca?* fluxes were de- termined in the presence of murexide by a spectrophotometric assay with a stopped-fiow apparatus, and Ca’* action potentials of cells were assayed by intra- cellular microelectrode recording. We found by each method of assay that logarithmically dividing control cells have little or no voltage-sensitive Ca?* channel activity; however, prolonged el- evation of cellular cyclic AMP activation of adenylate cyclase of cells with PGE), or by inhibition of cyclic nucleotide phosphodiesterase with dibutyryl cyclic AMP or theophylline, gradually results in the acquisition of functional voltage- sensitive Ca** channels by cells. Assay of Ca2* action potentials elicited by elec- trical stimulation of single cells with in- tracellular microelectric recording showed that most untreated NG108-15 or NBr10-A cells lack functional voltage- sensitive Ca’* channels. However, Ca** action potentials were found in 100 per- cent of the cells tested that had been treated for four more days with dibutyryl cyclic AMP. As shown in Fig. 3, ““Ca?* uptake by logarithmically dividing, control NBr10- A cells is not affected by depolarization of cells with 80 mM K*. However, cells that had been treated for 7 days with 10 uM PGE, and | mM theophylline or with 1 mM dibutyryl cyclic AMP re- spond to depolarization by 80 mM K* with a rapid influx of “Ca?* via voltage- sensitive Ca2* channels (34). Depolar- ization-dependent *Ca?* uptake is in- hibited completely by 1 x 10-*M D-600 (half-maximal inhibition was obtained with 9 x 10°7M D-600), an alkaloid known to inhibit voltage-sensitive Ca?* channels and slow Na* channels. “*Ca?* uptake also is inhibited by La’*, Co?*, and Ni?* ions. Exposure of NG108-15 cells to PGE, increases cellular cyclic AMP levels within seconds; however, no immediate effects of PGE,, PGE, and theophylline, or dibutyryl cyclic AMP on voltage-sen- sitive Ca** channel activity were detect- ed. Half-maximal and maximal voltage- sensitive Ca?* channel activity were ex- pressed by cells that had been treated   PGE, + theophylline = N a T       1.00 ‘Abibutyy! cyclic AMP 0.75 0.50   0.25 Change in 45Ga2* uptake (nmole/mg-protein) Control cells     0 2 4 6 8 10 Time (minutes) Fig. 3. The effect of culture conditions on the expression of functional voltage-sensitive Ca2* channels of NBrl10-A cells. Uptake of *5Ca?+ due to activation of voltage-sensitive Ca?* channels of untreated logarithmically dividing control NBr10-A cells, cells cultured for 6 days with 1 mM dibutyryl cyclic AMP, or 10 »M PGE, and 1 mM theophylline. The cells were depolarized with 80 mM K* (in place of 80 mM Na‘). Values for *°Ca’* binding to cells or uptake at 5.4mM K*, which were not inhibited by 100 pM D-600 and were not mediated by voltage-sensitive Ca?* channels, were subtracted from the val- ues shown. Uptake of Ca?* dependent on cell depolarization was completely inhibited by 100 Af D-600. [Data from (34)] with PGE, and theophylline or dibutyry| cyclic AMP for 2 and 4 days, respective. ly. Relatively weak voltage-sensitive Ca?* channel activity appears in untreat. ed NBr10-A cells when cells form con- fluent monolayers. Thus, cell concentra. tion or adhesive interactions between cells also regulates the expression of voltage-sensitive Ca?* to some extent, Nitrendipine and other dihydropyri- dine derivatives inhibit voltage-sensitive Ca** channels of smooth muscle (43), striated muscle (44), and cardiac muscle (45), and specific binding sites for 37H. labeled nitrendipine have been found in these tissues and in brain (46). The ni- trendipine receptors are thought to be part of the voltage-sensitive Ca?* chan- nel complex, perhaps functioning as reg- ulators of channel activity. Kongsamut and Miller have shown that “°Ca?* uptake by NG108-15 cells mediated by voltage-sensitive Ca’* channels is inhibited by nitrendipine (47). We have confirmed this and find that NBr10-A cells are inhibited half- maximally by 3 nM nitrendipine. A sin- gle class of specific binding sites for 3H. labeled nitrendipine was found in mem- branes from NBr10-A cells that had been treated with PGE, and theophylline with a dissociation constant, estimated by Scatchard analysis, of 2 x 107'°M, which is similar to values reported for other tissues (43, 45-46). The maximum number of specific nitrendipine binding sites was estimated to be 61 fmole per milligram of NBri0-A membrane pro- tein, which is equivalent to approximate- ly 16,000 specific sites for nitrendipine per cell. In contrast, few or no specific binding sites for *H-labeled nitrendipine were detected in membranes from un- treated, logarithmically dividing NBrl0- A cells. These results show that cyclic AMP regulates the number of specific nitrendipine receptors per cell. Specific binding sites for >H-labeled nitrendipine also were not detected in membranes prepared from two lines of hybrid cells (SB21B-1 and SB37-B) that lack func: tional voltage-sensitive Ca?* channels and do not synapse with muscle cells. Cyclic AMP increases the probability of opening Ca’* channels of cardiac muscle cells (48); however, responses cyclic AMP are rapid and thus differ from the slow effects found with NBrl0- A cells. The molecular weights of nitrendipin¢ receptors in intact membranes of smooth muscle (49), transverse tubule ment branes of skeletal muscle, and cerebr cortex synaptic membranes (44) wet estimated by radiation inactivation targe! analysis to be 278,000, 210,000, and 210,000, respectively. Available infor- mation suggests that the nitrendipine re- ceptor complex is a glycoprotein with N- acetylglucosamine or sialic acid residues (or both) (50). Nitrendipine receptors of smooth and cardiac muscle were report- ed to be covalently labeled with a radio- active affinity label analog of nitrendi- pine, *H-labeled 2,6-dimethy1-3,5-di- carbomethoxy - 4 - (2 - isothiocyanato- phenyl)-1,4-dihydropyridine; labeled pro- tein then was solubilized and fractionat- ed. A peak of labeled protein with a molecular weight of 45,000 was identi- fied (49). These results suggest that the molecular weight of voltage-sensitive Ca?* channel in membranes is 210,000 to 778,000, that each channel is composed of two or more subunits, and that one subunit is a protein with a molecular . weight of 45,000, which binds nitrendi- pine. NG108-15 cells that had been grown with or without 10 wi PGE, were incu- bated with (>S]methionine to label the protein; the *S-labeled glycoproteins then were solubilized and fractionated by wheat germ agglutinin-, ricin-, or len- til-lectin column chromatography and by two-dimensional gel electrophoresis (51). Elevation of cellular cyclic AMP levels resulted in the disappearance of some *°S-labeled glycoprotein", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7yk5~wkvv_gzc6", "00000000-0000-0000-7F2D-A0EF7A242D21", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "A Biochemical Characteristic of Ascites Tumor Cells", "101584910X126", null, "1959", "December 1959", "This article was published while Marshall Nirenberg was a post-doctoral fellow of the American Cancer Society and includes previously unpublished results of experiments completed with J.F. Hogg on glycogen levels in ascites tumors.", "Articles", "Neoplasms,Phosphorylases", "Biographical Information", "6", "pages", "Text", "English", "Reproduced with permission of the American Society for Biochemistry and Molecular Biology.", "Copyright may apply", null, null, "Tue Jovrnat or BrovogicaL CHEMISTRY Vol. 234, No. 12, December 1959 Printed in U.S.A, A Biochemical Characteristic of Ascites Tumor Cells MARSHALL W. NIRENBERG* From the National Institute of Arthritis and Metabolic Diseases, National Institutes of H ealth, Public Health Service, United States Department of Health, Education and Welfare, Bethesda, Maryland (Received for publication, July 2, 1959) Glycogen and glycogen phosphorylase are almost ubiquitously distributed among mammalian tissues. The enzymatic activa- tion of phosphorylase is hormonally regulated, and the following scheme summarizes the major findings with liver phosphorylase (1, 2). ATP, Mgt* epinephrine __(?) | glucagon Adenosine-3’-5'-cyclic phosphate { ) 'dephosphophosphorylase : ikinase + ATP + Mgtt inactivating enzyme phosphorylase <2 dephosphophosphorylase Jt seemed reasonable to suppose that a study of phosphorylase and its activating enzymes in tumor tissues might yield some information bearing upon (a) the characteristically high rate of glucose utilization by tumors (3), and (b) the degree of control exerted by certain hormones over these enzymes in neoplastic cells. . In previous studies (4-6) various aspects of carbohydrate me- tabolism and its hormonal control were investigated in several strains of ascites tumors and an absence of glycogen was noted in one of the tumors. In further preliminary reports many different ascites tumors were shown to possess little or no glyco- gen and glycogen phosphorylase (7, 8). It seemed striking that cells with such large capacities to utilize glucose should be unable to degrade glycogen appreciably and should therefore be free from this hormonally controlled regulatory mechanism. The present report contains more com- plete information concerning these findings. EXPERIMENTAL The tumor strains and the strains of host mice used in this study are listed in Table I. The majority are ascites tumors; exceptions are the HeLa carcinoma grown in tissue culture and the Rous sarcoma, a solid tumor. The method of harvesting the ascites tumors has been presented previously (5). HeLa cells grown both on glass and in suspension were the generous gift of Dr. Harry Eagle. The HeLa cells were harvested by centrifuging the cells at 200 x g for 5 minutes. The cells were washed with Earle’s solution and were recentrifuged. Cells were homogenized at 0-5° by the following techniques. Sonic disintegration for 5 to 10 minutes with a Raytheon 10 ke. sonic oscillator; cell shearing in a motor-driven, all-glass Potter- Elvejhem homogenizer (Kontes Company); the application and * Postdoctoral Fellow of the American Cancer Society. rapid release of a pressure greater than 1500 Ib. per sq. in. of Nz in a small stainless steel tank at room temperature (9); hand homogenization in an all-glass Tenbroeck homogenizer (Kontes Company); and, agitation in a Nossal shaker (10) for 90 seconds. The degree of homogenization was followed microscopically in all experiments. Glycogen (Nutritional Biochemicals Company) was precipi- tated three times from ethanol before use. Dipotassium glucose 1-phosphate was obtained from Schwarz Laboratories. Glucose 1,6-diphosphate was a gift from Dr. Victor Ginsburg, crystalline glucagon from Dr. 0. Behrens. Glucose 6-phosphate dehydro- genase was prepared from ycast (11). p1-Epinephrine bitartrate was obtained from Winthrop Laboratories; some experiments were performed with epinephrine chloride solution (1:1000) ob- tained from Parke, Davisand Company. Caffeine was obtained from the Eastman Chemical Company. Glycogen was determined by the method of Stadie, et al. (12) and by the anthrone method (13). Phosphorylase was assayed routinely by the method of Sutherland and Wosilait (14). The Cori et al. phosphorylase assay (15) was used where stated. The method of Rall et al. (16) was slightly modified for dephos- phophosphorylase activation experiments and details are pre- sented with the experimental data. Phosphorylase inactivating enzyme was assayed by the technique of Wosilait and Sutherland (17). Protein was determined by the method of Bucher (18) with crystalline bovine albumin used as the standard. Purified preparations of dog liver phosphorylase, dephosphophosphorylase and dephosphophosphorylase kinase were the generous gift of Dr. Earl Sutherland. Histochemical assays were very kindly performed by Dr. Samuel Spicer. RESULTS Polysaccharide Content of Tumor Cells The total polysaccharide content of freshly harvested tumors is given in Table II. The average concentration of polysac- charide in each tumor was approximately 5 umoles of glucose equivalents per g of protein. This may be compared with normal rat liver containing approximately 1400 umoles of glu- cose equivalents per g of protein. No glycogen was found in tumor cells even after incubating cells aerobically for one hour in Krebs-Ringer-bicarbonate buffer containing 10% glucose.’ Since 3 to 8% of ascites tumor cell populations consisted of normal erythrocytes and leukocytes, it was of interest to de- termine whether polysaccharides were present in the normal cells, in the tumor cells, or distributed among both. Fresh suspensions of the Ehrlich carcinoma, hepatoma, and the Krebs- 1J. F. Hogg and M. W. Nirenberg, unpublished results. 3088 December 1959 2 carcinoma were stained with the periodic acid-Schiff reagent (19). Glycogen granules were visible in the normal polymor- phonuclear leukocytes contaminating the ascites tumor suspen- sions, and the glycogen could be removed by treatment with diastase. No glycogen could be demonstrated in the tumor cells. The tumor cells exhibited a light reddish, diffuse color after staining which did not disappear after diastase digestion. It is likely that the tumor cells contain very low levels of uni- dentified polysaccharides, probably mucopolysaccharides. The chemical analyses for polysaccharide contained by the mast cell tumor revealed somewhat higher levels than the other tumor cells. Bright red granules were found in the mast cell tumor after periodic acid-Schiff staining. The granules did not disappear after diastase digestion; hence the polysaccharide was not glycogen. Since this tumor strain is known to synthesize heparin (20) it seemed likely that the granules were aggregates of heparin. This assumption was tested by the use of the azure A metachromatic stain, specific for acidic polysaccharides. The granules gave a positive reaction. Therefore, a close correlation was obtained between the chemical and the histochemical assays. Phosphorylase Contents of Tumor Cells The phosphorylase contents of the tumor cells are presented in Table III. Normal mouse liver contains an active phos- phorylase, whereas the phosphorylase activity of the hepatoma, taken from the same animal was one-tenth to one-twentieth that of liver. Addition of 5’/-AMP did not result in an increased ac- tivity. Similar results were obtained with all of the tumors. Five homogenization techniques were applied in the hope that an increased activity could be obtained. In each case whole homogenates were used. In additional experiments, whole ho- mogenates were centrifuged at 100 x g for 2 minutes to remove debris, and the supernatant suspensions were used. No signifi- cant changes in phosphorylase activity resulting from different methods of homogenization could be found. Incubation of the reaction mixture for various intervals of time, up to 2 hours, had no effect upon phosphorylase activity. Assaying phosphorylase by the method of Cori et al. (15) did not result in an increased activity. Addition of MnCl, (10-° M), ATP (10° M), MgCh (2 x 10-* M), and UTP (10-* M) to separate reaction vessels also did not increase phosphorylase activity. The pH optima of both liver and muscle phosphorylase lie between pH 6 and 7 (14,21). The pH optimum of Ehrlich ascites tumor phosphorylase was 6.4 and was therefore similar to that of normal liver and muscle. The phosphorylase assay was validated by demonstrating the stoichiometry of the reaction (Table IV). The appearance ofa large amount of inorganic phosphate release from glucose 1- phosphate paralleled the net synthesis of glycogen in mouse liver and muscle. With Ehrlich ascites tumor homogenates, however, a small amount of phosphate was released from glucose 1-phos- phate, but no synthesis of glycogen could be detected. The phosphorylase reaction also was measured in the reverse direction, i. e., from glycogen to glucose 1-phosphate by incu- bating tumor homogenates with glycogen and determining the disappearance of the glycogen (Table V). Mouse muscle and liver homogenates catalyzed the rapid disappearance of glycogen; the rate of the reaction in hepatoma homogenates was approxi- mately one-tenth to one-twentieth that of liver. Essentially similar results were obtained with all tumor homogenates tested. M. W. Nirenberg 3089 Tase I Tumor Strains             Tumor Type Host Source Ehrlich earcinoma.| Ascites | Swiss mouse | Dr. Arthur Schade Krebs-2 carcinomal Ascites | Swiss mouse | Dr. Mark Woods Hepatoma-129-F (Reference 36)..| Ascites | C;sH mouse | Dr. Morris Belkin Lymphocytic leu- kemia-388-S..... Ascites | BALB/e x | Dr. Michael Potter dba mouse Plasma cell-70429 (Reference 37)..| Ascites | CsH mouse | Dr. Michael Potter Mast cell-815 (Reference 38)..| Ascites | BALB/c x | Dr. Michael Potter dba mouse Sarcoma-37....... Ascites | CFW mouse | Drs. Peter Eck, Margaret Ogara HeLa carcinoma. .| Tissue Dr. Harry Eagle cul- ture Rous sarcoma..... Solid Chicken Dr. W. Bryan Tas_Le II Total Polysaccharide Content of Tumor Cells   Total polysaccharide moles glucose equivalents/g protein Tissue   Hepatoma ascites.............. 00 sees e eee 4.1 Ehrlich carcinoma ascites.................- 0.48 Lymphocytic leukemia ascites.............. 6.1 Plasma cell ascites............. cece een eee 5.2 Mast cell ascites. .........0: cece cece ee eee 13.0 Krebs-2 carcinoma ascites...............56- 1.2 Sarcoma-37 ascites. ......... 0.6 e eee eee 1.2 HeLa carcinoma (tissue culture)............ 5.3   TasB_e III Phosphorylase activity of tumor and normal tissue homogenates Tumor cells and liver slices were washed once with 0.9% NaCl and were homogenized at 5° in an all-glass Potter-Elvejhem ho- mogenizer (Kontes Glass Company). The concentration of 5’- AMP was 2 umoles/ml reaction mixture, when present. Phos- phorylase was assayed by the method of Sutherland and Wosilait     (14). In some cases the results were checked by the assay of Cori et al. (15). Aumoles P;/10 min./mg protein Tissue ——— —AMP +AMP Mouse liver (normal)............- 1.57 1.56 Hepatoma ascites...............-- 0.125 0.0990 Ehrlich carcinoma ascites......... 0.081 0.0845 Lymphocytic leukemia ascites. . .. 0.114 0.116 Plasma cell ascites................ 0.0993 Mast cell ascites.................. 0.0613 0.0674 Krebs-2 carcinoma ascites......... 0.144 0.135 Sarcoma-37 ascites................ 0.140 0.134 HeLa carcinoma (tissue culture). . 0.116 0.129 Chicken muscle (normal)......... 1.63 7.23 Rous sarcoma.............+ee eee 0.165 0.204       3090 TasBLe IV Stoichiometry of the phosphorylase reaction Reaction mixtures contained 110 umoles of glucose 1-phosphate, 2.2 mg of glycogen, 220 umoles of NaF, 4 umoles of Na ethylene- diamine tetracetate, and whole tissue homogenate. Final volume was 2.8m], pH 6.1. Flasks were incubated for 20 minutes at 37° with shaking. Aliquots were taken for analysis at 0 and 20 min- utes. Inorganic phosphate was determined by the method of Fiske and SubbaRow (39); glycogen by the anthrone method (13).         7 APi A gl les/flask Tissue umoles/flask eycogenamoles/flas Mouse liver................. 39.5 32.2 Mouse muscle.............. 26.4 18.6 Ehrlich ascites tumor....... 3.8 —2.9 TABLE V Glycogen utilization by tumor and normal tissue homogenates Reaction mixtures contained 6 mg of glycogen (33.3 umoles glu- cose equivalents), 200 zmoles of inorganic phosphate, 100 ymoles of NaF, 2 umoles of 5’-AMP (when present), and whole tissue homogenate. pH was 6.1; final volume, 3.0 ml. Aliquots were taken for analysis at 0 and 60 minutes. Glycogen was determined by the method of Stadie e¢ al. (12).       A glycogen pmoles glucose equivalents/mg protein/hr Tissue —AMP +AMP Normal mouse muscle............. —6.6+ 0.3 | —6.0 + 0.3 Normal mouse liver............... —4.3 —4.2 Hepatoma ascites................. —0.3 —0.4 Ehrlich carcinoma ascites......... —0.2 _ Lymphocytic leukemia ascites..... 0 —0.3 Plasma cell ascites................ —0.3 —0.1 Mast cell ascites.................. —0.4 —0.3 Krebs-2 carcinoma ascites......... —1.0 -1i.1 HeLa carcinoma (tissue culture) .. +0.1 +0.3   Such phosphorylase activity as was found in the tumor ho- mogenates might have been due to the presence of normal eryth- rocytes and leukocytes. This possibility was tested by apply- ing a histochemical phosphorylase assay (22) to the Ehrlich carcinoma, hepatoma, and Krebs-2 carcinoma cell suspensions. A highly active phosphorylase, as judged by histochemical stain- ing, was found in the normal polymorphonuclear leukocytes; no phosphorylase activity whatsoever could be detected in the tumor cells. It seems likely, then, that most or all of the phosphorylase activity found in the tumor homogenates is due to the presence of small amounts of normal cells contaminating the tumor cell suspensions. It should be noted, though, that HeLa cells are obtained in pure culture, and small, but nonetheless significant, phosphorylase activity can be found. The low phosphorylase content of ascites tumor homogenates was validated in still another manner. The following spectro- photometric method was devised to assay glycogen phosphory]- ase. phophorylase Glycogen + P; glucose l-phosphate (1) Characteristic of Ascites Tumors Vol. 234, No. 12             -900 1 1 ; a BOO L +PHOSPHORY- _ S LASE ~ -7T00F - Ee 600 - 4 > .500 + E wn, b- - 2 400 Q 300; +5-AMP 1 _J L -5-AMP a -200 = .100+ MINUS J a. GLYCOGEN So 0 Ls < I 2 3 4 5 MINUTES Fie. 1. Spectrophotometric assay of Ehrlich ascites tumor phosphorylase. The reduction of TPN was followed at 340 mu. © crystalline muscle phosphorylase added; @ 0.2 zmoles 5’-AMP added; A minus 5’-AMP; °C] minus glycogen. The reaction mix- ture consisted of the following: 40 wmoles of tris(hydroxymethyl) aminomethane, pH 7.0; 10 zmoles of NaF; 10 zmoles of phosphate, PH 7.0; 20 umoles of glycogen (glucose equivalents) ; 0.005 »mole of glucose 1,6-diphosphate; 50 umoles of neutralized cysteine; 0.8 umole of TPN; glucose 6-phosphate dehydrogenase; and clarified tumor supernatant fluid (3.0 mg of protein). The final volume was 1.0 ml. phosphoglucomutase er glucose 1,6-diphosphate cysteine (2) Glucose 1-phosphate glucose 6-phosphate glucose 6-phosphate dehydrogenase   Glucose 6-phosphate + TPN 6-phosphogluconie acid + TPNH (3) The formation of TPNH was followed spectrophotometrically at 340 mu. The results of these analyses are given in Fig. 1. The data of Fig. 1 demonstrate that TPNH is rapidly formed when crystalline muscle phosphorylase is added to the reaction mixture. The formation of TPNH is dependent upon the addi- tion of glycogen. It should be noted that Ehrlich ascites tumor extracts contain 6-phosphogluconic acid dehydrogenase; there- fore, approximately 2 wmoles of TPNH are formed for each umole of glucose 6-phosphate consumed (5). A slight lag can be observed at the beginning of the reaction, but the rate of the reaction is proportional to time after the first minute. When glycogen was omitted from the reaction mixture little TPNH was formed, and this demonstrates in an independent manner, the low amount of endogenous glycogen present in tumor ho- mogenates. Addition of 5’-AMP had no effect upon the phos- phorylase activity of the tumor homogenates. The spectrophotometric phosphorylase assay of Ehrlich ascites tumor homogenates was compared with the phosphorylase assay based upon the disappearance of glycogen (Table V) and the phosphorylase assay of Sutherland and Wosilait (Table III). The values obtained with each method respectively /mg protein / 10 minutes were: 0.035 umole glucose 1-phosphate formed from glycogen, 0.033 mole glucose equivalents disappearing from December 1959 glycogen, and 0.081 ymole P; released from glucose 1-phosphate. Separate Ehrlich ascites tumor homogenates were prepared for each analysis, yet the results of the three different types of assays essentially agree with each other. It seems reasonable to con- clude that Ehrlich ascites tumor homogenates have 3 to 10% of the phosphorylase activity of normal liver or muscle and that most or all of the observed activity is derived from normal leukocytes present in the ascites suspensions. Ehrlich ascites tumor homogenates can utilize glucose 1-phos- phate. Supplementation with glucose 1,6-diphosphate and eysteine was necessary for optimal phosphoglucomutase activity; both were routinely added for phosphorylase assays. The results of Fig. 1 demonstrate that phosphoglucomutase is present and is not rate limiting in glycogen degradation by Ehrlich ascites tumor homogenates. Phosphoglucomutase has also been dem- onstrated in the Novikoff hepatoma (23). Phosphorylase Activity of Tumor of Viral Origin The phosphorylase content of a virus-induced tumor, the Rous sarcoma, and normal chicken muscle are presented in Table III. Both samples of tissue were removed from the same animal. The Rous sarcoma was a solid tumor and was not studied as com- pletely as the other tumors, but nonetheless low phosphorylase levels, comparable to the ascites tumors and the HeLa carcinoma, were found. Activation of Phosphorylase by Epinephrine and Glucagon Since epinephrine and glucagon are involved in the activation of phosphorylase, it seemed logical to determine whether either hormone could facilitate the activation of phosphorylase in hepatoma and Ehrlich ascites tumor cells. The addition of 50 pg/ml of epinephrine and 25 pg/ml of glucagon to mouse liver slices resulted in a marked and rapid reactivation of phosphoryl- ase. Additions of epinephrine and glucagon to hepatoma and epinephrine to Ehrlich ascites tumor cells had no effect upon phosphorylase activation. These data demonstrate the absence of this hormonally controlled enzymatic response in these tumors. Studies with Phosphorylase Activating System Since the tumor cells had negligible phosphorylase activities and were not responsive to epinephrine and glucagon, it was of interest to determine whether the phosphorylase activating en- zymes were present. The data of Table VI demonstrate that HeLa carcinoma, hepatoma, and Ehrlich carcinoma homogenates can convert dephosphophosphorylase to phosphorylase. Addi- tion of dephosphophosphorylase kinase had no effect upon phos- phorylase activation in HeLa and Ehrlich carcinoma homog- enates, but increased the phosphorylase activity of the hepatoma homogenate. The conversion of dephosphophosphorylase to phosphorylase was not proportional to the amount of homog- enate added, possibly due to the involvement of adenosine-3’-5’- cyclic phosphate in the over-all reaction. The activation of phosphorylase was dependent upon the presence of ATP. The results of Table VI demonstrate that the tumors possess a vigor- ous dephosphophosphorylase-activating system and a relative absence of dephosphophosphorylase. Phosphorylase Inactivating Enzyme The question may be asked, “Does the Ehrlich ascites tumor have a high phosphorylase inactivating enzyme activity?” The M. W. Nirenberg 3091 TaBLe VI Activation of Dephosphophosphorylase by HeLa, Hepatoma and Ehrlich Homogenates Reaction mixtures contained 4 pmoles of tris(hydroxymethyl) aminomethane, pH 7.4, 0.34 umole of ATP, 0.5 pmole of MgSO,, 0.0389 umole of epinephrine Cl., whole homogenate, and where indicated, dog liver dephosphophosphorylase and dog liver de- phosphophosphorylase kinase. 0.05 ml of the HeLa and hepatoma homogenates contained 2.37 and 1.90 mg of protein respectively. 0.15 ml of Ehrlich homogenate contained 9.04 mg of protein. Total volume was 0.2 ml. Reaction mixtures were incubated at 30° for 5 minutes. 1 ml of the phosphorylase reagent (14) contain- ing 2.0 wmoles 5’-AMP was then added, and the tubes were incu- bated at 37° for 20 minutes. Samples were deproteinized by tri- chloroacetic acid precipitation at 0 and 20 minutes.     No, | Home: Addition care Hepa- ‘care ml A pmoles P;/20 min. 1 | 0.05 | None 0.190} 0.033 2 | 0.05 |} + Dephosphophosphorylase} 6.48 | 0.850 3 | 0.10 | None 0.346; 0.549 4 | 0.10 | + Dephosphophosphorylase | 15.7 | 11.7 5 | 0.15 | None 0.768) 1.19 | 1.68 6 | 0.15 | + Dephosphophosphorylase | 19.7 | 15.8 | 13.2 7 | 0.15 | + Dephosphophosphorylase + Dephosphophosphorylase | 20.9 | 28.0 | 12.3 kinase 8 | 0.15 | + Dephosphophosphorylase ; 3.96 | 2.01 — ATP 9 | 0.15 | — Dephosphophosphorylase 1.90 + Dephosphophosphorylase kinase 10 | 0.15 | — Homogenate + dephos- 0 phophosphorylase + de- phosphophosphorylase ki- nase             conditions for assaying liver phosphorylase inactivating enzyme have been described (17). Purified preparations of dog liver phosphorylase were added to homogenates of normal mouse liver and Ehrlich tumor cells and the disappearance of phospho- rylase activity was measured at 10 and 20 minutes. The specific activity of the phosphorylase inactivating enzyme in homog- enates of dog liver is reported to be 1.2 to 1.6 (17). Under identical conditions the specific activity of this enzyme in mouse liver was 0.04 and in Ehrlich ascites tumor, 0.02. The low value obtained in mouse liver, as compared to dog liver possibly may be a species difference. These experiments demonstrate that mouse liver can inactivate phosphorylase at approximately twice the rate of Ehrlich ascites tumor. The possibility that the tumor extract contains a powerful inhibitor of phosphorylase can there- fore be excluded. DISCUSSION Tumors exhibit such diversity in form and type that it would seem highly unlikely to expect a relative absence of phosphorylase in all tumors. The results obtained with ascites tumors should not be extrapolated to other types of tumors. Phosphorylase has been demonstrated in two types of solid tumors (24); however, considerably decreased phosphorylase levels have also been at- tributed to a solid hepatoma (25). Since solid tumors contain 38092 variable numbers of normal cells such as connective tissue, as- cites tumors were used primarily in this study. One advantage of ascites tumors is the relative purity of cell type which can be obtained. Glycogen synthesis has been shown to proceed in a variety of tissues by Leloir and others (26-29) by an irreversible UDP glucose transferase reaction. This, rather than phosphorylase, may be the main route of glycogen synthesis. Dr. R. Wu? has found UDP-glucose transferase in HeLa cells. Under certain growth conditions the cells can accumulate glycogen; then low levels of phosphorylase, about 1% that of an equivalent amount of muscle, can be demonstrated. Since HeLa carcinoma homog- enates can rapidly reactivate added dephosphophosphorylase (Table VI), the rate-limiting factor appears to be the availability of dephosphophosphorylase. UDP-glucose transferase has not been looked for in ascites tumors. Although no stored glycogen can be found in these tumor cells, the absence of glycogen need not always go hand in hand with the absence of phosphorylase. The possibility exists that some cells contain UDP-glucose trans- ferase but lack phosphorylase. This situation might result in a marked accumulation of glycogen. Rat muscle and brain have little phosphorylase at birth, and after approximately 10 days the phosphorylase activities rise to adult levels (30). The phosphorylase content of rat liver 1 day after term also is greatly reduced.* Fetal guinea pig liver, how- ever, contains adult quantities of phosphorylase (31). Some, but not all embryonic tissues, therefore, have greatly reduced phosphorylase levels when compared to the corresponding adult tissues. Although both glycogen and phosphorylase are present in al- most all adult mammalian tissues, every cell type need not con- tain these substances. Histochemical studies have demon- strated, for example, the uneven distribution of phosphorylase activity among different cell types of a given tissue (22). Pre- liminary work with a virus-induced tumor, the Rous sarcoma, has revealed remarkably low phosphorylase levels when com- pared with normal chicken muscle taken from the same animal. Although this tumor is a sarcoma, chicken muscle may not be an adequate control, for the Rous sarcoma can arise from in- fected avian fibroblasts (32). Although no answer is available, the question should be raised, “Do certain types of normal cells such as fibroblasts also have low phosphorylase levels, and, if so, are ascites tumors derived primarily from these cell types?”’ The breakdown of glycogen in both liver and muscle is clearly regulated by a complex hormonal mechanism. The extremely rapid interconversion of dephosphophosphorylase and phospho- rylase in resting versus contracting muscle has been emphasized (33), and it is possible that the activation and deactivation of phosphorylase controls the release of distinct waves of glucose- 1-phosphate which can be converted quickly to lactate either with the concomitant production of pulses of ATP, if it is metab- olized via the Embden-Meyerhof pathway, or with the produc- tion of waves of TPNH if it is metabolized via the hexose mono- phosphate shunt. Although ascites tumor cells have exceedingly high rates of carbohydrate metabolism, they lack to a large extent this hormonal regulatory mechanism. The suggestion has been made (34) that some tumors become insensitive to certain controlling forces, such as hormonal regulation, through loss or inhibition of particular enzyme pathways. Transhydro- 2 Personal communication. *M. W. Niremberg, unpublished results. Characteristic of Ascites Tumors Vol. 234, No. 12 genase is present in normal liver but has not been found in a number of ascites tumors (35), including a hepatoma. Addition of epinephrine and glucagon to hepatoma cells in this study did not result in an increased level of phosphorylase, possibly because of the low amount of dephosphophosphorylase available. Since it is always difficult to validate a negative finding, such as the absence of an enzyme, an attempt has been made to in- vestigate thoroughly the parameters of the phosphorylase assay. It seems reasonable to conclude that the phosphorylase contents of the ascites tumors studied, such as the hepatoma, are very low when compared with normal liver or muscle. It is not pos- sible with the methods available to ascribe a total absence of phosphorylase to ascites tumors. It should be noted that the phosphorylase levels herein ascribed to ascites tumors undoubt- edly represent maximal values, for histological examination re- vealed high phosphorylase activity in normal leukocytes also present in ascites suspensions. No phosphorylase whatsoever could be detected histologically in the tumor cells. It seems striking that these tumors, utilizing monosaccharides at such rapid rates, should be relatively unable to degrade gly- cogen. In normal cells glycogen appears to serve the cell as a hormonally controlled reservoir, or buffer, for “energy” and sub- strates. Clearly, the tumor cells studied neither possess a car- bohydrate reserve nor have, to any appreciable extent, the con- trol mechanism which may release pulses of intracellular glucose 1-phosphate. It is not known what effect this may have upon the metabolism and economy of these neoplastic cells. SUMMARY The glycogen phosphorylase activities of seven types of ascites tumors, a tumor grown in tissue culture, and a solid virus-induced tumor were determined by chemical and histochemical tech- niques. Negligible phosphorylase activities were found com- pared to. normal control tissues. Although glycogen is present in some neoplastic cells, little or no glycogen could be found in the ascites tumors. The phosphorylase activating enzymes of three tumors were studied. All contained ATP-dependent phosphorylase activating enzymes but had negligible amounts of dephosphophosphorylase. No epinephrine or glucagon-induced activation of phosphorylase was observed. Tt is not known whether this enzymatic defect in ascites tumors can be extrapolated to other types of tumors. The relative absence of a hormonal mechanism regulating stored carbohydrate utilization in ascites tumors was discussed and was held in con- trast to the rapid degradation of monosaccharides by these tu- mors. Acknowledgments—The help of Dr. Samuel Spicer in perform- ing the histochemical assays, Dr. Earl Sutherland for generously supplying purified preparations of dog liver enzyme, and Dr. Harry Eagle for supplying HeLa cell cultures is gratefully ac- knowledged. The author wishes to express his appreciation to Dr. DeWitt Stetten, Jr., and the members of the Section on Intermediary Metabolism for their continued helpful advice and encouragement. REFERENCES 1. Rau, T. W., anp SutHertann, E. W., J. Biol. Chem., 232, 1065 (1958). 2. SUTHERLAND, E. W., AnD Ratu, T. W., J. Biol. Chem. 232, 1077 (1958). 3. WarnBura, O., Science, 123, 309 (1956). December 1959 = on 10. li. 12. 13. 16. 17. 18. 19. 20. Nirenpere, M. W., anp Hoae, J. F., J. Am. Chem. Soc., 78, 6210 (1956). _ Nrrenpena, M. W., anp Hoag, J. F., J. Am. Chem. Soc. 80, 4407 (1958). _ Nirensere, M. W., anp Hoaa, J. F., Cancer Research, 18, 518 (1958). . NrrENBERG, M. W., Federation Proc., 17, 283 (1958). . NrrENBERG, M. W., Biochim. et Biophys. Acta, 80, 203 (1958). . Frencu, C.S., AnD Mityer, H., in 8. P. Cotowick anp N. O. Kaplan (Editors), Methods in enzymology, Vol. I, Academic Press, Inc., New York, 1955, p. 64. Nossal, P. M., Australian J. Exptl. Biol. Med. Sci., 31, 583 (1953). Horecxer, B. L., anp Smyrnrotis, P. Z., in S. P. CoLowick AND N. O. KAPLAN (Editors), Methods in enzymology, Vol. I, Academic Pres, Inc., New York, 1955, p. 323. Srapign, W. C., HAUGAARD, N., anp Marsu, J. B., J. Biol. Chem., 188, 167 (1951). Canrout, N. V., Lonatey, R. W., anp Ros, J. H., J. Biol. Chem., 220, 583 (1956). SUTHERLAND, E. W., anp Wosiuait, W. D., J. Biol. Chem., 218, 459 (1956). . Cort, G. T., Inuincworts, B., anp KELLER, P. J., in 8. P. CoLowick. AND N. O. KAPLAN (Editors), Methods in enzy- mology, Vol. I, Academic Press, Inc., New York, 1955, p. 200. Rau, T. W., SUTHERLAND, E. W., aND BERTHET, J., J. Biol. Chem., 224, 463 (1957). Wostnarr, W. D., anp SUTHERLAND, E. W., J. Biol. Chem., 218, 469 (1956). Bucuer, T., Biochim. et Biophys. Acta, 1, 292 (1947). Linus, R. D., Histopathologic technic and practical histochem- istry, 2nd edition, Blakiston Company, New York, 1954, p. 120. Korn, E. D., J. Am. Chem. Soc., 80, 1520 (1958). M. W. Nirenb", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-83xs.g43e.cch4", "00000000-0000-0000-F7AF-1D232880864A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Comparison of the Tryptic Peptide of Wild Strains of Tobacco Mosaic Virus", "101584910X127", null, "1960", "December 1960", "Wittman determines the exact amino acid composition of different strains of the tobacco mosaic virus by splitting the viruses into RNA and proteins.  This work on determining the amino acid sequence would be cited by Nirenberg and others later as the bulk of amino acid substitutions found in TMV proteins did not occur in clusters, suggesting a non-overlapping code.", "Articles", "Tobacco Mosaic Virus,Peptides", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "4", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "DISCUSSION AND PRELIMINARY REPORTS G13 Comparison of the Tryptic Peptides of Wild Strains of Tobacco Mosaic Virus Mutants of tobacco mosaic virus (TMV) derived under experimental conditions from the normal TMV strain vulgare show only minute differ- ences, if any: one or only a few amino acids may be replaced by others. Besides these mutants a number of TMV-strains are known whose rela- tionship to vulgare is not clear because they were found in nature. These strains show many biological differences when compared with the normal TMY strain; it is therefore interesting to study what chemical differ- ences exist among them. One of these wild TMY strains is dahlemense, isolated by Melchers (1) from tomato plants about twenty years ago. In contrast to the mutants derived from vulgare, which all produce local lesions on Phaseolus vul- garis, dahlemense does not give lesions on this plant. It shows green sys- temic symptoms on “Samsun tobacco” and produces local lesions on “Java tobaececo’’. Its serological behavior is quite different from that of the vulgare strain or its mutants (2, 3). Figure 1 shows the electrophoretic mobility of whole virus and corresponding derived A protein of rulgare,                                           +08 =e—t | +06|—— wonne | +04|-—=o Qe BIE +a2|-— 3S ae ly 5 a0 — 7 8 9 3 -ae\\\\— -04\\\\— al oe “06 ; + —_ : \\\\ se teen g ~~ ---../] en F o AV \\\\ “Ye ° AF Woe -g8|—— _ LL. a AD Bal a ee ----4 L-—— I : a 10 | a-~-b----4 -~--D       Fic, 1. Electrophoretic mobility of vulgare (V). flavum (F), and dahlemense (D) and their corresponding A proteins (AV, AF, and AD) (ef. (9)). Reprinted from Virovocy, Volume 12, Ne. # December 1960 Copyright © 1960 by Academie Press Ine. Printed in CLS A. 614 DISCUSSION AND PRELIMINARY REPORTS flavum, and daklemense. From this it can be concluded that the number of acidic groups in dahlemense virus is higher than in rulgare and the munber of basic groups lower. The isoelectric point of pH 2.7 is lower than that of any other mutant or strain of TMV, The amino acid com- position has been previously studied by the DNP method (4), however, without splitting the protein into peptides. In order to determine the exact amino acid composition of dahlemense its tryptic peptides were studied. The virus was split into ribonucleic acid and protein, the protein was digested with trypsin, and the tryptic                                         15 T - Vulgare T o | _ Os j | eff ree 1 r 1 re “1 Q 30 60 30 120 150 180 210 240 270 300 Pia. 2. Elation curves of the tryptic peptides of ragare. Optical density after alkaline hydrolysis and ninhydrin reaction, plotted against number of fractions,   1S r Dahlemense | 10|----                        a it bo 1 L a 30 60 90 120 1 1 Ld L 1 i 1 4. 4 4 4 150 780 270 240 270 300   Fig. 3. Elation curves of the tryptic peptides of dahlemense, Optical density after alkaline hydrolysis and ninhydrin reaction, plotted against number of frac- tions. DISCUSSION AND PRELIMINARY REPORTS 615 Thr Ser Ala Val Met Jleu Leu together with peptide No Phe T1121 275 120] 7 | | [9 | 37070 |19]19 [19 |79| 7 | 7 [7724 72 V=Vulgare, D=Dahlemense          I ~ x ~ Fie. 4. Comparison of the tryptic peptides of eigare and dahlemense. peptides were separated by column chromatography; ligs. 2 and 3 show typical elution curves for radgare and daklemense. The peptides were purified if necessury by paper chromatography or high voltage elec- trophoresis and hydrolyzed, their amino acid composition was de- termined in an automatie amino acid analyzer. Details of the method have been deseribed previously (4, 6). Figure + shows the comparison of the tryptic peptides of vulgare and dahlemense. The composition of certain of the dahlemense peptides, e.g., No. EX with 7 amino acids, is very different from that of the correspond- ing rudgare peptides, whereas other peptides, e.g., VI with 10 amino acids, are exactly the same. It is interesting that daklemense peptide X con- tains an amino acid, namely methionine, which is not present in rulgare or in any of its many mutants, but only in other very distantly related TMY strains, e.g., Holmes’ ribgrass strain (7). In spite of the pronounced differences between the proteins of vulgare and dahlemense the number of amino acids within the protein chain of both strains is the same, namely 157. The number of peptides obtained from dahlemense is 10, compared with 12 from rulgare; this is because two arginines, at which amino acid trypsin splits the chain, are exchanged for two other amino acids, Dahle- mense peptide IL] with 20 amino acids corresponds to rulgare peptide TT] O16 DISCUSSION AND PRELIMINARY REPORTS with 15 plus peptide I with 5 amino acids. Similarly the C-terminal dahlemense peptide X with 24 amino acids (and no arginine and lysine) corresponds to the cxulgare peptide IV with 7 plus peptide X with 17 amino acids. By comparing the tryptic peptides of several TMV strains and by determining their N-terminal amino acid residues, it has been possible to elucidate the arrangement of most of the tryptic peptides within the protein chain of TMY (8). ACKNOWLEDGMENT The tecbnical assistance of Miss Ingrid Hindennach and Miss Brigitte Ostertag is gratefully acknowledged. REFERENCES 1. Mevcuers, G., Vaturwissenschaften 30, 48-49 (1942). 2, Friepricnu-Frexsa, H., Meccuers, G., and ScuramM. G., Biol. Zentr. 66, 187—- 222 (1946). 3. Aacnu, HG... 4. Naturforsch. 12b, 614-022 (1937). 4. Ascn, H.G.. Z. Naturforsch. 18b, 425-433 (1958). 5. Wrrrmann, H.G., and Braunirzer, G., Virology 9, 726-728 (1959). 6. Wirrmann, H. G., Z. Vererbungslehre 90, 463-475 (1959). y. Weranr, C. A. J. Biol. Chem. 171, 297-309 (1947). 8. Wirrmann, H.G., Virology 11, 505-508 (1960). 9. Kramer, [., and Wirrmann, H. G., Z. Naturforsch. 18b, 30-33 (1958). Max-Planck-Institut fiir Brologie H. G. WrtTMANN bt. Melchers Tiibingen, Germany Received October 21, £960.", "Wittmann, Heinz-Gunter", null, "Virology", "The Academic Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bfwp-i6ni_cnxh", "00000000-0000-0000-DA25-5691311E3180", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Comparison of the Tryptic Peptides of Chemically Induced and Spontaneous Mutants of Tobacco Mosaic Virus", "101584910X128", null, "1960", "December 1960", "Wittman compares the structural relationship between RNA and corresponding proteins in TMV.  As the infecting TMV-RNA induces the cell to produce new virus particles consisting of RNA and a specific protein, Wittman manipulates the sequence of RNA bases in order to study the effect on protein synthesis.  By elucidating the amino acid sequence of TMV and determining which amino acid can be substituted for which, Wittman's work is an important contribution to resolving the genetic code.", "Articles", "Tobacco Mosaic Virus,Peptides,Mutation,RNA,Amino Acids", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "4", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Reprinted from Vrrotoay, Volume 12, No. 4, December 1960 Copyright © 1960 by Academic Press Inc. Printed in U.S.A. Comparison of the Tryptic Peptides of Chemically Induced and Spontaneous Mutants of Tobacco Mosaic Virus The genetic information of tobacco mosaic virus (TMV) resides in the sequence of the bases within its ribonucleic acid (RNA). The infecting TMV-RNA induces the cell to produce new virus particles consisting of RNA and a specifie protein. There must therefore exist a structural re- lation between the RNA and the corresponding protein. One way to study this relation is to change the sequence of the RNA bases and to study the effect on the corresponding protein. Treatment of TMV-RNA with nitrous acid converts certain bases into others, e.g., uracil to cytosin (1); a change of one nucleotide is sufficient to produce a mutation (2). By variation of the experimental conditions it is possible to vary the statistically defined average number of nucleo- tide changes within the RNA. Investigation of the protein structure of these chemically induced mutants promises to give information on the coding problem between nucleic acids and the corresponding proteins. The results of a comparison of the tryptic peptides of 26 chemically 610 DISCUSSION AND PRELIMINARY REPORTS induced and spontaneous TMV mutants will be described here. Part of this work has been presented before the National Academy of Sciences, Washington, D. C. (3). Comparisons of the amino acid composition (4) and of the composition of the tryptic peptides (5) of the normal TMV strain and a mutant induced by nitrous acid treatment have been pub- lished. To solve the coding problem, it is essential to have a method capable of detecting with certainty a change in only one out of the 157 amino acids making up the TMV protein. Therefore the purified TMV was split into RNA and protein and (besides determining the amino acid composition of the virus protein in an automatic amino acid analyzer) the protein was digested with trypsin; the resulting 12 tryptic peptides were isolated and purified by column and paper chromatography and paper electrophoresis. Their amino acid composition was analyzed in amino acid analyzers according to Spackman et al. (6). The methods were essentially the same as those described previously in detail (4, 7). Figure 1 gives a scheme showing derivation of the mutants whose tryptic peptides have been studied. Those mutants designated by “Ni” were produced by nitrous acid treatment; all others were spontaneous mutants. The isolation of the spontaneous mutants A 7 to E83 was done in such a way as to reduce to a minimum the probability of getting more than one mutation step (8). This principle was not stressed as much in isolating the spontaneous mutants designated by Latin names [flarwm           @ b c qd e i Z k { m A             | vulgore |   o Ss ? Yu v w x y 2 PIR IE IEE eee]                         Fig. 1. Scheme of the TMV mutants studied. The differences in the amino acid composition of the tryptie peptides are given in the text. DISCUSSION AND PRELIMINARY REPORTS G11 (flav.), necans, (nec.), rertrescens (rev.), and reflareseens (refl.)], some of which were isolated many yeurs ago (9). The amino acid composition of the latter mutants has been analyzed before, without splitting the pro- tein into tryptie peptides (74). When ¢ is the time of treatment and 7 the time after which the in- fectivity decreases to Lc of the value before treatment [sce (2)], then for both NiXX. band NiXX 8,0 = d.sfor Ni 11, Ni 18, and Ni ay T t = 5.2 [see (1/)|; for Ni 54/45 and Ni 44/20, t 2.0; for Ni 54 °20.34, ; T = 6.5; and for Ni 101 to Ni 118, —_ 3.0. The 6 mutants Ni 101 to Ni T 118 and the 2 Ni XX/... were produced by treatment of the virus nucleoprotein with nitrous acid whereas all other Ni mutants were isoluted after nitrous acid treatment of the TMV-RNA. The mutants flavum, Ni XX/8, Ni 54/20/34, and FE 83 are yellow strains which can- not be differentiated by symptoms on “Samsun tobaceo”’. The analysis of the tryptic peptides of the 26 chemically induced and spontaneous mutants listed, gives the following results (cf. Fig. 1; pep- tides numbered as in (7)]: Steps ace, doe, fg. su, vow, ye no change Steps b, hyo. a: Asp — Ala in peptide NIT! Steps i,j: Asp 2 Gly in NOL Step k: Glu > Gly in VII Step 1: Asp > Gly in IX Step im: Ser > Phe in IV; Glu -» Valin NIT Step ns Pro > Leu in NIL step p: Ala + Phe > Val + Leu in NIT; Ser > Phe in IV Step q: Val + Leu > Asp + Phe in NT Step r: Leu > Phe in NOT Step t: eu — Thr in NI Step x: Asp > Lys in IV The results confirm the earlier finding (5) that there exist mutants which have the same amino aeid composition of their tryptic peptides al- though they show very striking differences in symptoms; this is true for both spontaneous and chemically induced mutants. This ean be ex- plained by assuming that the region within the TMV-RNA which codes for the 157 amino acids of the virus protein ix smaller than the 6500 nucleotides making up the TMV-RNA. Only when nucleotides within this region are changed docs an alteration of amino acids in the viral protein oecur, whereas changes of nucleotides outside this region result 1 Peptide No. NUL Ovith 41 amino acids) contains 4 Asp [see (7)]. ASP means aspartic acid or asparagine. Ghi means glutamic acid or glutamine. 612 DISCUSSION AND PRELIMINARY REPORTS in alterations that lead to different symptoms without changing the viral protein, By investigation of more nitrous acid mutants than have been studied hitherto, the number of nucleotides within the “gene” for the viral protein could be determined and the question answered how many nucleotides code for one amino acid. The elucidation of the total sequence of the 157 amino acids of the TMV protein (72) makes it possi- ble to pinpoint the alteration of a certain amino acid at a certain posi- tion within the protein chain. This opens up the possibility of seeing whether the points of amino acid replacement. are distributed randomly over the protein chain or whether there exist regions with a high fre- quency of replacement, and also of answering the question whether a certain amino acid can be substituted only by a limited number of others; this information would give additional help in resolving the code. ACKNOWLEDGMENT The technical assistance of Miss Ingrid Hindennach, Miss Brigitte Ostertag, and Miss Julita Huf is gratefully acknowledged. REFERENCES 7. Scuuster, H., and ScuramM, G., Z. Naturforsch. 13b, 697-704 (1958). 2. Gierer, A., and Munpry, KK. W., Vefure 182, 1457-1458 (1958). 3. Wirrmann, H. G., Symposiian on Genetic Determination of Protein Structure, Ann. Meeting Natl. Acad. Sei. Washington April, 1960. 4. Tstaira, A., and FRAENKEL-Conrat, H., Proce. Natl. Acad. Sct. U. S., 46, 636-642 (1960). §. Wittmann, H.G., 2. Vererbungslehre 90, 468-475 (1959). 6. Spackman, DD. H., Srerx, W. H., and Moors, 8., Anal. Chem. 30, 1130-1206 (1958). 7. WitrmMann, H. G., and BRAUNITZER, G., Virology 9, 726-728 (1959). 8. Me.cuers, G., unpublished data. 9. Meccousrs, G., Vaturwissenschaften 36, 48-49 (1942). 10, Aacu, H.G.. Z. Naturforsch, 18b, 425-433 (1958). it. Munpry, K.W., and Gierer, A., Z. Vererbungslehre 89, 614-4030 (1958). 12. ANbeRER, A., Utttic, H., Weper, IX., and Scuraw, G.. Nature 186, 922-925 (1960). Max-Planck-Institut fiir Biologie H. G. WittMaNnn Abt. Melchers Tiibingen, Germany Reeeived October 21, 1960", "Wittmann, Heinz-Gunter", null, "Virology", "The Academic Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fumb.jtpv-v32u", "00000000-0000-0000-F537-79D7D74EDF74", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Interaction and Mixed Aggregation of Proteins from Tobacco Mosaic Virus Strains", "101584910X129", null, "1960", "1960", "This article examines the differences in polypeptide chain structure in various forms of TMV.  The author explores the significance of fundamental differences in the composition and sequence of amino acids between the viruses and the fundamental similarity that allows them to \"associate near their respective isoelectric points in an orderly way.\"  The formation of mixed aggregates is also partly significant because Nirenberg used work on TMV protein configuration and polypeptide chain formation in different TMV strains as evidence for a non-overlapping code.. Sarkar suggested that \"Profiles in Science\" include the following statement regarding this article: \"This publication contains the first experimental data in support of a \"trimer configuration\" of TMV-protein, as postulated later by D.L.D. Casper (Adv. Protein Chem. 18:37, 1963) on theoretical considerations and crystallographic principles.  Further experimental proof was offered on the basis of osmotic pressure measurements by K. Banerjee and M.A. Lauffer (Biochemistry 5: 1957, 1966).\"", "Articles", "Tobacco Mosaic Virus,Peptides", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "10", "pages", "Text", "English", "Reproduced with permission of Verlag der Zeitschrift fur Naturforschung.", "Copyright may apply", null, null, "Sonderabdruck aus der ZEITSCHRIFT FUR NATURFORSCHUNG Band 15b, Heft 12, 1960 Verlag der Zeitschrift fiir Naturforschung, Ttibingen Interaction and mixed aggregation of proteins from Tobacco mosaic virus strains By Saryaprata Sarkar Interaction and mixed aggregation of proteins from Tobacco mosaic virus strains By Satyaprara Sarkar Aus dem Max-Planck-Institut fiir Biologie, Abt. Metcuers, Tiibingen, CorrensstraBe 41 (Z. Naturforschg. 15 b, 778 —786 [1960] ; eingegangen am 10. Oktober 1960) Proteins from four strains of TMV, namely vulgare, flavum, dahlemense and Holmes’ rib grass, were electrophoretically examined singly and in pairs at different hydrogen ion concentrations. In weakly alkaline media an average of six polypeptide chains of TMV-protein remain asso- ciated together, while constant dissociation and reassociation takes place. This dynamic state of equilibrium is responsible for transient reciprocal associations of proteins or polypeptide chains from the first three TMV-strains. On lowering the pry value, these interacting proteins form mixed aggre- gates with intermediate mobilities. Protein from the fourth strain, Holmes’ rib grass, when tested against the strain, vulgare, neither showed any interaction in alkaline media nor formed mixed aggregates. Factors determining the formation of mixed aggregates and the possible relationship among the four strains have been discussed. The protein-part of a single Tobacco mosaic virus 157* amino acids (Wirrmann and Braunirzer, 1959)? particle consists of 2130440 polypeptide chains whose sequence has been recently worked out? which are very probably identical with one another! (Annerur et al., 1960). (Franky, Caspar and Kuve, 1959). A polypeptide * chain of the common TMV strain, vulgare, has a According to a recent letter from Professor W. M. Sraney addressed to Professor G. Mrtcuurs a polypeptide chain molecular weight of about 17500 and consists of of TMV protein contains 158 amino acids and not 157 as found here earlier. This result could now be confirmed also by Dr, H. G. Wrrrmaxy. 1 R.E. Frayxun, D.L.D. Caspar and A. Kiva, in: Plant 2-H. G. Wirrmann and G. Brausrrzer, Virology 9, 726 [1959]. Pathology, Problems and Progress 1908—1958, Univ. of 5 A, Anperer, H. Unuic, E. Weer and G, Scurama, Nature Wisconsin, Pp. 447—461 [1959]. [London] 186, 922 [1960]. INTERACTION AND MIXED AGGREGATION OF PROTEINS The polypeptide chains of several other TMV strains differ more or less in the composition and sequence of their amino acids ** but they all possess one remarkable common property: they can asso- ciate near their respective isoelectric points in an orderly way producing cylindrical protein-coats, typical of intact TMV-particles which were studied electrophoretically by Kramer and WITTMann, 1958 8, as well as Kuzczkowsx1, 1959 7). The capa- city is inherent in the structure of the polypeptide chains themselves since cylinders of variable length are produced even without the presence of a ribo- nucleic acid core. At neutral and moderately alkaline py the TMV protein has often been reported to exist as small aggregates of six polypeptide chains with a mole- cular weight of about 100000 and has been given the name “A-protein” as it was obtained by alkaline splitting of TMV. The size of an aggregate is, how- ever, a function of such factors as py, ionic strength, temperature, protein concentration and so on. On high dilution, at high alkalinity or by the action of various organic solvents, the protein falls apart into single polypeptide chains which undergo easy denaturation (AnpERER, 19598, Ansevin and Laur- FER, 1959 9, Wirrmann, 1959 1°). On the basis of these observations, it seemed interesting to investigate: 1. Whether in a mixture of proteins from two different TMV strains an exchange of polypeptide chains occurs, and 2. whether mixed aggregates can be formed on acidification of such a mixture of proteins. These investigations were undertaken with a view to finding out how far an exchange of polypeptide chains and formation of mixed aggregates are deter- mined by the number of charged groups per poly- peptide chain and by their structural similarities and differences. Four strains of TMV, vulgare, flavum, dahle- mense and Holmes’ rib grass, were available, of C. A. Kntent, J. biol. Chemistry 171, 297 [1947]. H. G. Wirrmany, Vergleichende Strukturuntersuchungen an Tabakmosaikvirus-Stimmen verschiedenen Verwandt- schaftsgrades. Communication to the meeting of the Deut- schen Botanischen Gesellschaft, Kéln 1960 and Virology 12, No. 4 [1960]. 6 ©. Kramer and H.G. Wirtmann, Z. Naturforschg. 13b, 30 [1958]. 7 A. Kieczxowsk!, Virology 7, 385 [1959]. 8 A. Anperer, Z. Naturforschg. 14b, 24 [1959]. 779 which the amino acid compositions are now known (Wirrmann, 19605). Flavum and vulgare resemble each other very closely; each polypeptide chain of the former contains one aspartic acid less and one alanine more than the latter and most probably one amide group more than the latter on the basis of electrophoretic measurements (Kramer and Wirr- mann, 19581). As compared to both of them, dah- lemense shows several quantitative differences and contains one methionine per polypeptide chain which is not present in the other two strains 5, Holmes’ rib grass strain differs very widely from all the three above both qualitatively and quantitatively. Each polypeptide chain of Holmes’ rib grass contains several glutamic acid residues, three residues of methionine and one histidine, the last named being absent from the other three strains (Knicut, 1949 4, Wirrmann, 19605). Exchange of polypeptide chains and the forma- tion of mixed aggregates were detected by the method of free electrophoresis since the proteins of the four strains have characteristically different mobilities (Kramer and Wirrmann, 1958 8), Present observations speak for mutual interaction and mixed aggregation between proteins from closely related strains of TMV. I. Material and Methods Isolation of Virus. After multiplication on green- house grown tobacco plants each strain of virus was isolated in a pure form by the conventional method of differential centrifugation with alternate freezing and thawing. A preliminary removal of associated plant pro- teins from expressed sap was achieved either by heat- denaturation at 60°C for 10 minutes or by an isoelec- tric precipitation at py 4.3 (Commoner et al., 195017; Wirtmann 1959 13). As the isoelectric point of Holmes’ rig grass strain lies near about 4.5 (Oster 1951 *4; Ginoza and Atkinson 1955 45), only a heat-denaturation treatment was adopted for the primary clearing of the virus containing plant-sap. Splitting of virus and purification of A-protein. About 10 cm? of a 1—3% virus suspension in dilute phosphate 9 A.T. Ansevin and M.A. Laurrer, Nature [London] 183, 1601 [1959]. H. G. Wirtmann, Experientia [Basel] 15, 174 [1959]. E. Kramer and H. G. Wirrmany, Communication No. 2-25, Proceedings of the 4th International Congress of Bioche- mistry, Vienna 1958. 12 B. Commoner, F.L. Mercer, Pu. Meru and A. J. Zorn, Arch. Biochem. Biophysics 27, 271 [1950}. 19H, G. Wirrmany, Z. Vererbungslehre 90, 463 [1959]. 14 G. Oster, J. biol. Chemistry 190, 55 [1951]. 18 W. Gunoza and D. E. Arxtnson, Virology 1, 253 [1955]. 780 buffer (m/50) of py 7 was dialysed with stirring against one litre of glycine-NaCl-NaOH buffer of pu 10.3 — 10.4 (pu 10.6 in case of HR-strain) and an ionic strength of 0.1 at +4°C for 12 to 16 hours. The unsplit or in- completely split TMV particles still present were cen- trifuged down in the cold in a Spinco preparative ultra- centrifuge at about 60000¢ for 60 minutes, and the clear supernatant was dialysed for 12 to 24hours against glycine-NaCl-NaOH buffer of py 9.4 and ionic strength 0.04. The protein was purified by the method of countercurrent electrophoresis from the nucleic acid fraction at py 9.4 using the 10cm? standard cell of a “Phywe” electrophoresis apparatus. A potential gra- dient of 9—10 Volt/em for 8 to 10 hours was employed. As a criterion of purity, the ratio of absorption at 260 and 280 mu was kept as low as possible. The values for the proteins of the four strains abbreviated as AV, AF, AD and AHR were 0.54; 0.56; 0.58 and 0.53 respec- tively indicating that the protein-preparations were prac- tically free of nucleic acid (ef. Kramer and Wrrrmann 1958 ®), Concentration of protein was calculated from the ex- tinction at 280 my, since a good agreement exists be- tween Aggy and nitrogen content. A sedimentation ‘co- efficient of Syy FW 4 at py 8 was measured for each type of A-protein. Electrophoresis studies of A-protein mixtures at dif. ferent py-values, Electrophoretic mobility measurements were done at 3.2°C bath temperature in a “Phywe” electrophoresis apparatus. Errors in the magnification factor of the Schlieren camera and in the cross- sectorial area of Tiselius cell have been eliminated by using the sate limb of the same cell for all com- parative runs (AvBerty and Marvin 1950 1). To avoid smal] fluctuations in py or ionic strength a large volume of buffer solution was prepared for a whole series of experiments, kept at 4°C and checked for py and con- ductivity from time to time. AV, AF and AD solutions, each containing 0.4% pro- tein and AHR containing 0.25% protein were dialysed separately in Michaelis buffer of py 8.0 and of an ionic strength, /’=0.037. This ionic strength, which is half of that used by Kramer and Wittmann (1958) & was chosen to achieve greater numerical differences among ithe mobility values of the A-proteins and con- sequent better separation of the Schlieren peaks. The combinations and concentrations of the protein mixtures are given in Table 1. Each mixture was studied electrophoretically within one hour from the moment of mixing of the respective A-protein pair. After the runs the solution was recovered from the Tiselius cell and after standing for a week at 4°C reexamined electrophoretically. For aggregation experiments, the first three mixtures of table 1 were dialysed successively against Mich- aelis buffer of py 6.5, 5.5 and 5.0 (7 =0.037) for four hours each and then overnight against acetate buffer of py 4.92, = 0.037, all in a cold room at +4 °C. The 8 R.A. Arserty and H.H. Maarvin, J. phys. Colloid Chem. 54, 47 [1950]. S. SARKAR     Concentration ': . of each protein Mixture type Pu in the final mixture {gm/100 ml] 1 AV4+AF 5 800 | 0.2 2 AVI AD 8.0 0.2 3 AF4S AD 8.0 0.2 4a AV + 4HR 8.0 0.13 4b AV+ AHR 8.5 0.12       Table 1. Protein mixtures used for electrophoretic studies. mixture 4a was acidified slowly by dialysis against buf- fers of gradually lower py values and examined electro- phoretically at every stage. As AHR precipitated out easily at py 5.0 and also for reasons described later, the mixture 4b was dialysed directly against a Mich- ae lis buffer of py 5.2, /'= 0.037. The aggregated protein mixtures were split again into the constituent A-proteins by dialysis against glycine- NaCl-NaOH buffer of py 10.4 and their electrophoretic diagrams at py 8.0 and 8.5 were compared with their corresponding original mixtures. II. Results The ascending Schlieren pattern was sharper than the descending one but the deviation from enantiography was not too great when each protein was present alone. Anodic mobility values, expres- sed as (4/sec)/(V/cm) are given in table 2. Each value is the average of several determinations with a potential gradient of 7.5 to 9.5 V/cm. The values were remarkably uniform with fluctuations mostly within + 2%. The descending peak has generally a_ slightly lower mobility value, a phenomenon of common experience (ALBERTY, 1953 17), The ascending boundary of AHR at py 8.0 has, however, a lower mobility than the descending one. An explanation for this discrepancy has to be sought in the nature of the AHR molecule itself, since all other factors are kept constant. However, the higher mobility-value of the descending boun- dary of AHR at py 8.0 might be due to dissociation of the imidazole ring of histidine. This amino acid is not present in the other three virus strains. It is also worth noting that the difference between the mobilities in the two arms disappeared by simply raising the py-value to 8.5. 17 R. A. Auperty, in: The Proteins, vol. I A, edited by Neu- rath and Bailey, Academic Press 1953, P. 523. INTERACTION AND MIXED AGGREGATION OF PROTEINS Mobilities of the observed peaks with the mix- tures 1, 2, 3 and 4b (cf. table 1) are given in table 3 and the electrophoretic patterns are re- produced in fig. 1. In all mixtures, excepting AV + AHR, the mobilities of the observed Schlie- ren peaks are different from those of the pure com- ponents as presented in Table 2. Fig. 1. Ascending-limb electrophoretic patterns of TMV-pro- tein mixtures. V, F, D and HR stand for proteins from vul- gare, flavum, dahlemense and Holmes’ rib grass strains re- spectively. The first three diagrams are at py 8.0 while the V+#HR mixture is at py 8.5.   Protein type Pu U ascending | U descending AV : 8.0 0.46 0.46 AV 8.5 0.47 0.47 AF 8.0 0.37 0.36 AD 8.0 0.59 0.57 AHR, 8.0 0,67 0.71 AHR | 8.5 0.84 0.84       Table 2. Anodic mobilities (U) of A-proteins in Michaelis buffer, [=0.037, U=(u/sec)/(V/cm). 781 The Schlieren peaks were always much better resolved at the ascending boundary. Due to incom- plete separation and rapid spreading at the de- scending boundary. only one average mobility- value of this boundary has been given in some cases. From Table 3 it can be seen that: (1) mobi- lity of a particular peak remains practically un- changed even after 7 days’ standing of a protein mixture, (2) aggregation and resplitting does not affect the subsequent Schlieren pattern at pq 8.0 and 8.5, (3) a fall in total protein concentration from 0.4% to about 0.1% after aggregation and re- splitting hardly affects the mobility values and the Schlieren patterns. A comparison with table 2 indicates that (4) in all the three mixtures of AV’, AF and AD the mobility of the observed peaks are not exactly the same as the corresponding proteins when each is present alone; the two peaks appear to approach each other so to say, while (5) the mobilities of AV and AHR are exactly reproduced in mixture. Different degrees of flattening of the depicted. the sharpest AV + AHR and the most overlapping and spread- ing is AF + AD. On reducing the py value of the mixed solutions to 4.92 as described earlier, the proteins were found diagrams are being to aggregate as was evident from the opalescence of the samples. In table 4 are given the anodic mobilities (U = (4¢/sec)/(V/em)) of aggregated pro- teins both alone and in mixture at py 4.92. Agere- gated dahlemense protein moved as a single boun- dary while vulgare as well as flavum each formed two distinct peaks. According to Kramer und Wirr- mANN (19358) ® the faster peak, designated here as the main gradient. corresponds to rod-like aggre-     | i - Total pro- i ; ; : Mixture | tein cone. | PH Time after | U ascending U descending ype gm/100 ml] mains P AF + At 0.40 8.0 1 hour 0.40 + 0.44 0.37 + 0.41 AF+ A} 0.40 . 8.0 7 days 0.40 + 0.45 0.38 + 0.42 AF + AV 0.11 | 8.0 : 12 days * 0.39 + 0.44 0.43 AV+ AD 0.40 : 8.0 1 hour 0.51 + 0.56 0.53 AV + AD | 0.40 \\\\ 8.0 | 7 days 0.52 + 0.55 0.50 + 0.53 AV+AD 0.19 8.0 10 days * 0.52 + 0.56 0.50 + 0.53 AF+ AD | 0.40 8.0 1 hour 0.44 to 0.50 + 0.587 0.33:0.46 + 0.49+ AF+AD 0.40 30 | 8 days | 0.41 to 0.49 + 0.56+ 0.34 + 0.47 AF + AD 0.18 8.0 i 10 days * 0.41 to 0.48 + 0.57+ 0.35:0.46 + 0.51 AV + AHR 0.25 8.5 | 1 hour 0.47 + 0.85 0.45 + 0.83 AV+ AHR 0.25 | 8.5 : 7 days | 0.47 + 0.84 0.46 + 0.84       Table 3. Anodic mobilities of peaks observed in A-protein mixtures (4/sec)/(V/cm). * After aggregation and resplitting. * The slower peak is flattened and broader than the faster peak. 782 gates of dimensions comparable with intact TMV- particles, while the slower (= second) gradient cor- responds to discs and small incompletely aggregated units. The failure of better reproducibility as seen in table 4 may well be traced back to the high py-mobi- lity dependence at this py range (KRAMER and Wirrmann, 1958 §). ,     ae | U = (ulsec)/(V/em) at py 4.92 No. | Composition ascending j “descending | main second | main second peak peak j; Peak | peak 1 AF 0.77 0.68 | 0.64 | 0.65 2 AP 0.73 0.66 | 069 | — 3 AV 0.99 0.91 0.99 ; 0.97 4 AV 0.95 0.84 0.92 — 5 AD 1.09 _ 108 2 — 6 AD 1.09 ~ 110 | — 7 AF — AV 0.91 | 0.82 0.94 | 0.88 8 AV+AD] 097 | — 0.96 | — 9 AF+ AD ; O91 2 — 0.89 | —     Table 4. Anodic mobilities of aggregates of A-protein and their mixtures. Results indicate that in all the three cases mixed aggregates are produced. The formation of one single gradient from AF and AD with a mobility value almost exactly in the middle of those of the corresponding pure strains show that the two types of A-proteins possess sufficient similarity to be able to form mixed aggregates. The greater spreading of this gradient as compared to AV + AD (Fig. 2a and 2b) is a natural consequence of the fact that the difference in mobilities between AD and AF is greater than that between AV and AD. Therefore, even if the degree of heterogenity in composition remains the same, the mobility-value of the mixed   Fey   | OV ’ D+F sm AA. SS Fig. 2, Ascending-limb electrophoretic patterns of TMV-pro- tein mixtures at pH 4.9. m=main peak, s~second peak, other abbreviations as in Fig. 1. For explanation see text. 5S. SARKAR aggregates will be scattered over a wider range in case of AF + AD, In case of AV + AF, although two separate peaks are seen (fig. 2c), it is found that the main peak corresponding to protein cylinders has a mobility intermediate between the main AV and the main AF aggregates while the second peak corresponding to discs lies also intermediate between the correspond- ing second peaks of the pure aggregates (Fig. 3).               b<—ascending—>| |«—descending—> iar « = £ OO “| mq sti rl — <q S r mr, 8 = n sd = 990-7} —+ 44 8 ‘4 ‘SJ = 5 > 5 880 |-\\\\— - _ Mm 070 . ce _In. $ & 960                   D pey V yor F Feo ® pey V yor F rep Fig. 3. Diagrammatic representation of the relative positions of schlieren peaks produced by TMV-proteins both alone and in binary mixtures at py 4.9, after an arbitrary but equal time of electrophoresis under identical conditions. Abbreviations as in figs. 1 and 2. For explanation see text. It is, however, interesting to note that whenever 4D is present, the united aggregates move as a single boundary. The tendency of AD-units to unite to long cylinders seems to be so strong that the free existence of AV and AF discs (cf, Kramer and Wirrmann, 1958%) is rendered difficult. In any case, the absence of peaks corresponding to pure AF and pure AD aggregates and very probably also of pure AV aggregates in the mixed solutions at pu 4.9 speak strongly for the existence of mixed aggregates, The turbidity observed in each mixed solution at Pu 4.9 was as far as possible removed by centrifug- ing at about 7000 g for 30 minutes in the cold and the opalescent supernatant was examined electro- phoretically. The peaks observed maintained the same contour and mobility as before. After the run, INTERACTION AND MIXED AGGREGATION OF PROTEINS each solution was dialysed against glycine-NaOH buffer, py 10.4 to split the mixed aggregates again into their constituent A-proteins as described be- fore. Electrophoresis in Michaelis buffer py 8.0 exhibited again the presence of two peaks of ap- proximately equal concentrations as judged by their area; the mobilities have already been given in Table 3. In sharp contrast to the formation of mixed ag- gregates among vulgare, flavum and dahlemense proteins, those of vulgare and Holmes’ rib grass strain maintained their identities even in intimate mixture. As the py value of a mixture of AV and AHR was lowered stepwise (sample 4a in Tab. 1), the AHR units aggregated into rods early. At pu 9-9 there are at first three gradients with mobili- ties 0.39, 0.75 and 1.10, which correspond to vul- gare A-protein, aggregated HR protein and aggre- gated vulgare protein respectively (Table 5). After about 14hours of standing, the slowest peak cor- responding to free AV practically disappeared, the fastest assumed a larger area showing the forma- tion of more vulgare-aggregates, while the middle one (HR-aggregate) remained unchanged (Fig. 4). On further lowering the py value to 5.0 the solution became too turbid for electrophoretic runs.       | U = (u/sec)/(V/em) i | AV | AHR 8.0 | 0.46 0.85 6.5 | 0.46 0.86 6.1 0.45 : 0.85 5.5 | 0.39 and 1.10* 0.75% 5.5 0.40 and 1.14* 0.79* 5.0 too turbid |   | Table 5. Anodic mobilities (U) of observed peaks in a mix- ture of AV and AHR in Michaelis buffer of different pH values. J'=0.037. (Results of sample 4 a from table 1.) * Aggregated. We Fig. 4. Ascending-limb electrophoretic patterns at pH 5.5 showing the gradual transformation of vulgare protein (V) into vulgare-aggregates (Vn) of higher mobility in the pre- sence of aggregated Holmes’ rib grass protein (HRn). The lower pattern was observed 14 hours after the first run (upper diagram). For explanation see text. 783 Obviously, no mixed aggregates were formed. During such a slow and stepwise lowering of the py, the aggregation of HR-protein is complete before vulgare-protein approaches its isoelectric point. To bring about quick aggregation, therefore, a mixture of AV and AHR at py 8.5 (sample 4b of table 1) was dialysed directly against Michaelis buffer of py 5.2 of ionic strength 0.037. On analysis, two sharp peaks were obtained in both arms of the Tiselius cell with mobilities corresponding to pure vulgare- and pure HR-aggregates (Fig. 5 and Table 6), the deviation being well within the range of experimental errors. Both the gradients are sym- metrical and the Schlieren pattern returns to the base line between peaks indicating the absence of mixed gradients having intermediate mobilities.   +t Fig. 5. Ascending-limb electrophoretic pattern of a mixture of vulgare- and Holmes’ rib grass proteins at prj 5.2.   U = (u/sec)/(V/em) Sample wo ee pooccwnsae= ascending descending AHR alone 0.67 0.65 AV | alone 1.09 | 1.06 AV+t AHR 0.67 and 1.08 0.68 and 1.05       Table 6. Anodic mobilities of AV and AHR at py 5.2. III. Discussion The protein mixtures between vulgare, flavum and dahlemense in weakly alkaline media undergo varying degrees of interaction. Among the various types of protein-protein interactions, a simple dis- sociation and association of peptide chains has been reported from time to time, whereby either relati- vely stable complexes are formed or the different forms remain in a state of dynamic equilibrium. If the six polypeptide chains of .AV and AF could dissociate freely and combine reciprocally with one another, the different mixed units would possess characteristic mobilities as listed below (Table 7), considering that the preferred size of the aggregates remains at six-polypeptide chains and that in such smal] aggregates all the charged groups of each type of polypeptide chain are electrokinetically active.   784 . | Anodic mobility Configuration U == (u/sec)/(V/om) a) 6AV 0.460 b)SAV +1AF 0.445 c)4 AV +2AF 0.430 d)3AV+3AF 0.415 e)2AV +4 4F 0.400 fh lAV+5AF 0.385 g)64F 0.370     Table 7. Calculated mobilities of various mixed aggregates of vulgare and flavum proteins at py 8.0. There are only two observed peaks with mobili- ties which correspond to b and f. It is difficult to visualise why only these two configurations should accumulate preferentially. The mobility values of AV +AD and AF +AD mixtures do not conform well to such 5:1 combinations and the electro- phoretic diagrams speak more for a dynamic equi- librium. Continuously interacting protein mixtures cause boundary anomalies in sedimentation and electro- phoretic studies, which have been summarised and classified on the basis of the velocity constants of the forward and reverse reactions by Atperty and Marvin (1950) '®, Lonesworta (1959)18 and Brown and Timasnerr (1959)! among others, Fretp and Ocsron (1955)?° using human hemoglobin showed that the spreading of a boundary will be greater as the difference of velocities between the forms and their mean lives are greater. These boundary ano- malies should not, however, be confused with the more common types of anomalies encountered with high protein concentrations in comparatively low ionic strengths of the buffer as pointed out by various workers (Svensson, 1944 2!), Jounston and Oasron, 1946 7*, Loncswortu, 1947 23, and ALBERTy, 1948 24). The non-existence of such factors under the conditions of the present investigations is cor- roborated by the absence of boundary anomalies with AV + AHR mixture and by the constancy of mobilities over relatively wide ranges of protein concentrations. An understanding of the dynamic state of equi- librium between the protein mixtures under con- 8 L.G. Loxeswonts, in: Electrophoresis, edited by Milan Bier, Academic Press 1959, Pp. 91 —136. 19 R.A. Brown and S.N. Timasnerr, in: Electrophoresis, edited by Milan Bier, Academic Press 1959. Pp. 317 — 367. 0 E. O. Fintp and A. G. Oesroy, Biochem. J. 60, 661 [1955]. 21H. Svensson, Ark. Kem., Mineralog. Geol., Ser. A 17, 14 f1944]. S. SARKAR sideration demands a knowledge of the mode and rate of dissociation of the proteins. This is difficult, since —as already stated in introduction — the size of an aggregate depends upon various interacting factors. Ultracentrifugal studies on dissociation of TMV-protein into single polypeptide chains have not provided definite clue as to whether the dissociation proceeds through definite steps or not (cf. ANSEVIN and Laurrer, 1959°%, Wirrmann, 195919), With- out, therefore, attempting to ascribe definite mobi- lity values for particular mixed aggregates in weakly alkaline media by postulating particular dis- sociation types as 6P = 3P+3P, 6P24P+2P or 6P 25 P+1P where P stands for a single poly- peptide chain, it can be stated that wherever mixed aggregates of transient existence are formed, it is evidenced by a flattening of the electrophoretic dia- grams and by an approaching together of the respec- tive Schlieren peaks. A simple case would be the one where a vulgare “A-protein’”-unit associates with one of flavum, thus forming shortlived “double- A-protein-units” of intermediate mobility. The marked deviation from enantiography in the two arms of the Tiselius cell is characteristic of reaction-boundaries. The descending boundaries could not be used to calculate exact mobilities for evident reasons but the ascending values were well reproducible. According to Svensson (1946)?5 “the generalisation that all disturbances are smaller on the descending side is no doubt erroneous and the omission of data from the ascending side involves an unjustifiable waste of experimental material”. In contrast to the mobility-values of AV + AF mix- ture at py 8.0 where both schlieren peaks appear to approach each other, it is remarkable that with AD + AF mixture the mobility of the faster peak corresponds very closely to that of pure AD while there is no peak corresponding to pure AF on the ascending side. The difference in mobility between AD and AF is rather large and, therefore, on the ascending side, where the particles move out of the solution into the buffer above, some of the faster units will have a chance to escape the zone of rapid dissociation and reciprocal association. Once out of 2 2 J.P. Jounstox and A. G. Ocsron, Trans. Faraday Soc. 42, 789 [1946]. 3 L. G. Lonasworru, J. physic. Chem. 51, 171 [1947]. °4 R.A. Avserty, J. Amer. Chem. Soc. 70, 1675 [1948]. ®5 H. Svensson, Ark. Kem., Mineralog. Geol, Ser. A 22, 10 [1946]. INTERACTION AND MIXED AGGREGATION OF PROTEINS the zone, the faster particles can gain their own mobility and thereby the front end of the schlie- ren diagram can move with a mobility equal to that of the pure faster component. The situation in the descending arm would be the reverse. On this side there is actually a small but clearly isolated peak corresponding to pure AF-units with a mobility 0.33 to 0.35 (Fig. 6). ee <_—— Fig. 6. Descending-limb electrophoretic pattern of a mixture of flavum- and dahlemense-proteins at pH 8.0. Arrow indicates the direction of migration. The proteins of vulgare and Holmes’ rib grass strains do not obviously exchange their polypeptide chains with each other, though each of them is sub- ject to reversible dissoziation. Both in weakly alka- line media and at py ~ 5 the observed peaks main- tain the characteristic mobilities of pure vulgare- and pure Holmes’ rib grass-proteins. An explanation for the formation of mixed aggre- gates on lowering the py value of binary mixtures of AV, AF and AD described before is superfluous on the basis of the interactions observed in weakly alkaline media. Obviously, with an increase of the H-ion concentration, large aggregates are formed in which polypeptide chains of different strains are packed together in various possible combinations. On simple statistical grounds, particles of greater heterogenity will be more frequent than relatively homogeneous aggregates and thereby the observed peak will depict an intermediate mobility. Somewhat similar observations with hemocyanins from three different species of snails were reported by TiseExius and Horsratz (1939) 76, Asymmetric recombina- tion of different types of human and canine hemo- globins, on rapid neutralisation of a mixture of acid-split half-molecules has been reported by Rosinson and I'rano (1960) 7\", Although mixed aggregation between proteins from vulgare, flavum and dahlemense strains of TMV have been observed, no such association was found between proteins of vulgare and Holmes’ rib 26 A. Tisetivs and F.L. Horsrau, J. exp. Medicine 69, 83 [1939]. 27 FE, Rostnson and H. A.Irano, Nature [London] 185, 547 [1960]. 785 grass strains. The polyp", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-avvk_pk9d_bnre", "00000000-0000-0000-8686-EC1BCD151786", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Some Characteristics of a Cell-Free DNAase Sensitive System Incorporating Amino Acids into Protein", "101584910X130", null, "1961", "1961", "This abstract explains the experimental process by which Nirenberg and Matthaei demonstrated the cell-free system's incorporation of amino acids into protein.", "Abstracts (summaries)", "Amino Acids,Deoxyribonucleases", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of the Federation of American Societies for Experimental Biology.", "Copyright may apply", null, null, "300-305 PROTEINS; POLYPEPTIDES $91 Biochemistry SOME CHARACTERISTICS OF A CELL-FREE DNAase SENSITIVE SYSTEM INCORPORATING AMINO ACIDS INTO PROTEIN. J. Heinrich Matthaei*™ and Marshall W. Nirenberg”. National Institutes of Health, Bethesda, Md. Extracts of E. coli W3100 prepared by grinding cells with alumina actively incorporate Cl4-valine into protein. The extracts were centrifuged at 20,000 x g for 20 minutes, Remaining intact cells and debris were removed from the super- natant suspension by centrifuging at 30,000 x g for 60 minutes. Ribosomes were obtained by centrifuging the supernatant sus- pension for 2 hours at 105,000 x g. For maximm incorporation of Cl4-valine into protein reaction mixtures require ATP, Mg++ an ATP generating system, a complete amino acid mixture, ribo- somes and a 105,000 x g supernatant solution. Incorporation of cl4-valine into protein proceeds at a rapid rate for 15 minutes at 37°, Incorporation is markedly inhibited by 50 pg. chloramphenicol per mi, 10 ug. per ml. DNAase inhibited approximately 70% of the incorporation whereas an equivalent amount of RNAase was completely inhibitory. Inhibition by DNAase cannot be reversed by addition of polyanions. Addition of a DNAase digest of salmon sperm DNA had no effect upon incorporation of cl4.valine into protein. Although DNAase markedly inhibits amino acid incorporation into protein, it ia not known whether intact DNA is necessary for this process.", "Nirenberg, Marshall W. ; Matthaei, Heinrich", null, "Federation Proceedings", "Federation of American Societies for Experimental Biology", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-uzz3_gmqg.geaj", "00000000-0000-0000-462D-76BB93638DE4", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Chemically Synthesized Deoxypolynucleotides as Templates for Ribonucleic Acid Polymerase", "101584910X132", null, "1963", "September 1963", "Khorana's methods for synthesizing nucleic acids were a prerequisite for the final solution of the genetic code.  Additionally, Nirenberg regularly made reference to Khorana's contributions, which eventually earned him a share of the Nobel Prize in 1968.  This article provides the results from the types of synthesizing efforts instrumental in his winning the Nobel.", "Articles", "Polynucleotides,DNA,RNA", "Translating the Code of Life and the Nobel Prize, 1962-1968", "6", "pages", "Text", "English", "Reproduced with permission of the American Society for Biochemistry and Molecular Biology.,Reproduced with permission of Julius Adler.", "Copyright may apply", null, null, "Tue JournaL or Biotogican CHEMISTRY Vol. 238, No. 9, September 1963 Printed in U.S.A. Chemically Synthesized Deoxy polynucleotides as Templates for Ribonucleic Acid Polymerase* Arturo Fanascut,t Junius ADLER, anp H. G. Kuorana From the Departments of Biochemistry and Genetics and the Institute for Enzyme Research, the University of Wisconsin, Madison 6, Wisconsin (Received for publication, April 25, 1963) The enzymatic synthesis of ribonucleie acid from the four ribonucleoside 5’-triphosphates in the presence of deoxyribo- nucleic acid has been documented in a number of laboratories (1-11). The reaction is catalyzed by an enzyme called RNA polymerase and the DNA determines the composition and the sequence of nucleotides in the synthesized RNA according to the base-pairing principle first recognized for DNA by Watson and Crick (7-11). The RNA synthesized in this manner stimu- lates the incorporation of amino acids into protein (12-15). A deoxypolythymidylate fraction obtained by chemical syn- thesis (16) was tested previously by Hurwitz et al, (7, 11) and was found to bring about the synthesis of ribopolyadenylic acid. In the present work, purified synthetic deoxypolynucleotides of known size and sequence have been used to study further the mechanism of action of RNA polymerase. The results reported herein show that deoxyoligonucleotides as small ag the penta- nucleotide dT; can serve as templates for the synthesis of ribo- polyadenylate.' The rate of the synthetic reaction increases with an increase in the size of the deoxypolythymidylate until a chain length of 14 is reached, and this polymer was actually more active than DNA. The product synthesized from deoxy- polythymidylate of various sizes was invariably very much larger than the size of the template. Several lines of evidence showed that the enzyme initiated the synthesis of new chains, rather than causing esterification to the 3’-hydroxyl end of the deoxy- polynucleotide chains. Finally, in these simpler systems, the incorporation of the ribonucleoside 5/-triphosphates again fol- Jowed the Watson-Crick base-pairing principle, although some exceptions were noted. A brief report of these findings has already appeared (17). * Supported by grants from the National Institutes of Health, the National Science Foundation, Life Insurance Medical Re- search Fund, and the Graduate School of the University of Wis- consin. { Fellow of the International Laboratory of Genetics and Bio- physics, Naples, Italy. Present address, Department of Bio- chemistry, Stanford University Medical School, Palo Alto, Cali- fornia, ? All of the chemically synthesized polynucleotides used in the present work carry a 3'-hydroxyl group at one end and a 5’-phos- phate group at the opposite end. For convenience, this class of homologous polynucleotides is simply designated in the text by the nucleoside initial with a subscript indicating the number of nucleosides in the chain. For example, deoxy-pTpTpTpTpT is dTs and deoxy-pCpCpCpCpCpC is dCs, ete. A, G, C, U, and T stand for adenosine, guanosine, cytidine, uridine, and thymidine. EXPERIMENTAL PROCEDURE RNA polymerase was purified from Escherichia coli according to the procedure described by Chamberlin and Berg (10). #. cold phosphodiesterase (18) was a gift from Dr. I. R. Lehman. C™- Ribonucleoside 5’-triphosphates were purchased from Schwara BioResearch, Inc. Deoxyribopolynucleotides—Deoxy cytidine, deoxyadenosine, and deoxyguanosine oligonucleotides were prepared by published procedures (19-21). The homologous deoxythymidine poly- nucleotides dT, to dT, were prepared as described previously (16, 22), and CJabeled thymidine polynucleotides were pre- pared by Mr. W. J. Connors by adaptation (23) of these pub- lished procedures (16, 22). Pure thymidine polynucleotides dTy. to dTy, and a fraction containing members higher than dTy4 were prepared from the 1 M triethylammonium bicarbonate fraction obtained in an experi- ment described previously (Khorana and Vizsolyi, Table I, and Fig. I (16)). The latter fraction, as mentioned earlier (16), was a complex mixture of polynucleotides containing apparently a high proportion of oligonucleotides linked together by pyrophos- phate bridges between the terminal 5’-phosphomonoester groups, with general structure pT (pT) .pT “4 \\\\ pT (pT) pT The mixture (110 optical density units at 267 my) was treated in dry pyridine (2 ml) with 0.5 ml of acetic anhydride for 3 days in order to selectively cleave the pyrophosphate bonds, and the reaction mixture was worked up as described earlier (22). Chro- matography on a DEAE-cellulose (carbonate form) column (50 X 1.2 em) gave a series of peaks which were processed by the standard method developed earlier (16). As a result of cleavage of the pyrophosphate bonds, more than 50% of the ultraviolet- absorbing material was present as a mixture of oligonucleotides smaller than dTj. The higher homologues were then applied to paper along with previously characterized dT,; as marker. The dTy», dT,3, and dT traveled with progressively decreasing Hp values when chromatographed for 2 weeks in descending n-propy! alcohol-concentrated ammonia-water (55:10:35). Acetylation of 3'-Hydrozyl End Groups in Penta- and Undeca- thymidylic Acids—An aqueous solution of ammonium salt of the polynucleotide (2 zmoles of thymidine) was passed through a column (1 X 2 em) of Dowex 50 ion exchange resin (H+) and 3080 September 1963 the total effluent and washings evaporated after addition of pyridine (1 ml). To the residue was added triethylamine (0.05 ml) and pyridine (2 ml) and the solution was re-evaporated with vacuum from an oil pump. The residue was rendered anhydrous by repetition of evaporation after addition of dry pyridine. Finally, dry pyridine (0.5 ml) and acetic anhydride (0.2 ml) were added and the sealed reaction mixture kept in the dark at room temperature for 4 hours. Water (2 ml) was then added and the total solution kept for 2 hours at room temperature. It was then evaporated under reduced pressure to an oil which was extracted with dry ethyl ether several times. The insoluble polynucleotide material was obtained as a fine solid deposit on the wall of the round bottom flask. It was dissolved in water and the aqueous solution was lyophilized. The solid residue was made up to 0.2 ml with water. Resistance of Polynucleotides Bearing Terminal 3'-O0-Acetyl Groups to E. coli Phosphodiesterase—An exonuclease purified from E. colt has been shown by Lehman (18) to degrade deoxy- ribopolynucleotides in a stepwise manner from the end bearing a, 3’-hydroxyl group. The reaction produces deoxyribonucleo- side 5’-phosphates until the chain length is reduced to the dinucleotide (18). In a control experiment, 0.17 pmole of the tetranucleotide d-pTpTpTpT was incubated at 37° in a 0.2-ml incubation mixture in the presence of 0.02 ml of 1 m Tris buffer (pH 7.5), 0.02 ml of 0.1 m magnesium chloride, and 100 units (18) of enzyme. Degradation to d-pT and d-pTpT was com- plete in under 2 hours as determined by paper chromatography of aliquots in descending ethy] alcohol-0.5 m ammonium acetate buffer, pH 3.8 (7:3, v/v). Incubation of the 3’-O-acetyl d- pTpTpTpTpT under identical conditions up to 4 hours showed complete resistance of the oligonucleotide to the enzyme. Ina second experiment the use of a 3-fold higher concentration of the enzyme preparation under the above conditions showed like- wise the absence of any degradation. On the other hand, the degradation of oligonucleotides bearing 3’-hydroxyl groups pro- ceeded normally in the presence of the 3'-O-acetyl derivatives, showing that the latter were not inhibitory. Assay of RNA Polymerase—The reaction mixture was exactly the same as that described by Chamberlin and Berg (10), except that synthetic deoxypolynucleotides usually replaced DNA. When DNA was used as primer, the C4“ RNA synthesized was measured after precipitation with perchloric acid, exactly as described by Chamberlin and Berg (10). When synthetic deoxypolynucleotides were used, .a new assay was devised in order to be able to detect any low molecular weight ribopolynucleotides that might be too small to precipitate in acid. The reaction was stopped with 0.02 ml of concentrated ammonium hydroxide and the entire mixture was then deposited in one pipetting onto the origin of a 2.5- x 57-cm strip of DEAE- cellulose? paper (Whatman DE-20). Descending chromatog- raphy was carried out in 0.8 mM ammonium formate for 23 hours. Under these conditions polynucleotides as small as ribotetra- adenylate remain at the origin, whereas unused nucleoside tri- phosphates move away. The strip was then left to dry in air. The area containing the C™-ribopolynucleotides (from 2.5 em below to 2.5 cm above the origin) was cut out, folded in half at the origin, placed folded up in a scintillation vial containing sol- vent (3 g of 2,5-diphenyloxazole and 100 mg of 1,4-bis-2-(5- phenyloxazolyl)benzene per liter of reagent grade toluene), and 2 The form DEAE- refers to diethylaminoethyl-. A. Falaschi, J. Adler, and H. G. Khorana 3081 counted at 2-80 (875 volts) and window settings of 10, 50, and 100 in a Packard Tri-Carb liquid scintillation spectrometer. Determination of Size of Ribopelynucleotides—After the area containing the C'-ribopolynucleotide had been counted as de- scribed above, it was cut into small pieces and incubated in 2 mi of 0.8 Nn NaOH for 20 hours at 37° to hydrolyze the ribopoly- nucleotide. The liquid was then filtered and chilled in ice. It was neutralized by adding slowly small amounts of dry Dowex 50 (H*) resin; after each addition, about 5 minutes with occasional stirring were allowed before the pH was measured. When the pH reached 7, 5 wl of 1 nN NaOH were added in order to avoid any dephosphorylation which could occur if the pH became less than 7 during the subsequent manipulations. The supernatant liquid, combined with 0.1 N ammonium hydroxide washings of the resin, was concentrated by lyophilization and spotted on a strip, 2.6 em X 57 em, of DEAE-cellulose paper. Descending chromatography in 0.2 m ammonium formate was carried out for 6 hours until the front reached the end of the paper. This served to separate added (as markers) adenosine, adenosine 3’-phosphate, and adenosine 2’(3’) ,5’-diphosphate. The dried strip was cut at 1-cm intervals and these pieces were put into scintillation vials and counted as described above. The ratio of radioactivity in adenylic acid to adenosine or in adenylic acid to the adenosine diphosphate was considered the average size of the ribopolynucleotide. RESULTS Deoxypolythymidylate as Template for Synthesis of Ribepolyadenylate Effect of Chain Length on Rate of Synthesis—The initial rate of incorporation of adenylate varied with the size of the deoxypoly- thymidylate at saturating concentrations of each polymer, as shown in Fig. 1. With dT; no activity was observed under the conditions used, and with dT, there was occasional activity. But dT; always brought about significant, although small, incorporation of adenylate. With further increase in size the effectiveness of the polymers then rose, at first slowly up to dT7, then with a big leap upward between lengths of 8 and 11. The activity reached   ° ¢ nN + PORATED/IOMIN. 10) mMOLES INCOR          4 6 8 190 l2 4 16 CHAIN LENGTH Fic. 1, Effect of chain length on the activity of deoxypoly- thymidylate. The experimental procedure for measuring the incorporation of adenylate from Cl-ATP is described under “Assay of RNA Polymerase.”’ 3082 Tasie I Efficiency of DNA and deoxypolythymidylate in stimulating RNA polymerase The experimental procedure is described under “Assay of RNA Polymerase.”   Incorporation of nucleotide   Polymer Only C'4-ATP UTP, and present CATE present   mumoles/10 min Calf thymus DNA................... 14.1 0.82 Heated calf thymus DNA*........... 4.8 3.05 Deoxypolythymidylate, 1 m fraction. / 20.0 AD ace e ees | 34.0   *Thymus DNA was heated by immersion in a boiling water bath for 10 minutes, then chilled in ice. a maximum at dT, and dTi;. Table I shows that dTy, is actu- ally more active for ribopolyadenylate synthesis than DNA is for the synthesis of RNA in the presence of all four nucleoside triphosphates. Hurwitz et al. (7, 11) had already shown that the 1 M triethyl- ammonium bicarbonate fraction referred to above is active for the synthesis of ribopolyadenylate. We have now confirmed the activity of the same 1 m fraction and have found it to be about 60% as active as dTy, (Table I). This lesser activity is probably due to the presence of less active polymers or inhibitors in the mixture. As mentioned above, the 1 m fraction contains polymers of various sizes larger than dTy, and also oligonucleo- tides linked together by pyrophosphate bridges. It seems very likely that such pyrophosphate compounds are inhibitors for the priming action of polynucleotides. RNA polymerase will catalyze the synthesis of ribopoly- adenylate when DNA is present together with ATP as the only nucleoside triphosphate (10). Denatured DNA is a preferred primer for this activity (24).3 Table I shows a confirmation of these results and a comparison of DNA with the activity of the 1 m fraction and dTy,. For the synthesis of ribopolyadenylate dT, is the most active polymer. In order to be sure that the comparison of the results with polynucleotides of different sizes was meaningful, it was neces- sary to check that the primer did not undergo degradation during the incubation with RNA polymerase (see also below). C4-Labeled dTz was used in the usual reaction mixture except that ATP was omitted. The mixture was incubated as usual and then put on a DEAE-cellulose carbonate column (0.6 x 27 cm) and cluted with a linear gradient of triethylammonium bicarbonate from 0 to 0.48 m (total volume, 500 ml). More than 99.7% of the radioactivity appeared as a single peak in the posi- tion corresponding to dT;. This ruled out the possibility of detectable breakdown of the polymer by the enzyme preparation. Effect of Chain Length on Saturating Concentration of Tem- plate—A saturating concentration was determined for each poly- mer that stimulated the synthesis of ribopolyadenylate in the experiment of Fig. 1. For dT;, dT, and dT, the results are plotted in Fig. 2 according to the method that Lineweaver and Burk have used for substrates (25). The polymer concentra- tions which gave half-maximal rates with dT7, dTu, and dTi.4 3M. Chamberlin and P. Berg, unpublished observations. Synthetic Deoxypolynucleotides as Templates for RNA Polymerase Vol. 238, No. 9 were found to be 50 X 1076, 20 x 1078, and 2.0 x 107° m, re- spectively. Increasing the size of the polymer not only increases the maximal velocity of the reaction (as shown also by Fig. 1) but decreases very strikingly the concentration of polymer re- quired for half-saturation. Apparently the affinity of the deoxy- polythymidylate for the enzyme increases markedly with size be- tween dTy and dT. Size of Ribopotyadenylate Formed—The C'-ribopolyadenylate formed in the presence of deoxypolythymidylate of various sizes was hydrolyzed with sodium hydroxide to C-adenosine, C'adenosine 2'(3’)-phosphate, and a C-material that resem- bled adenosine 3’,5’-diphosphate but was not further char- acterized. The details are described under “Experimental Pro- cedure.” The ratio of radioactivity in adenylic acid to adenosine and in adenylic acid to the adenosine diphosphate was taken to be the average size of the ribopolyadenylate. Table IT lists the results for the size of the products formed from dT;z, dTs, dT», dTu, and dTy4; for smaller thymidine oligonucleotides too little product was available to provide significant results. The estimate of the chain length of the products is only approximate, owing to the inaccuracy of counting on DEAE paper the small amounts of radioactivity in the adenosine and in the adenosine diphosphate region. The most striking feature of the results is that the product is of a much larger size than the deoxypoly- thymidylate. No marked differences are apparent between the sizes of the products obtained with different sized deoxypoly- thymidylates. Evidence for Noninvolvement of Terminal 8'-Hydroxyl Groups of Polythymidylate in Ribopolyadenylate Formation—During chromatography of the total alkaline hydrolysate of the ribopoly- adenylate (see above), no significant amount of radioactivity           ‘aT 7 A 1S Lob 0.5- dT) 005 010 O15 020 025 15 B dT AOL LL dy v 14 05 1 ! l 1 05 os 5 20 25 Cc Fic. 2. Reeiprocal plot of the effect of concentration of deoxy- polythymidylate on the incorporation of adenylate. A compares dT; and dTu; B compares dT and dT. The concentration of polymer is expressed as millimicromoles of polymer (not nue- leotide equivalents) per ml; v, millimicromoles of adenylate incorporated from ATP per 10 minutes, as measured by the DEAE-cellulose paper assay described under ‘‘Assay of RNA Polymerase.” September 1963 remained at the origin of the chromatogram where the deoxyribo- polythymidylate remains adsorbed. This result indicated that ribopolyadenylate synthesis was not initiated by esterification of the terminal 3’-hydroxyl group in deoxypolythymidylate to form a phosphodiester linkage. Such a linkage would have been resistant to alkaline hydrolysis and a product of the general structure (Diagram I) would have resulted. T T T A OH \\\\ P P P P. P Diagram I To test further whether the 3’-hydroxyl group is essential in the synthesis of ribopolynucleotides, 3’-O-acetyl-dT,, was pre- pared for testing as a primer. Table III (Lines 1 and 3) shows that 3’-O-acetyl-dT,; is about as active as dT. The concen- tration of 3’-O-acetyl-dT, required to half-saturate the enzyme was very similar to the concentration already found for dT. This diminished the possibility that a small amount of unacety- lated dT}, was the active component in the 3’-O-acetyl-dT 1, prep- aration. Furthermore, 3’-O-acetylthymidylate was found to be Tas ie II Size of ribopolyadenylate formed from deoxypolythymidylate tenvplates The experimental procedure is described under ‘‘Size of Ribo- polyadenylate Formed.”                 Radioactivity Chain length Polymer Ad : ; ; diphosphate Adenyiy | Adenosine | Bya | B/C | Average c.p.m, aT, 29 2,550 96 73 27 50 dTs 80 10,100 135 125 74 100 dT, 70 4,680 32 67 146 107 dT 1 34 3,590 86 101 46 7 dT 14 114 9,080 63 79 144 111 TaBLe III Acetylated deoxypolythymidylate as template for RNA polymerase Treatment with #. coli phosphodiesterase was carried out at 37° in a 0.15-ml reaction mixture containing 100 units (18) of enzyme, 0.02 ml of 1 m Tris buffer (pH 7.5), 0.01 ml of 0.1 m mag- nesium chloride, and 0.4 umole of nucleotide equivalent of poly- nucleotide. Then 0.02-ml aliquots of this were added to an RNA polymerase reaction mixture without prior inactivation of the diesterase, since it could be shown that the presence of diesterase does not interfere with the synthesis of ribopolyadenylate.       Polymer Treatment with | Incorporation of phosphodiesterase ; adenylate from ATP | mumoles/30 min dT LLe eee eee _ | 2.30 C6 + 0.04 3'-O-acetyl-dTir... 200.00. ..0.00. - | 2.37 3/-O-acetyl-dTi......-...000--. + 1.32   A. Falascht, J. Adler, and H. G. Khorana 3083 TasLe IV Deoxypolycytidylate as template for synthesis of rtbopolyguanylate The experimental procedure is deseribed under “Assay of RNA Polymerase.”’     Incorporation Incorporation of Polymer of guanylate from GTP adenylate from ATP myumole/10 min myumoles/10 min dCs <0.05 dCs <0.05 dCs <0.05 dC, 0.15 dCio 0.17 dT, 3.2   stable when incubated with the RNA polymerase preparation; this served to exclude any acylase activity. In order to ensure that none of the unacetylated dT, was present, the preparation of the acetylated polynucleotide was preincubated with the #. coli phosphodiesterase. Acetylation of the 3’-hydroxyl group in thymidine oligonucleotides confers resistance toward this enzyme (see above). Table III (Lines 2 and 4) compares dT), and 3'-O-acetyl-dTy; after treatment with phosphodiesterase. This enzyme abolishes nearly all of the activity of dT\\\\, whereas most of the activity of 3’-O-acetyl-dTy; remains resistant to phosphodiesterase. It may be concluded that 3’-O-acetyl-dT,; is active for the synthesis of ribopolyadenylate, and that a free 3’-hydroxyl group of deoxypolythymidylate is not essential for ribopolyadenylate synthesis to oceur. It follows that addition of adenylate to the 3/-hydroxyl end of deoxypolythymidylate is not a necessary part of ribopolyadenylate synthesis. Deoxypolycytidylate as Template for Synthesis of Ribopolyguanylate Incubation of deoxypolycytidylate with RNA polymerase and GTP led to the formation of ribopolyguanylate, and the rate of the reaction depended on the size of the deoxypolycytidylate (Table IV). Whereas a chain length of 5, 6, or 8 proved too small to be effective, dC, and dC, showed significant activity. The dependence of the reaction on the concentration of dC Is shown in Fig. 3; half-saturation occurred at 5.4 x 10-8 a. A comparison of the deoxypolycytidy late series with the deoxypoly- thymidylate series shows that lower homologues are more effec- tive in the latter series (Table IV) but a lower concentration of the deoxypolycytidylate is required for saturating the enzyme. Deoxypolycytidylate of much higher molecular weight than used here, prepared from the deoxypolycytidylate-deoxypolyguanylate polymer (26), has been shown by Chamberlin and Berg to be active for the synthesis of ribopolyguanylate (27). Experiments with Deoxypolyguanylate and Deoxypolyadenylate In the deoxypolyguanylate series, only dGg was tested. It proved to be inactive for the incorporation of nucleotide from CTP. This may be explained by the finding that even small homologues of deoxypolyguanylate have a high tendency to form aggregates of very large molecular weight (28). Chamber- lin and Berg (27) have reported that deoxypolyguanylate pre- pared from the deoxypolycytidylate-deoxypolyguanylate poly- mer is also inactive for the incorporation of cytidylate. 3084   40 3.0- I/V 20+ oO     |   al 0.2 0.3 I/CONCENTRATION OF POLYMER Fie. 3. Reciprocal plot of the effect of concentration of dCi) on the incorporation of guanylate. The experimental procedure is described under ‘‘Assay of RNA Polymerase.’’ The concentra- tion of polymer is expressed as millimicromoles of polymer (not nucleotide equivalents) per ml; v, millimicromoles of guanylate incorporated from GTP in 10 minutes, as measured by the DEAE- cellulose paper assay described under ‘Assay of RNA Polymer- ase,’’ TaBLe V Specificity of incorporation of nucleotide The experimental procedure is described under ‘‘Assay of RNA Polymerase.”’   Nucleotide incorporated Polymer     ATP only GTP only | CTP only |ure only   | mumoles/10 min Poly dT, 1 m fraction 8.7 |<0. 05-0.65/ <0.05 | <0.05 dC io <0.05 0.69 <0.05 | <0.05 Poly dA, 1 m fraction 1.4 <0.05 <0.05 | <0.05 dGe | <0.05   Deoxypolyadenylate homologues of sizes up to dAs and a 1 M fraction that contained a mixture of homologues larger than dAg failed to show significant incorporation of nucleotides from UTP. Possibly, conditions other than those used so far might be effec- tive for the synthesis of ribopolyuridylate. Specificity of Incorporation of Nucleotides In the presence of the 1 m fraction of deoxypolythymidylate, no significant incorporation of CTP or UTP was observed in 10 minutes, under conditions in which 8.7 mmoles of adenylate from ATP were incorporated (Table V, Line 1). Surprisingly, 0.65 mumole of guanylate from GTP was incorporated under the same conditions. As the enzyme preparation became older, the incorporation of guanylate disappeared at a time when the in- corporation of adenylate had decreased by only 60%. Furth, Hurwitz, and Anders in one experiment (7) also noted a small incorporation of guanylate. Deoxypolycytidylate brought about specific incorporation of guanylate (Table V, Line 2). With dC,o, 0.69 mymole of nucleo- tide was incorporated from GTP but there was no significant incorporation of nucleotide from ATP, CTP, or UTP. With the 1 m fraction of deoxypolyadenylate, the incorporation of 1.4 mumoles of nucleotide from ATP was unexpectedly ob- served (Table V, Line 3); in this experiment there was no signifi- cant incorporation of nucleotide from UTP, CTP, or GTP. Synthetic Deoxypolynucleotides as Templates for RNA Polymerase Vol. 238, No. 9 DISCUSSION The present work has demonstrated that short chain deoxy- polynucleotides serve as templates for the synthesis of ribopoly- nucleotides in the presence of RNA polymerase. The effective- ness of the homologous members in the reaction increases with an increase in chain length, the maximal rate in the case of the polythymidylate series being reached with dT. It is note- worthy that the maximal rate here obtained was higher than the rate normally obtained when all the four ribonucleoside triphos- phates and DNA are used. The saturating concentrations of the thymidine polynucleotides decreased with an increase in chain length and the enzyme showed high affinity even for the short chain length dTy. A major point of interest established by the present work has been that the ribopolynucleotide synthesis does not begin by adding nucleotides to the terminal 3’-hydroxyl group of the deoxyribonucleotide. This conclusion is based primarily on two findings: alkaline hydrolysis of the ribopolyadenylate product leaves no detectable adenylate in the deoxypolythymidylate, and dT carrying a 3’-O-acetyl group is still effective in the synthesis of ribopolyadenylate. Throughout this paper we have referred to the deoxypolynucleotides as ‘‘templates” for RNA polymerase rather than “primers” because the synthesis of the product in- volves the formation of new complementary chains instead of elongation of chains. Since addition to the end of the deoxypoly- nucleotide is not necessary, it may be that replication can begin anywhere along the template; this has significance at the bio- logical level, for it allows the synthesis of messenger RNA from any one of the genes in a DNA molecule without requiring syn- thesis from all. Although in the present work the Watson-Crick type of base- pairing was ordinarily observed, the slight incorporation of deoxy- guanylate in the presence of thymidine polynucleotides was noted, an observation which has also been made previously (7). A further noteworthy exception, which merits further study, was the formation of ribopolyadenylate when deoxyribopolyadenyl- ates were used as templates. The reaction resembles the previ- ously documented ribopolyadenylate-primed synthesis of ribo- polyadenylate (29), and its biological significance remains unknown. Under the conditions tested no polyuridylate syn- thesis occurred when deoxypolyadenylate was used as template. The failure is perhaps due to the lack of appropriate conditions, since the polyriboadenylate-dependent polyuridylate synthesis has already been found to be very sensitive to temperature and to the presence of a critical concentration of manganous ions (30). RNA polymerase brings about the synthesis of ribopoly- adenylate when DNA is present and ATP is the only substrate (10, 24). The reaction proceeds best when denatured DNA is used (24). The size of the ribopolyadenylate formed has been estimated to be in the range of 60 to 70 (10) and 400 (24). To explain that the expected short runs of polythymidylates in DNA could bring about the synthesis of much larger sized ribopoly- adenylate, Chamberlin and Berg (10) postulated a “slippage” mechanism whereby a run of AMP residues would slip along the sequence of thymidylate residues, leading to an elongation of the polyadenylate chain. The present work indicates that it would take a run of only five to seven thymidylates in DNA in order to synthesize ribopolyadenylate of a much larger size. All evidence points to the involvement of a single enzyme for the DNA-dependent synthesis of RNA and of ribopolyadenylate September 1963 (24) Since the synthesis of ribopolynucleotides observed here is so similar to the DNA-dependent polyriboadenylate synthesis, and since the same purified enzyme (10) was used here, it seems most probable that the syntheses studied in this work are due actually to RNA polymerase rather than some contaminating enzyme. The assay used in the present work involved separation of the product from the unreacted labeled nucleoside triphosphate by anion exchange chromatography on a DEAE-cellulose paper. Oligonucleotides as short as the tetranucleotide could be sepa- rated from the nucleoside triphosphates under the conditions used. This assay was developed to detect low molecular weight polynucleotides, which could be missed by the usual assay (10) that depends on acid insolubility of polynucleotides. The assay may be generally applicable in studies on polynucleotides, for example in determining the initial events in DNA and RNA synthesis. A related method based on the use of DEAE-cellulose paper has been previously reported by Bollum (31). The ribopolynucleotides obtained by the use of chemically synthesized deoxypolynucleotides and RNA polymerase may be expected to bring about the specific incorporation of amino acid into protein when added to an amino acid-incorporating system (32, 33). For example, the ribopolyadenylate synthesized here would be expected to lead to incorporation of lysine (33). SUMMARY Short chain thymidine polynucleotides serve as templates for the synthesis of ribopolyadenylate in the presence of RNA poly- merase. The effectiveness as template increases markedly with size. Thus, thymidine pentanucleotide shows detectable ac- tivity whereas the maximal activity is reached with tetradeca- nucleotide, the latter being more active for ribopolyadenylate synthesis than thymus DNA is for ribopolyadenylate or RNA synthesis. The product formed from the different sized tem- plates has in each case an average chain length of 50 to 100. The synthesis of ribopolyadenylate has been shown not to involve addition to the 3’-hydroxyl ends of polythymidylate. Deoxypolycytidylate larger than the octanucleotide brings about the synthesis of ribopolyguanylate. Deoxypolyadenylate has failed so far to give synthesis of polyuridylate. With deoxy- polyadenylate the synthesis of ribopolyadenylate is noted. Acknowledgment—We wish to acknowledge the technical assistance of Miss Rachael Gettle. REFERENCES 1. Weiss, 8. B., anp GiapstonE, L., J. Am. Chem. Soc., 81, 4118 (1959). 2. Wuiss, 8S. B., Proc. Natl. Acad. Sct. U. S., 46, 1020 (1960). 3. Hurwitz, J., BRESLER, A., AND Drrincer, R., Biochem. and Biophys. Research C", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-d2vt_aria.najd", "00000000-0000-0000-09A2-B4FDE4E36801", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Approximation of Genetic Code Via Cell-Free Protein Synthesis Directed by Template RNA", "101584910X133", null, "1963", "[January-February 1963]", "This article offers a solution to the technical problems of a direct biochemical approach to deciphering the genetic code.  Comparison of the nucleotide sequence of a gene with the amino acid sequence of its corresponding protein remained problematic as the sequence of nucleotides in each coding was initially unknown.  The authors demonstrate with experimental evidence that there are only a certain number of possible nucleotides per code word.  Additionally, by using tobacco mosaic virus RNA to direct the cell-free synthesis of a protein resembling TMV, the experiment suggests at least part of the genetic code appears to be universal.", "Articles", "DNA,RNA,RNA, Messenger", "Translating the Code of Life and the Nobel Prize, 1962-1968", "7", "pages", "Text", "English", "Reproduced with permission of the Federation of American Societies for Experimental Biology.", "Copyright may apply", null, null, "Approximation of genetic code via cell-free protein synthesis directed by template RNA MARSHALL W. NIRENBERG, J. HEINRICH MATTHAEI,! OLIVER W. JONES, ROBERT G. MARTIN, AND SAMUEL H. BARONDES National Heart Institute, National Institutes of Health, Bethesda, Maryland Arnoucx THE FIELD OF GENETiIcs attained a high degree of sophistication years ago, the molecular basis of genetic information storage and retrieval lagged behind. Only within the last 10 years has the chemical structure of DNA been established, mainly through the work of Chargaff et al. (3), Wilkins et al. (29), and Watson and Crick (28). Some 8 years ago Gamow (5) proposed a theoretical code and thereby stimulated a great deal of interest in this problem. The direct biochemical ap- proach, that is, comparison of the nucleotide sequence of a gene with the amino acid sequence of its corre- sponding protein, posed technical problems of great magnitude. Some of these difficulties have been circum- vented by the experimental approach summarized here. DEVELOPMENT OF CELL-FREE ASSAY FOR MESSENGER RNA C'*-amino acid incorporation into protein was studied in a cell-free E. coli system patterned generally after the systems established by Tissiéres, Schlessinger, and Gros (25), also by Lamborg and Zamecnik (g) and by Spiegel- man and his associates (24). A major difficulty in the study of cell-free protein synthesis in £. coli systems had been the necessity for preparing fresh enzyme extracts for each experiment. Techniques were not available for stabilization and storage of these enzyme extracts com- parable to those available for mammalian systems. E. coli extracts were found to be stable in the presence of mercaptoethanol and could be dialyzed and_ stored frozen without undue loss of activity (15). Independently, Tissiéres et al. (25), Kameyama and Novelli (8), and Nisman and Fukuhara (20) reported an inhibition of amino acid incorporation by deoxyri- bonuclease. We also observed such inhibition and studied its characteristics (15), for here appeared to be a cell-free system, in which DNA directed the synthesis of protein. The effect of deoxyribonuclease on C¥-valine incorpora- tion is shown in Fig. 1. In the absence of deoxyribonu- clease, C™-valine was rapidly incorporated into protein. 1NATO Postdoctoral Fellow. 55 Deoxyribonuclease completely inhibited incorporation occurring after 30 min of incubation (15). This type of experiment suggested that the deoxyribonuclease-in- hibited system might be dependent on the availability of messenger RNA. Crude messenger RNA fractions were found to stimulate amino acid incorporation and proved to be a requirement for cell-free protein synthesis (17, 18). This technique afforded a highly sensitive, cell-free assay for messenger RNA and provided the rationale for all of our subsequent work. EFFECT OF SYNTHETIC POLYNUCLEOTIDES IN DIRECTING PROTEIN SYNTHESIS Discovery of polynucleotide phosphorylase by Grun- berg-Manago and Ochoa (7) made available to us chemically defined polyribonucleotides which were assayed for messenger activity in the cell-free E. cols system (17, 18). Figure 2 shows the effect of the syn- thetic polynucleotide, polyuridylic acid,? on incorpora- tion of C-phenylalanine. In the absence of poly U, very little phenylalanine was incorporated into protein. Addition of 10 pg of poly U to a 1-ml reaction mixture resulted in a 1,000-fold increase in phenylalanine in- corporation. The synthesized protein was purified by extraction of precipitated washed protein with 33% HBr in glacial acetic acid followed by precipitation with H,O. The synthesized protein was shown to have unusual solubility and stability properties characteristic of au- thentic polyphenylalanine (18). The effect of poly U in directing the incorporation of 18 other C*4-amino acids into protein was studied. Marked specificity for phenylalanine was observed; however, a small stimula- tion of leucine incorporation was observed (3% that of phenylalanine) (14). The data of Fig. 3 show that the template activity of poly U was dependent on its molecular weight (14). 2The following abbreviations are used: poly U, polyuridylic acid; poly A, polyadenylic acid; octa A, octaadenylic acid; tetra A, tetraadenylic acid; poly C, polycytidylic acid; TMV, tobacco mosaic virus. 56 FEDERATION PROCEEDINGS Volume 22   64009, T 2000 T T t T   48,000-     1500;- T 32,000 T 1000 T COUNTS / MIN/mg PROTEIN         a + ONAase a T COUNTS /MIN /mg PROTEIN     ¥ 500 16,000)       MINUTES 1 L —o—»- + POLY Fic. 1. Cell-free E. coli assay for messenger RNA: ® Minus deoxyribonuclease, A 10 yg de- oxyribonuclease, Ml 500 yg of 1 crude messenger RNA prepared from yeast ribosomes added at 4 indicated time. For details see refs. 15 and 18. FIG. 2. Stimulation of Cl-L- phenylalanine incorporation by polyuridylic acid. @ Minus poly- 4 uridylic acid, & [0 yg poly- uridylic acid added. For details see ref. 18.   MINUTES PN   0 20 40 60 60 9° IS 45 75 FIG. 3. Relationship between   100 |   600 mumspul | 80 60 () 1 400} CPM. 40 T my MOLE [pU/FRACTION AMINO ACID INCORPORATION 40 CPM /my MOLE 200 1 20 T % 20       0 e 4   +19 0   molecular weight of polyuridylic acid and its activity in stimu- lating C4-phenylalanine incorpo- ration into protein. Polyuridylic acid was separated into fractions of different sizes by sucrose den- sity gradient centrifugation. Each fraction was then assayed for its ability to stimulate C'+-phenyl- alanine incorporation. For de- tails see ref. 14. FIG. 4. Relationship between guanylic acid content of different poly UG preparations and their their abilities to direct C'-valine, CJeucine, and C'-eryptophan Phenylatanine Voline 4, Leucine 4 Tryptophon   | 1} J   7 9 I 5 = BOTTOM 1S 0 10 FRACTION NUMBER TOP—» Larger molecules of poly U were separated from smaller molecules by centrifugation through a sucrose gradient. The heavier poly U molecules were considerably more active than the lighter fractions. Poly U of molecular weight of 50,000-100,000 was very active in this system; however, the minimum molecular weight necessary for activity is unknown. Stoichiometry experiments demonstrated that phen- ylalanine incorporation was proportional to the con- centration of poly U in the proper range. The data of Table 1 demonstrate the quantitative relationship between phenylalanine incorporated and poly U present in reaction mixtures. Results of three different experi- ments are presented. About 1.5 mymoles of uridylic acid residue in poly U were required to direct incorpora- tion of 1.0 mumole of phenylalanine (14). These data alone cannot be used to determine the number of uridylic acid residues in a phenylalanine coding unit, for addi- tional results, discussed later, suggest that a molecule of poly U may function catalytically, by directing the synthesis of a number of molecules of polyphenylalanine. Since one or more uridylic acid residues in poly U appeared to be the RNA code word which corresponded to phenylalanine, application of this experimental technique to determine other code words and the general % GUANYLIC ACID CONTENT OF POLY UG 20 30 40 50 incorporation into protein rela- tive to Cl-phenylalanine  in- corporation. TABLE 1. Relationship between C4-L-phenylalanine incorporated and polyuridylic acid present myumole pU myumole pU (in mumole C!-L-   Exp. No, Polyuridytic Acid) phenylalanine mumole cx phenylalanine Lf 11.35 g.12 1.24 2 17.7 10.25 1.73 3 25.7 16.9 1.52 In each experiment the amount of polyuridylic acid present was limiting. Final volume was 0.5 ml. Reaction mixtures were incubated for 60 min at 37 C as described elsewhere (14). characteristics of the genetic code appeared reasonable (17, 18). Randomly ordered polynucleotides containing different bases were tested by Ochoa and his collabora- tors (ta, 10, 11, 22, 23) and by ourselves (13, 14) to see if such polymers would code for different amino acids. In Table 2 are presented the effects of randomly ordered polynucleotides on amino acid incorporations. The composition of the different polynucleotides was ob- tained by base-ratio analysis. Since cach preparation of polynucleotide differs in molecular weight, it is difficult to compare directly the activity of one polynucleotide with another. Therefore, the figures in the main part of the table represent the per cent of an amino acid incor- January-February 1963 TABLE 2. Amino acid incorporation into protein stimulated by randomly mixed polynucleotides         Polynucleotides and Base Ratios Amino Acid UA ue UG U oes U wn U rs U _ 0.87 10 = 0.39 U _ 0.76) Ao 0.050] G = 0.152| G = 0.291 A= 0.13 |C = 0.61 |G = 0.24 C = 0.116] C = 0.502] A = 0.034 Phenyl- 100 100 100 100 100 100 alanine Arginine ° 0 rl 0 49.3 2.9 Alanine 1.9 ° ° 1.0 40.4 0.9 Serine 0.4 | 160 3.2 3.6 | 170 2.3 Proline o 285 oO ° 188 ° Tyrosine 13 ° ° 8 1.0 8.6 Isoleucine 12 1.0 1.0 4.8 5-4 8.4 Valine 0.6 ° 37 0.4 29.8 75 Leucine 4-9 | 79 36 5.1 | 157 44 Cysteine 4-9 o 35 ° 5-4 46 Tryptophan 1.1 ° 14 ° 1.6 23 Glycine 4.7 ° 12 0.5 9.7 15 Methionine 0.6 ° ° 0.6 1.5 8 Glutamic 1.5 ° ° 1.2 0.44 6.2 acid               Figures in main part of the table represent incorporation of any amino acid compared to phenylalanine incorporation ex- pressed as percentages (mumoles amino acid incorporated/mp- moles phenylalanine incorporated X 100). Approximately 25 ug of each polynucleotide was added to each 0.5-ml reaction mixture; these are described elsewhere (14). Samples were in- cubated at 37 C for 15 min. Incorporation of C1*-phenylalanine in counts per minute due to the addition of polynucleotides UA, UC, UG, UAC, UGG, and UGA were 731, 2,900, 714, 804, 2,144, and 2,744, respectively. The reproducibility of the above per- centage figures was +3. porated compared to phenylalanine incorporation stimulated by the same polynucleotide. Marked poly- nucleotide specificity in stimulating incorporation is apparent. A degenerate code is one in which two or more dif- ferent coding units can direct the incorporation of the same amino acid into protein. Since leucine can be coded by both poly UC and poly UG, the code, with respect to leucine, is degenerate (13, 14). Different preparations of poly UG containing in- creasing percentages of G were synthesized and assayed (Fig. 4). The relative amounts of valine, leucine, and tryptophan incorporations, compared to phenylalanine incorporation, was determined for each polynucleotide. Leucine and valine incorporation was proportional to the amount of G in poly UG. However, significant tryptophan incorporation occurred only when a high proportion of G was present. These data suggest that the coding units for both leucine and valine contain one G, that the coding unit for tryptophan contains two G’s, and that each preparation of poly UG contained equal numbers of coding units for leucine and valine. Similar experiments were performed with other amino acids and quantitative estimates were obtained of the number of G, C, or A residues per coding unit. These results are summarized in Table 3. The proba- bility of any sequence of three nucleotides occurring in randomly ordered polynucleotide of analyzed base GENETIC MECHANISMS 57 ratio can be calculated and may be compared with the probability of obtaining a sequence of UUU. The theoretical probabilities of obtaining such triplets, relative to UUU, are compared in Table 3 with the experimentally obtained data. Data obtained experi- mentally agree strikingly well with theoretical predic- tions. Such quantitative data demonstrate that the number of G’s, A’s, or C’s in coding units can be de- termined experimentally. The sequence of nucleotides in each coding unit is not known. The current status of the code is similar to that of an anagram, where the letters of coding units have been determined, but the sequence is unknown. Although the uridylic acid content of RNA viruses is not excessive, a surprisingly high proportion of U has been found in coding units thus far. This dichotomy cannot be explained at the present time. However, it is probable that only coding units containing U have been selected for by the assay method and that additional degeneracies without U will be found. The question may be asked, ‘‘Do the code words agree with known genetic data?” In Table 4 coding results are compared with nitrous acid-induced amino acid sub- stitutions in tobacco mosaic virus protein. Only amino acid substitutions found more than once have been cited. TMV protein contains 158 amino acids, and the entire sequence is known. Treatment of RNA with nitrous acid results in deamination of nucleotides (12) and hence mutations (6). Most nitrous acid-induced amino acid replacements should be due to the replace- TABLE 3. Summary of code words* Coding Unit Amino Acid Composition Theoretical Experimenta} Phenylalanine UUU... 100 100 Arginine UGG... 67 49 Alanine UGG... 67 40 Serine UUC... 157 160 (UCG) Proline UCC... 244 285, Tyrosine UUVA... 13 13 Isoleucine UUA... 13 12 Valine UUG... 32 37 Leucine UUG... 32 36 (UUC) 157 79 Cysteine UUG... 10.6 10.6 (UGG)? Tryptophan UGG... II 14 Glycine UGG... 1 12 Methionine UGA... 2 8 Glutamic acid UGA... 2 6 Lysine UAA... ? ? * Sequence of nucleotides in code words is not specified. Coding ratio is not known definitively. However, assuming a triplet code, the probability of a triplet occurring in a poly- nucleotide, relative to UUU, may be calculated from the base- ratio data presented in Table 2. For example, if poly UG hada base ratio of 3U/1G, the probability of obtaining the sequence UUU would be 34 & 34 X 3g = 2%. The probability of ob- taining the sequence UUG would be 34 XK 34 X 14 = %. Thus, 3 UUU would occur for 1 UUG and, assuming the frequency of UUU to be 100%, the frequency of UUG would be 33%. Dots after each ‘‘word’’ are used to indicate the possible presence of additional uridylic acid residues. 58 FEDERATION PROCEEDINGS TABLE 4. Comparison of nitrous acid-induced replacements in tobacco mosaic virus protein with the nucleotide composition of RNA coding units       iad Mutant Nucleotide C . | ee trains* Amino Acid Nucleotide Composition ossible Replacement | of Corresponding | Nysleotide A B I 2 Ser UUC... c J L J Phe UUU... U I 2 Glu UAG... A 1 1 1 Gly UGG... G I 2 Prol UCC... Cc J L 1 Leu UUC... U 2 Isoleu UDA... A J L L Val UUG... G 5 Arg UGG... Cc 4 L L Gly UGG... G           * The mutant amino acid replacement data cited and ob- tained either by A, Tsugita and Fraenkel-Conrat (30), or B, Wittmann (26). ment, during virus replication, of either a cytidine by a uridine or an adenine by a guanine (4). All of the amino acid replacements with the exception of the last are the result of a conversion of either a C to a U or an A toa G. Such results demonstrate confirmation between amino acid replacement data and proposed nucleotide compositions of coding units (14). Amino acid replacements in corticotropins, insulins, and cytochromes of different species have been examined also. Over 90 % of these acid substitutions corresponded to a replacement of only one nucleotide per coding unit. Such data sug- gest that at least part of the code may be universal. Nonoverlapping and overlapping codes would be read as follows: UUUUUU UUUUUUU         Nonoverlapping Overlapping Brenner on theoretical grounds ruled out overlapping triplet codes because of the restrictions such codes would impose on nearest-neighbor frequencies of amino acids (2). If the code were overlapping and one nucleotide in a coding unit were replaced with another due to mutation, amino acid substitutions might be expected to occur in clusters. The bulk of amino acid substitutions found in TMV protein do not occur in clusters, and these data suggest that the code is of the nonoverlapping type (26, 30, and personal communication from Tsugita and Fraenkel-Conrat). A number of polynucleotides which do not contain U have been tested (Table 5). Poly AC at the indicated base ratios determined by analysis, were found to direct small amounts of threonine and proline into protein. Non-U-containing polynucleotides of different base ratios, or more sensitive assays, may be necessary to demonstrate additional degeneracies. Volume 22 When poly U is mixed with poly A, double- and triple- stranded helices are formed which are completely in- active in directing polyphenylalanine synthesis. The effects of oligo A and poly A on polyphenylalanine synthesis are presented in Fig. 5. The stability of the oligo A-poly U helix is influenced strongly by the chain length of oligo A. Thus octa A forms more stable helices with poly U than tetra A. It may be seen that octa A and poly A are very effective inhibitors of polyphenylala- nine synthesis. In the reaction mixtures used, triple- stranded helices would be formed (U-A-U). A 99% inhibition of polyphenylalanine synthesis was observed at stoichiometric poly A concentrations, that is, 2 U’s in poly U per 1 A in poly A. In contrast, addition of poly C had little effect on polyphenylalanine synthesis. Many mechanisms for enzyme repression can be en- visioned with strands of RNA base pairing with parts of DNA or complimentary RNA. It will be of critical importance to determine whether the type of repression demonstrated in vitro with poly U and poly A may occur also in vivo. FATE OF MESSENGER RNA H*-poly U was prepared and its fate in reaction mix- tures was determined (1). Sucrose density gradient centrifugation experiments revealed that H*-poly U became associated with a polydisperse peak of ribosomes which sedimented faster than 70S ribosomes. Almost no poly U was found on 70S ribosomes. After 3 min of incu- bation approximately 90% of the poly U disappeared and was recovered as mononucleotides. The rest of the poly U still was associated with 100S ribosomes. After 10 min of incubation most of the poly U had been hy- drolyzed to mononucleotides (90% were 5’-mononu- cleotides, 10% were 2’, 3’-cyclic mononucleotides or 3’-mononucleotides.) These data show that poly U is bound to 1008 rather than 70S ribosomes. In addition, the observed rapid breakdown of poly U suggests that TABLE 5. Effects of non-U-containing polynucleotides on amino acid incorporation Minus Poly- C4.1-Amino Acid nucleotide AGz:t AGug:r AC 5:1 AC wos:r Phenylalanine 10 10 9 10 10 Valine 10 9 8 10 8 Serine 20 20 20 15 18 Methionine 10 10 9 10 Il Histidine 10 5 IL 10 10 Leucine 30 30 25 28 30 Lysine 10 10 15 13 Lo Aspartic acid 10 10 10 12 10 Alanine 10 10 It 10 10 Cysteine 20 10 10 20 10 Glycine 10 10 I 10 10 Glutamic acid 10 20 lo 10 20 Proline 10 10 Il 10 70 Isoleucine ro 10 10 10 It Tyrosine 10 10 8 10 9 Threonine 10 10 9 30 10 Arginine 10 10 9 8 & Figures represent the incorporation of C'-amino acids in uumoles. Base ratios were obtained by analysis. January-February 1963 Fig. 5. Repression of poly- GENETIC MECHANISMS on ©     phenylalanine synthesis by oligo and poly A. 12.5 mymoles of 140 uridylic acid residues in poly U and the indicated amount of oligo A, poly A, or poly C were incubated at 24C for 15 min be- fore adding to reaction mixtures. 12.5 mymoles of uridylic acid in poly U alone stimulated incorpo- ration of 2,250 counts/min of C-phenylalanine into protein. ‘Thus 2,250 counts/min repre- sents 100% incorporation. FIG. 6. Comparison between rate of polyphenylalanine syn- thesis and rate of H’-poly U de- uradation. Reaction mixtures used for assay of precipitable -poly U contained H*-poly U and G-phenylalanine. Reaction mixtures used for assay of phenyl- alanine incorporation contained °           80 a oO + CLPHENYLALANINE INCORPORATED > o 20 Poly U+ Poly A : =   Poly U + Di-A @ Poly + Pole Poly U+ Tetra-A     3 LoS T ‘ mHMOLES TCA PRECIPITABLE C!4 PHENYLALANINE /ml @—~@         CG\"-phenylalanine. The 2 reac- o 12.5 tion mixtures, each in a total volume of 2 ml, were incubated for 40 min and 0.2-ml aliquots were removed at the indicated time intervals and were transferred lo either 2 ml of 10% trichloracetic acid at 4C for assay of phenyl- only the remaining 10-20% directs protein synthesis. ‘These data and the stoichiometry data of Table 1 sug- gest that poly U functions catalytically; that is, each poly U molecule directs the synthesis of more than one polyphenylalanine molecule (14, 1). Figure 8 illustrates an experiment performed with unlabeled poly U and C\"-phenylalanine. After 3 min of incubation, phenylalanine incorporation occurred only un 1008 ribosomes. After 15 min of incubation, insoluble protein, presumed to be polyphenylalanine, and poly- phenylalanine associated with both r1ooS and 70S ribosomes had been formed. These data are in complete accord with the findings of Risebrough, Tissiéres and Watson (21), who have reported that rapidly synthesized RNA in intact cells becomes associated first with 1008 ribosomes. MECHANISM OF POLYPHENYLALANINE SYNTHESIS C-phenylalanine-sRNA was prepared enzymatically, and C!-phenylalanine was found to be transferred to protein without excessive dilution in the presence of a large pool of unlabeled phenylalanine. The transfer required poly U, ribosomes, purified transfer enzyme (16), GTP, and a GTP-generating system (19). The function of GTP is unknown, and undoubtedly several tnzymes are required for the over-all synthesis. Such txperiments demonstrate that sRNA is an intermediate in polyphenylalanine synthesis; thus, the initial enzy- matic reactions are as follows: phenylatanine- activating enzyme   L-phenylalanine + ATP (x)   AMP-phenylalanine + P-P 25 my MOLES [pA] or [pC]   aE 3 mpMOLES ALCOHOL PRECIPITABLE URIDYLIC ACID RESIDUES/ml 4-4 1 ° 1 | | I | | ° 37.5 0 10 20 30 40 . DURATION OF INCUBATION (MINUTES) alanine incorporation into protcin or into 0.2 ml of magnesium acetate and 0.4 ml of absolute ethanol at 4C for analysis of pre- cipitable H®-poly U (1). 2.00 160 Not incubated   4 120 + 80 440 25k o eee 0 200     3 Minutes 70S — $00 1.50 300 COUNTS / MINUTE 1.00 o—~ 0.0. 260my   a----& TRITIUM, 10 Minutes 1.00 F- 50 CPM “ b tee Bottom I 5 10 15 20 25 TUBE NUMBER       Fic. 7. Association of H’-poly U with ribosomes. Reactions were begun by addition of 20 umoles uridylic acid residues in H*-poly U to each 0.25 ml of reaction mixture. After incubation at 35C for indicated times, samples were briefly chilled in an ice bath and o.2-ml aliquots were layered on top of sucrose gradients at 3C. Centrifugation and analyses of each fraction were performed as described elsewhere (1). 60 FEDERATION PROCEEDINGS              q T I T T 2.00 + 800 3 Minutes 708 4 1.50 600 1.00 400 = = a 3 50 200 & ow 254 2° So 0 o °? > 200 ~| 800 ‘ _ 1 t 1.50 600 * 1.00 —| 400 1 1 _ 1 50 + 200 25 ga 4 0 0 | I 5 10 15 20 25 Bottom TUBE NUMBER Fic. 8. Incorporation of C!‘-phenylalanine into protein on ribosomes. A regular reaction mixture was prepared except that C#-phenylalanine (7 % 108 counts/min umole) was used. 20 mumoles of uridylic and residue in H*-poly U were added to 0.25- ml reaction mixtures, which were incubated at 35C for the indi- cated times. 0.2-ml aliquots then were layered on sucrose gradients at 3C. Centrifugation and analyses of each fraction were performed as described elsewhere (1).       T T t T T T T T T T 3200} 4 2800+ 4 2400F a, 4 5 Fic. g. Stimulation of C-L- 2000+ = valine incorporation into protein 2 by tobacco mosaic virus RNA. ook 3 4 0.25-ml reaction mixtures were ° incubated at 37C for 90 min be- 00+ 4 fore deproteinization as de- scribed elsewhere (19). soo}- 4 400}- 4 ng THY RNA Pop ii tof it toy fb   20 60 100 140 eo   Volume 22 phenylalanine- activating enzyme AMP-phenylalanine + sRNA (2)   phenylalaninesRNA + AMP Poly U ribosomes transfer enzyme 3 Phenylalanine-sRNA 343 4 (3) polyphenylalanine + sRNA MESSENGER ROLE OF VIRAL RNA The effects of TMV-RNA in directing protein syn- thesis in this system were studied in collaboration with Drs. Tsugita and Fraenkel-Conrat of the Virus Labora- tory, Berkeley, California (27). The effect of TMV-RNA on C'-valine incorporation into protein is shown in Fig. 9. Proportionality between valine incorporation and TMV-RNA added, in the proper range, was demon- strated. Part of the C'-protein formed a specific pre- cipitate with TMV antisera. Also, part of the C4 protein formed could be purified from reaction mixtures with unlabeled carrier TMV protein by repeated iso- electric precipitations and DEAE column chromatog- raphy. Virus reconstitution experiments showed that the C’*-protein product strongly and specifically inhibited virus reconstitution. However, the small amount of virus which did reconstitute in the presence of added unlabeled TMV protein and RNA contained small but significant amounts of C-protein product. TMV protein is an excellent one to characterize, for its entire amino acid sequence is known and digestion with trypsin converts it into 12 peptides of established amino acid composition and chromatographic properties. The C'-protein synthesized under the direction of TMV-RNA was purified from reaction mixtures with added unlabeled carrier TMV protein and was treated with trypsin. The peptides formed after digestion with trypsin were isolated by Dowex 1 column chromatog- raphy. Each peptide was subjected to amino acid analysis and its radioactivity was counted. The data of Table 6 summarize a small portion of these results (27). Seventeen peptides were isolated and ana- TABLE 6. Analysis of peptides obtained from C'-protein synthesized in the presence of either C'\\\\4-phenylalanine or C¥-tyrosine under direction of TMV-RNA . . CH-Phenylalanine, C4.Tyrosine, Peptide No. Amino Acid Composition counts/min counts/min 2 Thro.» Glur.9 Wali.o (Val) (Arg) 8 oO 3 Thro.1 Ser:., Glug. Proe.; Wale; Pher.o (Try, Lys, Arg) 73 5 Ir Aspr.o Thro.» Sero.s Glyz.o Tyri.o (Arg) 10 44 5 Vali.o Tyry.0 (Arg) 3 39 8 ASp2.7 Thrs 9 Glu3.7 Pro,» Ala. Valo.s Tleug.7 Leut.o (leu, Arg) 3 4 Asp2.o Sero.s Pro..o Phei.s (Lys) 96 (\" Reaction mixtures, described in detail elsewhere, contained TMV-RNA and either C-phenylalanine or CH-tyrosine. After a go-min incubation at 37 C, ribosomes were removed by centrifugation, and the 100,000 X g supernatant solutions were dialyzed extensively. Unlabeled, carrier TMV protein was added and then was purified from supernatant solutions by repeated isoelectric precipitations and DEAE column chromatography. Purified TMV protein was digested with trypsin and 17 peptides were sepa rated by Dowex 1 column chromatography. Amino acid content and radioactivity of each peptide were determined. When yeast messenger RNA was added to reaction mixtures in place of TMV-RNA, the peptides did not contain significant radioactivity. In some Cases, peptides were purified further by paper electrophoresis. Data concerning only 6 typical TMV-peptides are presented here. See ref. 27 for complete details. lanuary-February 1963 \\\\vzed; however, space permits presentation of only part of the data. Each peptide in Table 6 is a characteristic UMV peptide. TMV-RNA directed C'-phenylalanine Imi not C-tyrosine into phenylalanine-containing peptides; whereas TMV-RNA directed C'-tyrosine, but not C'-phenylalanine, into tyrosine-containing peptides. Although the counts were low, duplicate experiments gave similar results. In addition, several peptides were purified further by paper electrophoresis und still were found to contain radioactivity. The N-terminal amino acid of TMV protein is acetylated und the N-terminal peptide did not contain the expected (\"-tvrosine, possibly because acetyl CoA was not added to reaction mixtures. This is the only chemical difference detected so far. Thus, it appears justifiable to conclude that TMV-RNA directs the synthesis of a nonacetylated protein similar to TMV protein in this cell-free E. coli system. Such results strongly suggest that a large part of the code may be universal. SUMMARY Messenger RNA fractions were found to direct protein synthesis in cell-free EZ. coli extracts. This system afforded a sensitive assay for natural and synthetic messenger RNA and was used both to determine characteristics of messenger RNA and to decipher part of the genetic code. Polyuridylic acid specifically directed the synthesis of polyphenylalanine via an sRNA intermediate. The activity of poly U varied with its molecular weight; REFERENCES 1. Baronpes, S. H., anp M. W. Nirensera. Science 138: 810, 1962. 1a. Basitio, C., A. J. Wansa, P. Lencve, J. F. SPEYER, AND S. Ocuoa. Proc. Natl. Acad. Sei. U.S. 48: 613, 1962. . Brenner, S. Proc. Natl. Acad. Sci. U.S. 43: 687, 1957- . Cuarcarr, E., R. Lirsuirz, C. Green, AND M. E. Hopes. J. Biol. Chem. 192 : 223, 1951. . Freese, E. Brookhaven Symp. Biol. 12:63, 1959. . Gamow, G. Nature 173: 318, 1954. . Gterer, A., AND K. W. Munpry. Nature 182:1457, 1958. GRuNBERG-Manaoo, M., anv S. Ocuoa. J. Am. Chem. Soc. 77: 3165, 1955. . Kamevama, T., anp G. D. Novewti. Biochem. Biophys. Res. Commun. 2: 393, 1960. g. Lamsore, M. R., anp P. C. Zamecnix. Biochim. Biophys. Acta 42: 206, 1960. to. Lencyeu, P., J. F. Spever, C. Basitio, anp S. Ocnoa. Pree. Natl. Acad. Sci. U.S. 48: 282, 1962. 11. Lencye., P., J. F. Speyer, anp S. Ocuoa. Proc. Natl. Acad. Sa. U.S. 47: 1936, 1961. 12. Levine, P. A., anD R.S. Tipson. J. Biol. Chem. 111: 313, 1935. 13. Martin, R. G., J. H. Martuaer, O. W. Jones, anp M. W. NIRENBERG. Biochem. Biophys. Res. Commun. 6: 410, 1962. 14. MartHagl, J. H., O. W. Jones, R. G. Martin, AnD M. W. NIRENBERG. Proc. Natl. Acad. Sci. U. S. 48: 666, 1962. 15. Marruaet, J. H., anp M. W. Nrrenzere. Proc. Natl. Acad. Sci. U.S. 4.71580, 1961. won Oo wap GENETIC MECHANISMS 61 longer polynucleotide chains were more active than shorter ones. Evidence was presented which suggested that each poly U molecule acted catalytically and directed the synthesis of more than one molecule of polyphenylalanine. Single-stranded polynucleotides were shown to have messenger RNA activity but not double- or triple-stranded polymers. Polyphenylalanine synthesis was repressed specifically by poly A, which forms hydro- gen-bonded helices with poly U. Longer oligo A chains formed more stable helice", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yy8b~94ms.rye4", "00000000-0000-0000-73F6-D6501F7C795F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Regulation of Calcium Uptake in Neuroblastoma or Hybrid Cells - A Possible Mechanism for Synapse Plasticity", "101584910X134", null, "1979", "[1979?]", "This abstract presents the findings from one of a series of studies demonstrating the value of neuroblastoma research.  This study suggests that because calcium uptake is regulated by cyclic AMP it is possible to have defective cells or receptors that interfere with the process and lead to potential long term health risks.", "Abstracts (summaries)", "Neuroblastoma,Hybrid Cells,Synapses,Neuronal Plasticity", "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of the Federation of American Societies for Experimental Biology.", "Copyright may apply", null, null, "476 BIOCHEMISTRY OF DEVELOPMENT AND DIFFERENTIATION I (1298-1303) 1298 BIOCHEMISTRY REGULATION OF CALCIUM UPTAKE IN NEUROBLASTOMA OR HYBRID CELLS - A POSSIBLE MECHANISM FOR SYNAPSE PLASTICITY. A. Rotter®, R. Ray* and M. Nirenberg. NIH, Bethesda, Md. 20014 Thirteen neuroblastoma or hybrid cell lines with or with out defects in stimulus-dependent acetylcholine release and synapse formation (Wilson, S., et al. (1978) Fed. Proc. 37, 2819) were tested for Kt-dependent 45ca2+ uptake. NBrlOA hybrid cells (synapset) grown for days with 1 mM dibutyryl cAMP (Bt2cAMP) and incubated with 5.4 or 85.4 mM Kt accumu- late 2.5 and 5.0 nmoles of 45ca2t+/5 min/mg protein, respec— tively. Methoxy-verapamil inhibits Kt-dependent 45¢a2t uptake >95% (1059 = 2x10-7 M) but has no effect on basal 45ca2+ uptake. Sca2+ uptake also is inhibited by 10 mM La3t, Co2+, ni2t, Mn2+ or Sr2+ but not by 10 wM tetrodotoxin, 20 mM tetraethylammonium or 1 mM 3,4-diaminopyridine. Logarith- mically dividing NBr10A cells grown without Bt cAMP do not respond to Kt by accumulating 5ca2+ put can be shifted to a responsive state by treatment for 7 days with BtgcAMP or 10 pM PGE] (an activator of adenylate cyclase) and 1 mM theophylline. Examination of 12 other cell lines grown with BtgcAMP revealed 2 classes of synapse defects: (1) defects in Kt-dependent 45ca2t uptake and (2) defects in another unidentified step required for synapse formation. These results show that the Ca + uptake is regulated and that cell lines with or without defects in Ca + uptake can be generated. The results suggest that cAMP is required for the acquisition of xt-dependent Catt uptake thereby regulating synapse formation and efficiency. TUESDAY, PM", "Nirenberg, Marshall W. ; Ray, Radharaman ; Rotter, Andrej", null, "Federation Proceedings", "Federation of American Societies for Experimental Biology", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dve9_xsep-j96r", "00000000-0000-0000-DFCF-C9686703644B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Desensitization of Adenylate Cyclase to Prostaglandin E1 or 2-Chloroadenosine", "101584910X135", null, "1981", "1981", "Nirenberg and the Biochemical Genetics team at NIH worked on neuroreceptor sensitivity in the late 1970s and early 1980s.  This article confirms and extends earlier findings with the hormone signaling system cyclic AMP and the insoluble enzyme system adenyl cyclase.", "Articles", "Adenosine,Adenylyl Cyclases,Prostaglandins E,Neuroblastoma,Hybrid Cells", "From Neuroblastoma to Homeobox Genes, 1976-1992", "7", "pages", "Text", "English", "Reproduced with permission of the American Society for Pharmacology and Experimental Therapeutics.", "Copyright may apply", null, null, "MOLECULAR PHARMACOLOGY, 20:585-591 Desensitization of Adenylate Cyclase to Prostaglandin E, or 2-Chloroadenosine JAMES G. KENIMER’ AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205 Received February 20, 1981; Accepted June 8, 1981   SUMMARY The hypothesis was examined that prolonged activation of adenylate cyclase can result in a decrease in the specific activity of the enzyme, much as prolonged inhibition of adenylate cyclase gradually leads to an increase in the specific activity of the enzyme. Activation of adenylate cyclase of NG108-15 neuroblastoma-glioma hybrid cells by prostaglandin E, resulted in the gradual loss of basal adenylate cyclase activity as well as enzyme activity stimulated by prostaglandin E), 2-chloroadenosine, NaF, or both pros- taglandin E, and guanyl-5’-yl imidodiphosphate. Exposure of NG108-15 cells to 8-Br cyclic AMP also resulted in the loss of basal, prostaglandin Ej-stimulated, and 2-chlo- roadenosine-stimulated adenylate cyclase activities. Cyclohexamide had no effect on prostaglandin E)-dependent desensitization of adenylate cyclase, but inhibited recovery of enzyme activity from the desensitized state. In contrast, exposure of NG108-15 cells to 2-chloroadenosine resulted in the rapid loss of response to 2-chloroadenosine with a half- life of 1.8 hr, but prostaglandin E,-stimulated and basal enzyme activities decreased only slightly.   INTRODUCTION NG108-15 cells possess opiate receptors (1, 2), musca- rinic acetylcholine receptors (3, 4) and alpha-adrenergic receptors (5) which mediate inhibition of adenylate cy- clase (ATP pyrophosphate lyase-cyclizing; EC 4.6.1.1). Thus, exposure of cells to morphine (1), carbamylcholine (6), or norepinephrine (7) reduces cellular cyclic AMP levels; however, exposure of cells to the receptor ligand for 10-24 hr gradually results in an increase in adenylate cyclase specific activity. Thus, cyclic AMP levels of cells slowly return to the control value. Withdrawal of the inhibitory ligand unmasks the elevated enzyme activity and results in a prolonged 4- to 10-fold increase in cellular cyclic AMP levels. Cells thus develop an apparent toler- ance to and dependence upon morphine, carbamylcho- line, or norepinephrine with respect to maintenance of cellular cyclic AMP levels. The hypothesis that activation of adenylate cyclase may lead, conversely, to a reduction in adenylate cyclase activity is examined in this report. Preliminary results by Dr. Shail Sharma? showed that treatment of NG108-15 cells with PGE,’ for several days resulted in decreases in A preliminary report of this work has been published [Fed. Proc. 37:1539 (1978)]. ; ' Present address, Division of Drug Biology (HFD-413), Pharmaceu- tical Research and Testing, Food and Drug Administration, Washing- ‘on, D. C. 20204. 8 K. Sharma and M. Nirenberg, unpublished observations. “The abbreviations used are: PGE,, prostaglandin E,; PGAi, pros- 585 basal and PGE)-stimulated activities. These observations are confirmed and extended in this report. MATERIALS AND METHODS: Cell culture and homogenate preparation. Culture conditions for NG108-15 hybrid cells (subcultured 14-20 times) have been described (8); the growth medium con- sisted of DMEM (Grand Island Biological Company Cat- alogue No. 430-2100), 5-10% fetal bovine serum, 100 nm hypoxanthine, 1 umM aminopterin, and 16 um thymidine. Each flask (75 sq cm surface area) was inoculated with 5.0-7.5 x 10° cells in 15 ml of growth medium. The medium was replaced on the 3rd day and each day thereafter. A confluent layer of cells (approximately 1.5 X 10’ cells, 15 mg of cell protein per flask) was obtained on the 6th or 7th day of incubation. Desensitization experiments usually were initiated 5-6 days after cells were plated, when cell layers were approximately 80% confluent. The medium was changed at zero time as indicated in the figures and tables, and cells were incu- bated without further change of medium unless specified. Each monolayer of cells was washed twice with 15 ml (each wash) of isotonic salt solution (150 mm NaCl, 5.4 mM KCl, 0.17 mm Na2HPO,, 0.22 mm KHePOx,, 0.11 mm   taglandin Ai; PGF:., prostaglandin Fz; ClAdo, 2-Cl-adenosine; Bts cyclic AMP, N*,O?-dibutyryl cyclic AMP; Bt, cyclic GMP, N*,0?- dibutyryl cyclic GMP; Ro20-1724, 4-(3-butoxy-4-methoxybenzyl)-2-im- idazolidinone; Gpp(NH)p; guanyl-5’-yl imidodiphosphate. 0026-895X /81/030585-07$02.00/0 Copyright © 1981 by The American Society for Pharmacology and Experimental Therapeutics. All rights of reproduction in any form reserved. 586 KENIMER AND NIRENBERG CaCl, and 25 mm D-glucose, pH 7.4, 340 mOsmoles/kg). Then 10 ml of an isotonic salt solution without Ca** ions, adjusted to pH 6.6 (150 mm NaCl, 5.4 mm KCI, 0.17 mM NasHPO,, 0.22 mm KH2PO,; and 25 mm D-glucose) were added; after 5 min at 24°, the cells were dissociated by sharply tapping the flasks. The cells were centrifuged for 4 min at 1000 x g, and the cell pellets were frozen in Dry Ice and stored over liquid nitrogen. The cells were thawed, suspended in a cold solution containing 25 mM Tris-HCl (pH 7.5) (approximately 7.5 mg of protein per ml), and homogenized with 10 strokes of a Dounce ho- mogenizer with a size A pestle; 0.5-ml portions of the homogenate were frozen rapidly and stored in the vapor phase of a liquid nitrogen freezer. Homogenates were thawed immediately before use. Protein was determined by a modification of the method of Lowry et ai. (9) with bovine serum albumin as the standard. Adenylate cyclase assay. Adenylate cyclase activity was determined by a modification (10) of method C of Solomon et ai. (11). Each 100-pl reaction mixture con- tained 50 mm Tris-HCl (pH 7.5); 5 mm MgCl; 20 mM creatine phosphate, disodium salt; 10 units (71 yg of protein) of creatine phosphokinase; 1 mM [a-\"PJATP, tetrasodium salt (2 uCi); 0.5 mm cyclic AMP; G-*H-la- beled cyclic AMP (approximately 10,000 cpm); 0.56 mM Ro20-1724; 0.25% ethanol; and 50-200 yg of NG108-15 homogenate protein. Reaction mixtures were incubated for 6 min at 37° unless otherwise indicated. Under these conditions *2P-labeled cyclic AMP synthesis was propor- tional to the time of incubation for at least 20 min in the presence or absence of PGE, or ClAdo. Each reaction mixture was deproteinized by the addition of 0.9 ml of cold 6% trichloroacetic acid. The tubes were centrifuged at 1800 x g for 20 min and each supernatant solution was added to a Dowex AGSOW-X4 column. The cyclic AMP fraction from the column was eluted onto an alumina column and eluted from the alumina with 4 ml of 0.1 mm imidazole-HC1 (pH 7.5) into a counting vial. Values re- ported are the means of duplicate or triplicate determi- nations; most replicate values differed by less than 10%. RESULTS As shown in Fig. 1, PGE; stimulated NG108-15 ade- nylate cyclase activity. However, continued exposure of NG108-15 cells to 25 um PGE; gradually resulted in desensitization of adenylate cyclase to PGE; (Fig. 1A) and ClAdo (Fig. 1B) and also reduced the specific activity of basal adenylate cyclase (Fig. 1B). The specific activity of adenylate cyclase in homogenates prepared from un- treated, control cells also decreased; however, a larger decrease was always observed when cells were treated with PGE.’ Basal and PGE,-stimulated enzyme activi- ties, expressed as percentage of control values, decreased exponentially with half-lives of approximately 7.5 and 6 hr, and pseudo-first order rate constants for PGE,-depen- dent loss of enzyme activity of 1.6 x 10~° sec™’ and 3.2 4 Decreases in adenylate cyclase specific activities obtained with homogenates from untreated, control cells incubated for various periods probably resulted from replacement of the culture medium at zero time. When only 50% of the medium was replaced at zero time, adenylate cyclase activities of cells incubated for 8 hr did not change appreciably (data not shown). @ PGE, STIMULATED BASAL AND 2-Cl- ADENYLATE ADENOSINE STIMULATED CYCLASE ADENYLATE CYCLASE fo) y T T T T t T ,     pMOL cAMP/MIN/mg PROTEIN 20   0 f 1 ! { 6 12 18 24 0 6 #12 #18 24 HOURS CULTURED WITH OR WITHOUT PGE, Fic. 1. Effects of culturing NG108-15 cells in the presence or ab- sence of PGE, on basal, ClAdo-stimulated, and PGE,-stumulated adenylate cyclase activities Cells were cultured for 0-23 hr in the presence of 25 ym PGE, (®, Wl, VY) or in the absence of PGE, (O, DO, V). At the times indicated the cells were harvested and frozen. The data shown in A and B are from the same experiment. Homogenates were prepared and assayed for adenylate cyclase activity in the presence of 10 uM PGE, (A) or 50 uM ClAdo (B); basal enzyme activity shown in B also applies to A. Each value shown in the inset to B was obtained by dividing the mean basal, PGE,-dependent, or ClAdo-dependent adenylate cyclase specific activ- ities found with homogenates of PGE;-treated cells by the correspond- ing control values at each time; thus, the ordinate of the inset represents the percentage of the control values obtained with homogenates of untreated cells at each time. The abscissa (inset) represents hours of incubation of NG108-15 cells with PGE).   x 107° sec™', respectively (Fig. 1B, inset). The decrease in ClAdo-stimulated adenylate cyclase specific activity was biphasic, suggesting that the response to ClAdo was lost by a more complex kinetic mechanism than that found for basal or PGE,-stimulated adenylate cyclase (Fig. 1B, inset). Exposure of NG108-15 hybrid cells to 0.1 mM ClAdo resulted in time-dependent decreases in ClAdo-stimu- lated adenylate cyclase specific activities (Fig. 2B); how- ever, treatment of cells with ClAdo had little or no effect on basal or PGE)-stimulated adenylate cyclase specific activities (Fig. 2A). The half-life of the loss of respon- siveness of adenylate cyclase to ClAdo was 1.8 hr; enzyme specific activity decreased exponentially with an est! mated pseudo-first order rate constant of 1.1 x 10 sec (Fig. 2B, inset). The specific activities of basal and stim- ulated adenylate cyclase from control cells decreased somewhat during the course of the experiment; howeve!. ClAdo-dependent adenylate cyclase activities of cells treated with ClAdo decreased to <2% of the value ob- tained with untreated control cells. The effects of exposing NG108-15 cells to different concentrations of PGE, or ClAdo on adenylate cyclase activities are shown in Table 1. The lowest concentration of PGE, tested that resulted in desensitization of ade- nylate cyclase was 0.25 um. Almost maximal desensitiz4- tion was obtained with 2.5 um PGE,. Culture of NG10& PGE,-INDUCED DESENSITIZATION OF ADENYLATE CYCLASE BASAL AND PGE, 2-Cl- ADENOSINE         STIMULATED STIMULATED ADENYLATE CYCLASE ADENYLATE CYCLASE T T T T T y T T 2 140 iu) = — © 120 roll iva « oo. - 2 100 5 € © 10 $ PGE = 80 1 iu 5 ° = a s 60 <q Oo | _ 40 $ a 20 BASAL 0 | L Ll | 6 12 18 24 0 6 12 #18 «24 HOURS CULTURED WITH OR WITHOUT 2-Cl-ADENOSINE Fic. 2. Effects of culturing NG108-15 cells in the presence of ClAdo on basal, ClAdo-stimulated, or PGE,-stimulated adenylate cyclase activities Cells were cultured for 0-23 hr in the presence of 100 pm ClAdo (M8, A, ¥) or the absence of ClAdo (CG, ©, VY) and harvested at the times shown. The data shown in A and B are from the same experiment. Homogenates were prepared and assayed for basal adenylate cyclase activity and for activity in the presence of 10 pm PGE, (A), or 50 uM ClAdo (B). Each value shown in the inset was obtained by dividing the mean ClAdo-dependent adenylate cyclase specific activity found with homogenates prepared from ClAdo-treated cells by the mean ClAdo- dependent adenylate cyclase specific activity found with homogenates from untreated control cells at that time. The abcissa of the inset represents hours of treatment of NG108-15 cells with ClAdo. 15 cells with ClAdo resulted in marked desensitization to ClAdo at each concentration tested (0.50-50 um ClAdo), but only a small reduction in PGE,-stimulated adenylate cyclase activity was observed. Basal adenylate cyclase activity decreased 21-38% after treatment of cells with ClAdo. In other experiments (not shown), treatment of cells with ClAdo had little or no effect on basal adenylate cyclase specific activity. Treatment of cells with 50 uM ClAdo in the presence of 1 or 5 mM theophylline, an adenosine receptor antagonist and an inhibitor of cyclic nucleotide phosphodiesterase, prevented the ClAdo-de- pendent loss of responsiveness of adenylate cyclase to ClAdo. Treatment of cells with 1 or 5 mm theophylline alone had no effect on adenylate cyclase specific activity. Theophylline had no effect on PGE,-induced desensiti- zation of adenylate cyclase (data not shown). The effects of 10 um PGE, 25 mm Naf, or 10 uM PGE, and 1 mm Gpp(NH)p on rates of “P-labeled cyclic AMP synthesis in homogenates prepared from NG108-15 cells cultured for 16 hr in the absence of PGE, or with 2.5 uM PGE, are shown in Fig. 3A and B, respectively. Basal, PGE,-stimulated, NaF-stimulated, and PGE,- and Gpp(NH)p-stimulated adenylate cyclase activities were approximately 50% lower in homogenates from cells treated with PGE, than in homogenates from untreated cells. The effects of 1, 3, 5, or 10 mm NaF were similar to those observed with 25 mM Naf, and the effect of 5 um Gpp(NH)p was similar to that found with 1 mm 587 TaBLe i Adenylate cyclase activity in homogenates prepared from NG108-15 cells treated with different concentrations of PGE, or ClAdo NG108-15 cells were cultured in the presence of the indicated con- centrations of PGE, or ClAdo for 16 hr. Homogenates were prepared and assayed for basal, PGE, -stimulated, and ClAdo-stimulated adenyl- ate cyclase activities.       Expt. Cell ia aa (16 Adenylate cyclase assay No addi- 10 uM 50 uM tion PGE, ClAdo pmoles “P-labeled cyclic AMP/ min/mg protein 1 None 5.9 60.6 27.4 PGE, (pM) 0.0025 6.4 614 29.7 0.025 5.6 59.1 28.2 0.25 4.0 42.1 21.2 2.5 2.5 24.7 11.5 25.0 3.9 23.4 12.7 2 None 119 78.9 32.2 PGE, (um) 0.25 7.8 39.0 18.3 2.5 4.4 26.6 13.5 25.0 3.7 22.3 15.4 ClAdo (um) 0.5 9.4 80.1 18.8 5.0 TA 78.0 11.2 50.0 8.2 70.0 7.5 Gpp(NH)p (not shown). The demonstration that Gpp(NH)p inhibits PGE,-stimulated adenylate cyclase activity in NG108-15 homogenates confirms previous ob- servations (12). NG108-15 hybrid cells were cultured with 1 mm 8-Br cyclic AMP, 1 mM Bt2 cyclic AMP, or 1 mM Btz cyclic GMP for 4 or 20 hr to determine whether elevation of cellular cyclic nucleotide levels affects adenylate cyclase activity (Table 2). Treatment of cells with 8-Br cyclic     1400 (— T T T (@CONTROL CELLS T T (@® CELLS CULTURED WIT PGE ; |           pMOL cAMP/mg PROTEIN 4 8 12 16 0 4 8 12 16 MINUTES Fic. 3. Effects of PGE,, NaF, or Gpp(NH)p and PGE, on adenylate cyclase activity in homogenates prepared from control (untreated) or PGE,-treated NG108-15 cells Cells were cultured for 16 hr in the absence of PGE, (A) or in the presence of 25 um PGE, (B). ©, Basal adenylate cyclase activity; A, 25 mo NaF; V, 10 xm PGE; 0, 10 um PGE, and 100 pm Gpp(NH)p. 588 KENIMER AND NIRENBERG TABLE 2 Adenylate cyclase activity in homogenates of NG108-15 cells cultured with cyclic nucleotides or compounds that affect cyclic nucleotide levels NG108-15 cells were incubated with the compound indicated for the times shown. Homogenates were assayed for adenylate cyclase activity. Expt.   Cell treatment Adenylate cyclase assay     Addition Hr No 10pm S50 pM addi- PGE, ClAdo tion pmoles *P-labeled cyelic AMP/min/mg protein 1 None 4 14.0 95.3 46.2 1 mM 8-Br cyclic AMP 4 11.9 85.5 31.3 1 mm Bt» cyclic AMP 4 13.5 98.5 38.6 1 mM Bt, cyclic GMP 4 15.7 98.3 52.4 None 20 14.9 101.1 47.6 i mM 8-Br cyclic AMP 20 7.7 74.5 17.6 1 mM Bt» cyclic AMP 20 11.4 110.4 17.5 1 mM Btz cyclic GMP 20 14.4 91.0 51.0 2 None 16 8.6 68.5 27.6 25.0 um PGE, 16 47 24.6 15.1 25.0 um PGA) 16 6.3 53.7 22.8 25.0 um PGF 2, 16 6.8 63.8 27.6 0.5 mm Ro20-1724 16 6.9 72.4 24.0   AMP, which activates cyclic AMP-dependent protein kinase (13) and inhibits a low-Kn, phosphodiesterase (14), decreased basal, PGE;-stimulated, and ClAdo-stimulated adenylate cyclase activities. This observation suggests that PGE,-dependent inactivation of adenylate cyclase may be mediated by 8-Br cyclic AMP-dependent protein phosphorylation. In contrast, treatment of cells with Btz cyclic AMP, an inhibitor of a cyclic nucleotide phospho- diesterase (14, 15) and a relatively poor activator of cyclic AMP-dependent protein kinase (16), markedly desensi- tized adenylate cyclase to ClAdo but had little effect on basal or PGE:-stimulated adenylate cyclase activities. Bt cyclic AMP or a metabolite of Bt2 cyclic AMP resem- bles ClAdo with respect to its ability to desensitize NG108-15 cells, which suggests that Bt cyclic AMP may activate adenosine receptors. Bt2 cyclic GMP had little or not effect on basal, PGE:-stimulated or ClAdo-stimu- lated adenylate cyclase activities. Treatment of cells with 25 um PGA; for 16 hr resulted in small decreases in basal, PGE,-stimulated, and ClAdo-stimulated adenylate cy- clase activities, whereas treatment of cells with 25 um PGE,, or 0.5 mm Ro20-1724, a cyclic AMP phosphodi- esterase inhibitor, had little or no effect on basal, PGE,-, or ClAdo-stimulated adenylate cyclase activities (Table 2, Experiment 2). NG108-15 hybrid cells were cultured for 16 hr without PGE, or with 0.25, 2.5, or 25 ym PGE; homogenates were then prepared and assayed for adenylate cyclase activity in the presence of various concentrations of PGE; (Fig. 4A). The extent of desensitization of adenylate cyclase to PGE; was a function of concentration of PGE: used for cell treatment. Eadie-Scatchard plots are shown in Fig. 4B-D, and activation constants (Kac:) for PGE, and max- imal velocity (Vmax) values for PGE:-dependent adenyl- ate cyclase activities are shown in Table 3. Treatment of NG108-15 cells with PGE, resulted in increases in the activation constants for PGE,-dependent adenylate cy- clase activity (i.e., decreases in apparent affinity of PGE, for receptor) and decreases in the Vinax of PGE,-depen- dent adenylate cyclase. Similar results have been re- ported recently by Homburger et al. (17) with Cg glioma cells desensitized by isoproteronol. Cells were treated with 2.5 ym PGE; for 16 hr; the medium was then replaced with fresh medium without PGE, and incubation was continued for an additional 24 hr to determine the rate of recovery of adenylate cyclase responsiveness to PGE). At 4, 8, 12, and 24 hr after withdrawal of PGE; (20, 24, 28, and 40 hr of total incu- bation, respectively), homogenates were prepared, and basal, PGE;-stimulated, and ClAdo-stimulated adenylate cyclase activities were determined (Fig. 5A). On with- drawal of PGE:, adenylate cyclase activity gradually returned toward the control value; 50% of the enzyme activity lost because of PGE, was recovered within 8 hr, and 80% of the activity was recovered within 24 hr. The effect of PGE, concentration on adenylate cyclase activ- ity in homogenates prepared from untreated control cells cultured for 16 or 28 hr, from cells treated with 2.5 um PGE, for 16 hr, or from cells treated with 2.5 uM PGE                                      za i CONTROL | : om oO E z 0.25 uM PGE | = — 2 2.54M PGE, < ’ ° a a eed al _? $ 25.04M Pc, S piiiint pet ttt il | 10° 106 10° IPGE,], M NTRO 0.254M 400 CONTROL) 49 © PGE, 49 © ‘ 2 300 60 N, 8 \\\\ a a 200 40 6 | = z q 4 100+ \\\\ 20+ 5 J ° 20 40 60 80 10 20 30 40 5 10 15 20 AV = Vistimucaten) ~ Vieasaui Fic. 4. Effects of culturing NG108-15 cells with PGE, on the kinet- ics of PGE,-stimulated adenylate cyclase activity A. Cells were cultured for 16 hr without PGE, (V), with 0.25 uM PGE, (0), with 2.5 um PGE, (A), or with 25 um PGE, (O). Homogenate* were prepared and assayed for adenylate cyclase activity in the presence of various concentrations of PGE, (abscissa). B, C, and D. Eadie-Scatchard plots of the data from control cells gro¥” without PGE, (B), cells treated with 0.25 um PGE, (C), or cells treated with 25 um PGE, (D). The velocity, V, represents picomoles of “Pe labeled cyclic AMP formed per minute per milligram of protein: Al represents picomoles of “P-labeled cyclic AMP per minute per milli- gram of protein dependent on PGE; S represents micromolar PGE:. PGE,-INDUCED DESENSITIZATION OF ADENYLATE CYCLASE            TTT TT TTT Te     CONTROL Zz 16 hr ii ke 3 ec a oD — Zz 2 a 2 cg oO a a) 2 on        0 bo pp el 16 24 32 4 ° +O 108 107 106 10° HOURS [PGE,],M Fic. 5. Recovery of adenylate cyclase activity following removal of PGE, A. NG108-15 cells were cultured for 16 hr in the presence of 2.5 1M PGE, (@, MH, ¥) or absence of PGE, (©, O, V); at 16 hr the medium was replaced by medium without PGE). At the times indicated the cells were harvested and frozen. Homogenates were assayed for basal ade- nylate cyclase activity (O, @) and for stimulated activity in the presence of 10 ym PGE, (0, @), or 50 ym ClAdo (V, ¥). B. Symbols represent adenylate cyclase specific activities obtained with homogenates of NG108-15 cells from the experiment shown in A grown under the following conditions and assayed in the presence of the concentration of PGE, shown on the abscissa: O, cells grown for 16 hr without PGE;; V, cells grown for 16 hr with 2.5 ym PGE,, D, cells grown for 28 hr without PGE;; A, cells grown for 16 hr with 2.5 uM PGE, and for an additional 12 hr without PGE). for 16 hr and for an additional 12 hr in the absence of PGE, (28 hr in Fig. 5A) is shown in Fig. 5B. Activation constants for PGE, and Vinex values for PGE,-dependent adenylate cyclase are shown in Table 3. Treatment of cells for 16 hr with 2.5 wm PGE; resulted in an increase in Kee and a decrease in Vinax. The Vinax of PGE,-stimu- lated adenylate cyclase from control cells decreased be- tween 16 and 28 hr; however, the activation constant did not change appreciably. Treatment of cells with PGE, for 16 hr and withdrawal of PGE, for an additional 12 hr TABLE 3 Activation constants and maximal velocity values for PGE,- dependent adenylate cyclase activities shown in Fig. 4A-D and       Fig. 5B Fig. Addition to cells Kar Vinax Stage 1, Stage 2, 0-16 hr 16-28 hr pM pmoles PGE, cyclic AMP/min/ mg protein 4 None 0.18 72.8 0.25 pm PGE, 0.59 40.0 2.5 um PGE, 0.65 21.8 25.0 pm PGE: 1.52 16.7 5B None 0.22 102.4 2.5 um PGE, 0.73 41.8 None None 0.19 68.7 2.5 um PGE, None 0.32 58.6   589 TABLE 4 Effect of cycloheximide on PGE,-dependent desensitization of adenylate cyclase and on recovery of activity after PGE, withdrawal NG108-15 cells were incubated with the indicated compounds for the times shown. Homogenates were prepared and assayed for adenyl- ate cyclase activity. Where indicated, the concentration of cyelohexi- mide was 20 yg/ml of medium and the concentration of PGE, was 2.5 uM. “       Expt. Addition to cells Adenylate cyclase assay Stage Stage 2 No 10 ’ Mf 1, 0-16 16-24 hr addi- PGE, cones hr tion pmotes ®P. labeled eyelic AMP/min/mp — protein 1: Densensitization None None 13.2 99.7 40.5 None PGE, 54 34.0 187 None  Cyclohexi- 11.5 98.0 38. I mide , None  Cyclohexi- 4. 46.9 12.4 mide + PGE, 2: Recovery None  Cyclohexi- 10.3 76.0 28.1 mide , PGE,  Cyclohexi- 44 322 19.7 mide , PGE, — 5.1 23.5 18.0 PGE, None 5.7 50.0 27.9   resulted in an increase in Vmax and a . : Kact, 1.e. the values returned toward control ee \" The effects of cycloheximide on PGE)-dependent Ic of adenylate cyclase activity and on recovery of res on. siveness to PGE, following withdrawal of PGE x ; shown in Table 4. Cycloheximide had no effect on PGE, dependent loss of adenylate cyclase activity, but in. hibited the recovery of enzyme activity on withdrawal 7 f PGE). In other experiments not shown here, 16 pM acti nomycin D did not affect PGE:-dependent desensitiza. tion of adenylate cyclase or recovery of enz ivi t yme in the absence of PGE). aerivity DISCUSSION Repeated exposure of cells to a receptor activator ofte diminishes the extent of the responses mediated by that species of receptor (18-29). In most cases the response i lost only to the ligand used for desensitization; cells retain responsiveness to ligands for other species of receptors Yu and co-workers (28) have termed this phenomenon homologous desensitization. However, several investi a. tors (18, 27-29) have reported that exposure of cells to \"4 ligand for one species of receptor can result in a decrease in basal adenylate cyclase activity and/or loss of enzyme responsiveness to ligands for multiple species of recept. r [termed heterologous desensitization (28)]. Heterologo “ desensitization thus may be due to inactivation of either adenylate cyclase, molecules that functionally cou le receptors to adenylate cyclase, or to changes in mem. branes that affect the activities of the receptors and/« t other components that are part of the adenylate cyclase complex. Treatment of NG108-15 hybrid cells with PGE, results in decreases in basal adenylate cyclase activity and in 590 KENIMER AND NIRENBERG PGE,-, ClAdo-, NaF-, or Gpp(NH)p- and PGEy-stimu- lated activities. These results suggest that one or more components of the adenylate cyclase complex are inac- tivated when NG108-15 cells are treated with PGE. More than 95% of the specific binding sites for PGE; also are lost.’ Thus, PGE; receptors and molecules required for activation of adenylate cyclase may be lost coordi- nately. Tolkovsky and Levitzki (30) have reported that aden- osine receptors of turkey erythrocytes either are coupled permanently to adenylate cyclase or form long-lived in- termediates. Our results show that treatment of NG108- 15 cells with ClAdo results in rapid desensitization to ClAdo with little effect on basal or PGE,-dependent adenylate cyclase activities. The extent of activation of NG108-15 adenylate cyclase by saturating concentration of ClAdo is approximately 25-40% of that found with PGE, (12). The extent of desensitization of adenylate cyclase may be a function of the amount of cyclic AMP synthesized or the duration of the activated state. Assum- ing that the rate of cyclic AMP synthesis in intact NG108- 15 cells equals that found in homogenates, exposure of cells to PGE; for 360 min (the time required for 50% loss of PGE,-dependent adenylate cyclase activity) would result in the cumulative synthesis of 32 nmoles of cyclic AMP per milligram of protein above basal activity. Ex- posure of cells to ClAdo for 108 min (the time required for 50% loss of ClAdo-dependent adenylate cyclase activ- ity) would result in the cumulative synthesis of 4 nmoles of cyclic AMP per milligram of protein above basal activity. Thus, ClAdo-dependent adenylate cyclase cat- alyzes only 12.5% as much cyclic AMP synthesis com- pared with PGEi-dependent adenylate cyclase. The ap- parent absence of effect of ClAdo on responsiveness of adenylate cyclase to PGE, or on basal adenylate cyclase may be due to the lower extent of activation of the enzyme by ClAdo. Cycloheximide had no effect on PGE,-induced desen- sitization of adenylate cyclase, but markedly inhibited the recovery of adenylate cyclase activity from the de- sensitized state. In contrast, actinomycin D had no effect on either PGE)-induced desensitization of adenylate cy- clase or on recovery from the desensitized state. These results suggest that protein synthesis, but not mRNA synthesis, is required for recovery of PGE)-desensitized adenylate cyclase activity in NG108-15 cells. Cyclohexi- mide does not block the recovery of adenylate cyclase activity following beta-receptor-induced desensitization of the enzyme in frog erythrocytes (23) or in Ehrlich ascites tumor cells (26). Cycloheximide also does not affect desensitization or recovery of adenylate cyclase activity from the desensitized state in human astrocy- toma cells exposed to norepinephrine or PGE; (28). How- ever, cycloheximide or actinomycin D prevents the de- velopment of refractoriness to PGE» in cultured rat Graafian follicles (21). In conclusion we find that prolonged activation of NG108-15 adenylate cyclase by PGE; results in the prad- ual loss of adenylate cyclase activity, much as prolonged 5 J, G. Kenimer, manuscript in preparation. receptor-mediated inhibition of adenylate cyclase results in gradual compensatory increase in adenylate cyclase activity. We also find that exposure of NG108-15 to 8-Br cyclic AMP, which activates cyclic AMP.-dependent pro- tein kinase, results in the loss of adenylate cyclase. Fur- ther work is needed to determine whether the loss of adenylate cyclase is dependent on cyclic AMP-mediated phosphorylation of protein (31). REFERENCES 1. Sharma, S. K., W. A. Klee, and M. Nirenberg. Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc. Natl. Acad. Sci. U. S. A. 72:3092-3096 (1975). 2. Traber, J., G. Reiser, K. Fischer, and B. Hamprecht. Measurements of adenosine 3’:5’-cyclic monophosphate and membrane potential in neuroblas- toma * glioma hybrid cells: opiates and adrenergic agonists cause effects opposite to those of prostaglandin E.. F. E. B. S. Lett. 62:327-332 (1975). 3. Traber, J., K. Fischer, C. Buchen, and B. Hamprecht. Muscarinic response to acetylcholine in neuroblastoma x glioma hybrid cells. Nature (Lond.) 255: 558-560 (1975). 4. Burgermeister, W., W. L. Klein, M. Nirenberg, and B. Witkop. Comparative binding studies with cholinergic ligands and histrionicotoxin at muscarinic receptors of neural cell lines. Mol. Pharmacol. 14:751-767 (1978). 5. Sabol, S. L., and M. Nirenberg. Regulation of adenylate cyclase of neuroblas- toma X glioma hybrid cells by a-adrenergic receptors. I. Inhibition of ade- nylate cyclase mediated by a-receptors. J. Biol. Chem. 254:1913-1920 (1979). 6. Nathanson, N. M., W. L. Klein, and M. Nirenberg. Regulation of adenylate cyclase activity mediated by muscarinic acetylcholine receptors. Proc. Natl. Acad. Sci. U. S. A. 7521788-1791 (1978). Sabol, S. L., and M. Nirenberg. Regulation of adenylate cyclase of neuroblas- toma x glioma hybrid cells by a-adrenergic receptors. II. Long-lived increase of adenylate cyclase activity mediated by a-receptors. J. Biol. Chem. 254: 1921-1926 (1979). Nelson, P., C. Christian, and M. Nirenberg. Synapse formation between clonal neuroblastoma x glioma hybrid cells and striated muscle cells. Proc. Nail. Acad. Sci. U. S. A. 73:123-127 (1976). 9. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2xjc.29xk-pnpa", "00000000-0000-0000-7AB9-CF3536C9F15F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The NK-2 Homeobox Gene and the Early Development of the Central Nervous System of Drosophila", "101584910X136", null, "1995", "1995", "In this article, Marshall Nirenberg explains the \"logic that connects the genetic code to neurobiology is that information is processed in both genetic and neural systems.\"  This study of the NK-2 homeobox gene of the Drosophila fly examines the role of the gene's expression in the early development of the central nervous system of the embryo.  Strikingly, the article notes, \"The nucleus of the fertilized Drosophila embryo undergoes thirteen rounds of nuclear division in the first 130 minutes of embryonic development, resulting in an embryo that consists of a single cell with approximately 5,000 nuclei.\"", "Articles", "Central Nervous System,Drosophila,Genes, Homeobox ; Molecular Sequence Data,Amino Acid Sequence", "From Neuroblastoma to Homeobox Genes, 1976-1992", "19", "pages", "Text", "English", "Reproduced with permission of the New York Academy of Sciences.", "Copyright may apply", null, null, "The NK-2 Homeobox Gene and the Early Development of the Central Nervous System of Drosophila MARSHALL NIRENBERG,? KOHZO NAKAYAMA,’ NORIKO NAKAYAMA,’ YONGSOK KIM,’ DERVLA MELLERICK,? -LAN-HSIANG WANG,’ KEITH O. WEBBER,? AND RAJNIKANT LAD‘ 4 Laboratory of Biochemical Genetics National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892 ’ Department of Immunobiology Cancer Research Institute Kanazawa University Takaramachi 13-1 Kanazawa, Ishikawa 920, Japan © Laboratory of Molecular Cardtology National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892 4 Laboratory of Molecular Biology National Cancer Institute National Institutes of Health Bethesda, Maryland 20892 © Department of Psychiatry Hospital of the University of Pennsylvania Philadelphia, Pennsylvania My colleagues and I deciphered the genetic code gradually, over a period of five years, between 1961 and 1965. I then stopped working on the code and began working in the field of neurobiology. The logic that connects the ge- netic code to neurobiology is that information is processed in both genetic and neural systems. My interest in the NK-2 homeobox gene! of Drosophila stems from the observation that NK-2 is the earliest predominantly neural gene regulator that has been found thus far that is expressed in the ventrolateral neurogenic an- 224 NIRENBERG ¢t ai.: NK-2 HOMEOBOX GENE 225   FIGURE 1. Side view of stage 5 Drosophila embryo illustrates concentration gradients of proteins that regulate gene expression. The concentration of bicoid homeobox protein is high in the ante- rior (A) region and low in the posterior (P) region of the embryo. A concentration gradient of nanos protein is established in the posterior to anterior direction. A concentration gradient of dorsal protein is established in nuclei in the ventral (V) towards the dorsal (D) region of the em- bryo. lage, which gives rise to part of the central nervous system of the embryo. I will tell you what we know about the NK-2 gene and relate these findings to the early development of the Drosophila embryo and the central problem of understanding the principles that are used initially to construct part of the cen- tral nervous system of the embryo. The studies on NK-2 were performed by my colleagues, Yongsok Kim, Kohzo Nakayama, Noriko Nakayama, Dervla Mellerick, Lan-Hsiang Wang, Keith Webber, and Rajnikant Lad. The nucleus of a fertilized Drosophila embryo undergoes 13 rounds of nu- clear division in the first 130 minutes of embryonic development, resulting in an embryo that consists of a single cell with approximately 5,000 nuclei. Most of the nuclei move to the periphery during stage 4 (80-130 minutes after fertilization, nuclear divisions 10-133) and cell membranes form around each nucleus between 130 and 170 minutes after fertilization (stage 5). The anterior-posterior and ventral-dorsal axes of the embryo are established and different cell types are generated during stages 4 and 5 by the formation of concentration gradients of proteins that regulate gene expression (Fic. 1). A concentration gradient of bicoid homeobox protein is established in the anterior-posterior direction (high concentration in the anterior portion of the embryo, low concentration in the posterior portion**); and a concentration gradient of nanos protein is established in the posterior to anterior direc- tion.”-!0 Concomitantly, a concentration gradient of dorsal protein is estab- lished in nuclei, with the highest concentration of dorsal protein in nuclei in the ventral portion of the embryo and the lowest concentration in nuclei in the dorsal part of the embryo.!!-!4 These gene regulators and terminal gene 226 ANNALS NEW YORK ACADEMY OF SCIENCES regulators initiate the induction or repression of other genes that encode pro- teins that regulate gene expression and result in dynamically changing patterns of gene expression in different parts of the embryo, depending upon the con- centrations of gene regulators that the nuclei were exposed to. The anterior- posterior gradients of gene regulators result in the formation of vertical stripes of equivalent nuclei that were exposed to the same concentrations of gene reg- ulators, whereas the ventral to dorsal gradient of dorsal protein results in the formation of horizontal stripes of equivalent nuclei (for reviews see Refs. 15-18). In effect, the embryo is divided into a bilaterally symmetric checker- board of clusters of nuclei that express different combinations of genes for pro- teins that regulate genes. The position of a nucleus in the embryo therefore determines the initial developmental fate of the nucleus. In FIGURE 2 is shown the composite nucleotide sequence of NK-2 cDNA and genomic DNA and the deduced amino acid sequence of NK-2 protein.!° The NK-2 gene contains 3 exons and 2 introns; introns ] and 2 are approx- imately 1.68! and 3.1!° kb in length, respectively. 3 NK-2 protein contains two regions near the N-terminus that consist almost entirely of alternating acidic and basic amino acid residues or pairs of acidic and basic amino acid residues. The protein contains multiple Ala repeats, an Asn repeat, an acidic domain, followed by a homeodomain, which is not closely related to any other Drosophila homeodomain. The C-terminal region of NK-2 protein con- tains a 17—amino acid residue sequence of unknown function termed the NK- 2 box (amino acid residues 631-647) that has been highly conserved during evolution and a His-Ala repeat. The NK-2 gene was shown to reside on the sex chromosome at 1C]-5.1 The NK-2 homeodomain has been conserved during evolution. FiGURE 3A compares the amino acid sequence of the Drosophila NK-2 homeodo- main!!9 and NK-2-like homeodomains from Xenopus,?° mouse,?!-2? pla- naria,?3,24 leech,?> and tapeworm.© The similarity in amino acid sequence ranges from 95 to 67 percent. The mouse genome contains six copies of the NK-2 gene, presumably formed by gene duplication. The amino acid residues that comprise NK-2 homeodomain a-helices I, IH, and III were determined by NMR.’ Binding of a 77-amino acid residue protein that contains the NK-2 homeodomain to a high-affinity NK-2 binding site in DNA results in an increase in the length of a-helix III from 11 to 19 amino acid residues (from NK-2 homeodomain residues 42-52 to 42-60) and also increases the stability of the secondary structure of the homeodomain.?” Xenopus and mouse proteins with NK-2-like homeodomains also contain the highly conserved 17-amino acid residue NK-2 box after the homeodo- main (94-77% homology with the NK-2 box sequence of Drosophila NK-2 protein) (Fic. 3B). The Drosophila NK-3!:?° (bagpipe)?® homeodomain pro- tein also contains a sequence related to the NK-2 box (47% homology); how-   NIRENBERG et al.: NK-2 HOMEOBOX GENE 227     CCAGTTGTTGATGACACTTCAGTTTGGAATGT SGCTCGTK SCGTGCAACT! A RACGACTCGCGACCCTTGAACTATCGAACTTTCCCGGTGATAA ACTT. ARACTTAAGOCT AAATCGAACTATGT CTOGAATT TAAGTTGCCCT ACCAGGA TACCOGCTTAA PI CATCOCAGIGCT ACCCAGTAACTACACATARACATATI (CTI PCOCGTOCT     CCGAACACCGTAAAAGCTCCAA AACGCAAAAGTCCOCTC AATCAAACAGTOGTT A CA ur T CGTCGGOGTCCTTGGAGAGGACCCCCTCAAAGCGGGA TOGA GA TCGCGAGCGGGACAACAGCAGC GGT! CTGGGCAGCGCTGGCAGCTT GCCCGCATCGCCCCAAAGCGCTATCACGGTGA SAS LERTPSKEDRORERDNSSGLGSAGSLPASPQESEATTVS GTCCGTCCTCCCCAGCCACGCCGAAGGCCCOGCTSCCGACCT CAACEC CCT OBC: C SGCAAGGAGCGGCAGGAACGGC PSS PAT PKA PLPT ST PSLEREREBR BS DRE DR EDA KE RO EL BY ACGAGCGAGACAGAGATCACGAGAGATTCGCCGCAGT CTT CAGCACCGCTAGCACCACCUTGCCCACGAACACAAGTTCCAGT TCCGGATT GGCGCCCGAACAGCT 'CCGCATTCCGACGG ERDRDHERFAAVFSTASTTVPTNTS SSESGCLAPBEQLRIP TS CAGCGGA TTT CCGGGGCTCCACAGCATGAGTAGTCTTATGCTTCCATCGTCGGCGGCTST GECCGOCOCAGCA SEGSCCOCOT TPCT GCOS. 'GETCGCCCATCOTGC AAAPSGFPGLHSMSSLUMOEPS SRAVYVAAABRAA P FL PWS PILL y TECCECOSTGGAACCACGCCCTOCTACCAGCOGCCT TT TAT COGGCGGCCCT GCGAAACGCTTTGCCT OGCTTATTCGATOCAAAGGT GCOGTCOTCOCAACOCTCTGGCTICCATATAT PPWRNRHALULPAAFYPAALRNALPGLFOAKVP SSQREGPHI S COGACATCTTGAATTTGGAGGGCT.CTGAGCTGAAGAATOCAGCAGCTGCCGCCGCT GCTOCCACCCACCATGGCAGCGATTTGAGTCACCACT COGCCAGT GAGT CCACCAGTGGRCATC DILNLe#os &€&LKNAAAAAAAAAHHGSODLSHHSASESTSGHR GCOGCCAGGGATICT. CACACOTCGCCT TCGGCCGTETCGCCCA CGCCAGCGGGCGTTICCGCGGACGAGCACCACAATGGCA GCGGGACT GGGGGAGGA GCGBGAGANGCGGATCACCACA ¢qcSuHhTsPSAVSPTPAGVSADEHHNGSGTGGGAGEA D HHS   QCACCACCGAGCATCACGCCCCTCCTAGCCAT AGCAACAGCACCC CCAGCA! STCACCICCTC! \\\\GCAGCACCATC! SGGTGGCCCCAC TIEMHA PPSHE GOO PHY HOG PEEL LP Oo OO A VA P OL   IRCOCTTTEGCGCACCATCAGAGOGGCGAAGCCCAAAGCCACGCCCATGCCAATOCAGCOOCCGCCCA TCT GCTGGCCAGOCACAATSCA GERI TACGICT OCT OT GCE GCCBECCAAT PLA HHOSGEAQSHAHANAAAAHLL ASH NAAAAA AY AA GO Y ATCT GCCCAACCTGCCCAAGAACTTCCCGGGGAGCTIT: GGCGACGAGATGTCCTOGTACCACCACATGGCOCAAACTA TGCT GCA GCACTCGGGCAGGAGT ‘GCGTGGATCAAGGAGAACG DPNOLPKNFPGSFGDEXSSYHHHRAQTMLAHS GRSEAWI RENE q AGCTATACGGT. ACCCAGCAGCCOGCCA GT. CCGGA TAGCACCT CCCCAGTT ACCTOGGAA GTGTCGTACACCTACATT GGT TCCAACTSCCAGACAT COCCT GOCCTTTCCGGCGATTACA LYGcG?TQqgQPASPDSTSPVTSEVSYTYIGSNCQTSPALSEDY K AGAGCTACAGCOGGTCGGCCGATAGCGATGCACTATCCOTGGGCGACGCCCTGCACACCCTICCATGGA TCCTCT GGT AAT GGAAGT GCCGGAGGOGCT CCGA CGGCCCA TOCCCTACACA 5 YS RSADSDALSVGDRDALHTLHGSSEGNGSAGGAPTAHALHAYD     ACAACAATAATAATACGACAAACAACAATAACCACAGCCTGAAGGCL AC ‘OGT SCTCAACGA ‘CGACG ‘NNNNTOINNREBN HS LUKAEGINGAGSGHOD SL NE DG 1 ££ p12 p 120 240 360 4380 43 600 83 720 123 940 163 960 203 1080 243 1200 283 1320 323 1440 363 1560 403 1680 4a3 1800 483 1920 $23   ACGTGGACGACGCCGACGGCAGTGGCGUCGGGGAT GCAAAT GGA TCOGACGGT CTGCCAAA TARG bh AACGGAAGCGACGAGTCCTGTTCACCAAGGCGCAAACATATGAGCTGGAACGTC VRBDADGSGGGDANGSDGLPNWNKKRKERRVLF TKAQT Y EL ERA     GGTTTOGA CAACAACGTT ACT T GAGTGOCCCGGAAGGCGA GCACCT GGCCAGTT TGA TCCGCCTGACGCCGACCCA GGTGAAGATCTGGTTTCAAAACTA TCGCT ACARGACGAAGC GGG rRQOQRYLSAPERREHUASLUIRLT PT QV RK IWF QN HW ORY K T OK RA 2040 563 2160 603       CGCAAAACGAGAAGGGCTACGAGGGTCATCCTGGTCT ACT GCACGGCCATGCCACCCATCCGTA TCACCOCAGTGCCCTCCATCSLCC CGN CGTTCCAGTTCTGGTGAGGA Q EKRKGYEGHuHPGtLLHGHA TH PH HPS AL P     ACGGAAAGCCCTGCTTGGOCGATAGTT.CCARACTGGGAGCOGACTGCGT CT CCGTGTCATCAGCCACCGCCACCGCCA TGCAGAATOCCGCCGCOCATCACTTGGTTGCCCTAAA TOGA GERERPCLGDSSKLGADCVSVYVSSATATAMQNAAAH HL VALN GA CGGCCECCT AT CAACA TGCCGCTGCAGCGGCTOCCEGCOTTCACGCCCACGCCCATGCTCATACCCACOCCCACGGACACGGCCA TOCACACGCOCACGCCTAGA SESE C OCCT GST GST AA YQHAAAAAA G LHAHABHAHAHA HU GH GH P HAHA Q RA AWW P CCTAATATTOCTAGGAACTGGCATTCACGGGACTOGGGGCGGATAGGTOGA TAGTCAGGCCOGT GGAGCGATGAGCCAGAAGACCACGCCTCCAGATTCCCAGT GAAGCCAAGTAAAAGA : 2 TATACAATAATAATTGAAACACATACAAACCCCT ACT AGCGCTCAGT CTCCCGGACTTATCCTIGAGTCGTTTCTGTACATAGTTCATATATATARTCATATATATACCTAAATGACAT AAATTTATOCATTCCTOGATATCAGACCANTOCCCAT GACAAAAGAAAGCTACATTTCCTTCTABATAABAATCAATACTAGTGTCTCTTGTCAAGCOCATTT AAT CGCATAAAATTTA GAAGTAGATTCAT EET TPTTATETT TTT TTT TTAGCCCTAAGTCCAATCCCAAAGAT AAAATGTGTAAATATAATATAAAAATGAAGT AAA TCTGAAAAA TAGATTATTICAAAAGAGA AATAAATAATAGGAAATATGCAACTAAAAARAA FIGURE 2. The nucleotide sequence of NK-2 cDNA and the deduced amino acid sequence of NK-2 protein (from Ref. 19). The homeodomain is enclosed within a box. Other interesting amino acid sequences are underlined, such as an acidic domain before the homeodomain and the highly conserved NK-2 box sequence after the homeodomain (amino acid residues 631-647). An inverted triangle shows the position of an intron. 2280 643 2400 683 2520 723 2640 2760 2880 3000 3033 228 ANNALS NEW YORK ACADEMY OF SCIENCES                         Q-HELIX 3 A SPECIES Q-HELIX 1 QHELIX 2) Q=HELIX 3 sic a 1 11 22 28 38 42 52 60 HOMOL- . . : . . . : . OGY REF, NK-2 d KRKRRVLFTKAQTYELERRFRQQRYLSAPEREHLASLIRLTPTQVKIWFOQNHRYKTKRAQ 100 1, XeNK-2 x s M---R 95 20 Nkx-2,.2 m Ss M. R 95 21 Nkx-2.4 m SQ--V K--K M-H M--OA 83 21 Nkx-2.1 m R sQ--v K--K M-H M--OQA 82 21 Nkx-2.3 m R--P----SQ--VF K SLK~--$ R---C--QR 75 21, Nkx-2.5 m R=--P~---SQ--V------- Kroon en DQ---VLK--S-------~- R---C--OR 73 22 Nkx-2.6 m Q-~S----SQ--VLA---~- K T ALQ--S R---S-SQR 70 22 Dth-l =p ---=----- $-K-IL----H---KK N--G-S M---H 80 23, Dth-2 p R----I--SQ--I K--K N--N C--S- 82 23, Lox 10 1 R----I-~SQ--I K TF-G KSK 80 25 EgHbx-3 t QS----~-N-F-1ISQ--K---K----T-0--QE--HT~G----~-~~------ A--M--LF 67 26 B SPECIES % AAA HOMOLOGY REF. NK-2 d 26 SPRRVAVPVLVR-NGKPC 100 1, 19 XeNK-2 x ll ------------: D---- 94-20 Nkx-2.2 m 110 ------------: D---- 94.21 Nkx-2.3 m 140 Peewn-------: D---- 88S 21 Nkx-2.4 m 2300 terete nnn K'D---- 8821 Nkx-2.1 m 34 ----------- K*D---- 88 = 21 Nkx-2.5 m 13. PA--I-------- D---- 77 22 Nkx~-2.6 m 13. PA----~---- L-D---- 77 = 22 NK-3 (bagpipe) d 10 ASK--P-Q----ED-STT 47 1, 28-29 FIGURE 3. (A) Comparison of the amino acid sequence of the (d) Drosophila NK-2 homeo- domain with similar homeodomains from (x) Xenopus, (m) mouse, (p) planaria, (I) leech, and (t) tapeworm (from Ref. 19). The symbol (—) represents the same amino acid residue as NK-2. The amino acid residues of Drosophila NK-2 homeodomain a-helix 1, a-helix 2, and a-helix 3 were determined in NMR.2’ In the absence of DNA a-helix 3 extends from amino acid residue 42 through 52. However, binding of the NK-2 homeodomain to a high-affiniry NK-2 site in DNA increases the length of a-helix 3 (residues 42-60).2” (B) The NK-2 box is a highly con- served 17—-amino acid residue sequence that is found after the homeodomain in proteins related to NK-2. AAA represents the number of amino acid residues between the end of the homeo- domain and the beginning of the conserved NK-2 box sequence. The symbol (-) represents the same amino acid residue; (.) represents the absence of an amino acid residue. ever, a highly conserved NK-2 box was not detected in the planarian homeo- domain proteins, Dth-] or Dth-2. Northern analysis of poly A* RNA from Drosophila at various stages of de- velopment showed that NK-2 mRNA is present in highest concentration in 3-6-hr Drosophila embryos and then progressively decreases during further em- bryonic development.!? No NK-2 mRNA was detected in 0-3-hr Drosophila embryos; therefore, no maternal NK-2 mRNA was found. Larvae and pupae contain greatly reduced levels of NK-2 mRNA compared with that of 3-6-hr embryos; however, an increase in NK-2 mRNA was found in adult flies. FIGURE 4 shows the distribution of NK-2 mRNA in Drosophila embryos as a function of developmental age, determined by # situ hybridization. NIRENBERG et al.: NK-2 HOMEOBOX GENE 229 NK-2 gene expression is initiated during stage 4 in bilaterally symmetrical longitudinal stripes, one stripe on each side, in the ventral (i.c., medial) half of the ventrolateral neurogenic anlage (Fic. 4A). By stage 5, when the first cell membranes are forming around the nuclei, the stripes of nuclei that ex- press NK-2 extend from 0 to 90% of the embryo length and each stripe is 6 or 7 nuclei in width (Fic. 4B). NK-2 is also expressed in part of the proce- phalic neuroectodermal anlage, the endodermal anterior and posterior midgut anlagen, and the hindgut anlage. The ventral mesodermal primordium invag- inates during gastrulation, bringing the longitudinal NK-2 positive bands of neuroectodermal cells closer to the ventral midline, separated only by ventral midline mesectodermal cells, which do not contain NK-2 mRNA (Fics. 4C-F). At first the level of NK-2 mRNA in the band of NK-2-positive cells is fairly homogeneous; however, during early gastrulation clusters of cells with high levels of NK-2 mRNA appear that are separated by vertical stripes of cells, 1 or 2 cells in width, that contain lower levels of NK-2 mRNA, apparently due to repression of the NK-2 gene (Fics. 4E-F). Initially one cluster of cells with a high level of NK-2 mRNA is formed per hemisegment;, later two clusters of NK-2-positive cells appear per hemisegment (Fics. 4G—J). Germ band extension results in an increase in the length of the band of cells that synthesize NK-2 mRNA and a concomitant decrease in the width of the band to 2 to 3 cells per side (Fic. 41). Hence, the neuroectodermal cells that syn- thesize NK-2 mRNA give rise to medial and paramedial neuroblasts that con- tinue to synthesize NK-2 mRNA. Ganglion mother cells and neurons were found that express the NK-2 gene that perhaps are the progeny of neuroblasts that express the NK-2 gene. However, during later embryonic development, the abundance of NK-2 mRNA decreases in some neurons and is extinguished in others. Some neurons that express the NK-2 gene form commissures and others contribute to longitudinal connectives. FiGuRE 5 shows schematic diagrams of cross-sections of embryos at the cellular blastoderm stage (stage 5) before nuclei have been enclosed by cell membranes, and at the end of gastrulation (end of stage 7) after ventral me- sodermal primordium cells have invaginated. These cross-sections of embryos illustrate ventral-dorsal patterning during early development of the Drosophila embryo. The ventral to dorsal concentration gradient of dorsal protein!-!* ac- tivates the tvist3°-34 and snail3+3 genes in the most ventral nuclei, which correspond to the mesodermal anlage, and the NK-2 gene is activated in the ventral (medial) half of the ventrolateral neuroectodermal anlage. A gene reg- ulator specific for dorsal (lateral) neuroectoderm has not been identified thus far. Dorsal protein represses the decapentaplegic (dpp) gene,*’— which encodes a protein that is a homologue of TGF-8,°7#! and the zen-I and zen-2 homeo- box genes*2-6 in nuclei in the ventral and lateral parts of the embryo, but not in nuclei that become dorsolateral epidermoblasts or dorsal amnioserosa, 230 ANNALS NEW YORK ACADEMY OF SCIEN! CES   PO aad cid   NIRENBERG et al.: NK-2 HOMEOBOX GENE 231 respectively. Hence, the concentration gradient of dorsal protein establishes the ventral-dorsal axis of the embryo and divides the embryo into longitudinal bands of nuclei that have different developmental fates. After cell membranes form, the most ventral cells, the mesodermal anlage, invaginate, which brings the mesectodermal cells to the ventral midline. Neuroectodermal cells gradually segregate as neuroblasts between about 3.5 and 7.3 hours after fertilization*”~4° (Fic. 6). Eventually a monolayer of neuroblasts and glioblasts separated by ventral midline mesectodermal cells is formed above the epidermal cells. Doe*? has shown that thirty-one neuro- blasts or glioblasts delaminate per hemisegment, that each is a unique cell type, and thar the relative position of each neuroblast or glioblast in the set is determined. A Drosophila embryo contains 14 parasegments and additional segments in the head region. Approximately 800 ventrolateral neuroblasts or glioblasts segregate from the medial and lateral neuroectoderm per embryo and addi- tional neuroblasts and glioblasts are formed from mesectodermal cells.4? The pattern of neuroblasts is repeated in different segments possibly with some vari- ation. However, some genes that encode proteins that regulate gene expres- sion are known to be expressed only by cells in a single segment, or a few seg- ments. Although most of the proof is lacking, it is likely that many neuroblasts also express segment-specific gene regulators and that most of the neuroblasts per side eventually will be found to be unique cell types. Three longitudinal stripes of neuroblasts or glial precursors can be distin- guished on each side that are the precursors of neurons and glia of the ventral nerve cord: ventral midline mesectodermal cells that separate the right and left halves of the ventral nerve cord, medial neuroblasts or glial precursors that express the NK-2 homeobox gene, and lateral neuroblasts or glial precursors that have little or no NK-2 mRNA. The monolayer of neuroblasts that give rise to the ventral nerve cord is also divided along the anterior-posterior axis of the embryo into 14 paraseg- ments; most parasegments consist of posterior compartment neuroblasts that   FIGURE 4. Distribution of NK-2 mRNA in Drosophila embryos as a function of developmental age (from Ref. 19). The RNA probe used for ## situ hybridization was from the 3’-untranslated region of NK-2 cDNA; the probe did nor contain the homeobox. (A) Expression of the NK-2 gene is initiated during stage 4, the syncytial blastoderm stage. (B) Stage 5-6, side view. (C) Ven- tral view, stage 6, early gastrulation. (D) Ventrolateral view in late stage 6 embryo. (E) Side view of embryo; gastrulation is almost completed. Late stage 7, about 185 minutes after fertilization. Notice the apparent segmentation of the NK-2-positive region. (F) Late stage 7 illustrating ap- parent segmentation of NK-2-positive region. (G) Side view stage 9 embryo 3.7—4.3 hours after fertilization. Two clusters of neuroectodermal cells and/or neuroblasts that contain NK-2 mRNA can be seen per hemisegment. (H) Side view of stage 9-10 embryo. (1) Ventral view of stage 10 embryo. Two clusters of medial neuroectodermal cells and/or neuroblasts that contain NK-2 mRNA are present per hemisegment. (J) Stage 9-10 embryo, side view. 232 ANNALS NEW YORK ACADEMY OF SCIENCES EMIX STAGE 7 140 MIEN. FN IE Obst atte hoody at ee ODER. DOWSAL Tabs ele Lari Prelster atin! avn rtrte i DT AUIEO1On KO ]R] ALB! a NEUROECTODERT : 7 ree om y VENTRAL “an \\\\\\\\\\\\ NEUHOECTONER’ i air NESECTODERM   MESODERM FIGURE 5. Schematic drawings of cross-sections of embryos before gastrulation (stage 5) and after gastrulation (end of stage 7) to show the ventral neuroectoderm nuclei or cells (yellow) that express the NK-2 gene. 31 KINDS OF NEUROBLASTS OR GLIOBLASTS NEUROBLAST SEGREGATION ames a ane at Kraee esis yA ene hey Lene y ANTE FIORE NEUROBLASTS lela] GLIOBLASTS aM pier eran en aes rh Se ef Sie) Sena hve} ABOUT $00 VENTROLATERAL NEUROBLASTS ARE FORMED. MOST NEUROBLASTS PROBABLY ARE UNIQUE CELL TYPES.   FIGURE 6. Some of the neuroectodermal cells delaminate and form a monolayer of neuroblasts and/or glioblasts immediately above the epidermal cell layer. The figure on the deft is a schematic illustration of a cross-section of an embryo to show the monolayer of neuroblasts or glioblasts that delaminate from the neuroectodermal cell layer. Béve: ventral midline mesectodermal cells that express the sim gene. Yellow: medial neuroblasts that express the NK-2 gene. Red: lateral neuro- blasts and/or glioblasts. Upper right panel shows 31 kinds of neuroblasts or glioblasts that delam- inate per average hemisegment. Four of the delaminated cells have migrated to other positions; hence, only 27 delaminated cells are shown. Lower right panel: Ventral view of the monolayer of neuroblasts and/or glioblasts to illustrate ventral-dorsal and anterior-posterior patterns. The yellow medial neuroblasts and/or glioblasts express the NK-2 gene. NIRENBERG et al.: NK-2 HOMEOBOX GENE 233 express the engrailed (en) homeobox protein*?,5° and anterior compartment cells that do not express en. Hence the 31 neuroblasts or glial precursor cells that segregate per hemisegment are divided into four groups of cells, de- pending on the position of the cells and the expression of the NK-2 and en genes: medial anterior compartment cells that have high levels of NK-2 mRNA but no en mRNA, lateral anterior compartment cells that lack NK-2 and en mRNA, medial posterior compartment cells that have high levels of both NK-2 and en mRNA, and lateral posterior compartment cells that have en mRNA and low levels of NK-2 mRNA. Neuroblasts start to divide soon after they segregate from the neuroecto- dermal cell layer. Each neuroblast division gives rise to a small ganglion mother cell and a large neuroblast, which becomes smaller with each division (Fic. 7). Each ganglion mother cell divides only once and gives rise to two neurons. The first neuroblasts to originate divide about eight times, whereas the last neuroblasts divide five times.*”-*® Therefore, a single neuroblast may be the precursor of 10 to 16 neurons. The neuroblasts that express the NK-2 gene®! in a thoracic segment at the end of neuroblast segregation (late stage 11) are shown schematically in FicureE 8, It should be emphasized that the abundance of NK-2 mRNA changes dynamically during development. NK-2 is expressed by two longitu- dinal columns of medial neuroblasts on each side; however, the abundance of NK-2 mRNA usually is higher in the column of neuroblasts adjacent to the mesectodermal ventral midline cells than in the second column of neuro- blasts. All neuroblasts in the posterior compartment express the NK-2 gene; however, the lateral neuroblasts contain much less NK-2 mRNA than do the medial neuroblasts. Hence, a medial to lateral gradient of NK-2 mRNA is es- tablished in both the anterior and posterior compartments. The amount of NK-2 mRNA in neuroblasts 2-1], 2-3, 5-1, and 5-2 (that is, immediately after or before the posterior compartment) decreases during development, re- sulting in the formation of two clusters of neuroblasts that express the NK-2 gene per hemisegment, one cluster in the anterior compartment and the second consisting of posterior compartment neuroblasts. Some ganglion mother cells and neurons also express the NK-2 gene; however, the levels of NK-2 mRNA decrease markedly in some cells during later stages of embryonic development. These results show that about half of the ventrolateral neuro- blasts express the NK-2 gene and that medial neuroblasts contain higher levels of NK-2 mRNA than do intermediate or lateral neuroblasts. The pattern of expression of the NK-2 gene also was determined in various mutant lines of flies as a function of developmental age.5! The NK-2 gene was found to be activated initially by dorsal in the ventral half of the embryo. However, the NK-2 gene normally is not expressed in the mesodermal anlage because of repression by snail, in the mesectodermal anlage because of repres- sion by sim, or in part of the lateral neuroectodermal anlage or dorsal epi- 234 ANNALS NEW YORK ACADEMY OF SCIENCES   FIGURE 7. POSTERIOR NN e=asiO) ; , rere Nal COMPARTMENT vTatsh zen (X) t dpp IN omanleiney GRADIENT L (X) 1 dorsal \" sim snail twist   Figure 8. FIGURE 9. NE URL Or=) od cero ra. ee LO lety: team =a Odea tuts) -DORSAL NEUROECTODERM -VENTRAL NEUROECTODERM MESECTODERM -MESODERM   Ficure 10. NIRENBERG et al.: NK-2 HOMEOBOX GENE 235 dermal anlage because of repression mediated by dpp. Both dorsal and twist were found to be required to activate the NK-2 gene in the hindgut pri- mordium and the posterior midgut primordium. During stage 4 dorsal activates the twist, snail, and NK-2°! genes (Fic. 9). Dorsal represses dpp and the zen genes in the ventral and lateral portions of the embryo; however, the concentration of dorsal is too low for effective re- pression of dpp or zen genes in the dorsolateral and dorsal portions of the em- bryo, respectively (for reviews see Refs. 52-54). snail is expressed only in the most ventral nuclei, which comprise the mesodermal anlage.55-5° sim is acti- vated in both the mesectodermal and mesodermal anlage, but sim is expressed only in the mesectodermal anlage because of repression by snail.3457-6° The NK-2 gene is activated in the mesodermal, mesectodermal, ventral neuroec- todermal, and part of the dorsal neuroectodermal anlagen, but is repressed by snail in the mesodermal anlage, by sim in the mesectodermal anlage, and by a gene regulator that has not thus far been identified whose repression is mediated by dpp in the dorsal neuroectodermal anlage.®! mist is expressed in the mesodermal, mesectodermal, and the ventral portion of the ventral neuro- ectodermal anlagen.°!-62.35 fist protein activates the snad gene in the meso- dermal primordium*® and the NK-2 gene in the ventral portion of the neuro- ectoderm.5! snail represses the sim and NK-2 genes in the mesodermal anlage, 5-59.60 while sim represses the NK-2 gene in the mesectodermal an- lage.*! The hierarchical organization of gene regulation results in the appear-   FIGURE 7. Neuroblast division. A neuroblast (green) divides 5-8 times. Each neuroblast di- vision is unequal and gives rise to a slightly smaller neuroblast and a much smaller ganglion mother cell (ed). Each ganglion mother cell divides once, giving rise to two neurons (yellow). FIGURE 8. A ventral view of neuroblasts in a thoracic segment at the end of stage 1] is shown using the neuroblast nomenclature of Doe.49 Neuroblasts shown in color express the NK-2 gene (from Ref. 51). The varying darkness of the neuroblast color from brown to mn represents the relative abundance of NK-2 mRNA; for example, in order of decreasing abundance of NK-2 mRNA, we see brown (neuroblast 4-1), orange (4-2), yellow (5-1), pale yellow (5-2), and tan (2-1). No NK-2 mRNA was detected in black neuroblasts. Medial neuroblasts closest to the ventral mid- line usually contain more NK-2 mRNA than do neuroblasts in a more lateral position in that vertical row. All posterior compartment neuroblasts express the NK-2 gene. A medial to lateral NK-2 mRNA gradient is present. FIGURE 9. Regulation of NK-2 gene expression deduced from the patterns of expression of the NK-2 gene in various mutant lines of flies (from Ref. 51). An arrowhead represents gene ac- tivation, while a terminal dar represents gene repression. (X) corresponds to an unidentified re- pressor mediated by dpp. FIGURE 10. Side view of an embryo showing the ventral-dorsal pattern of the anlagen indi- cated and the hierarchically organized regulation of NK-2 gene expression.5! An arrowhead cor- responds to gene activation, while a terminal dar represents repression. dpp indirectly mediates repression of the NK-2 gene in dorsal neuroectoderm, via (X), an unidentified repressor. The NK-2 gene is repressed by sim in the mesectodermal anlage and by snail in the mesodermal anlage. 236 ANNALS NEW YORK ACADEMY OF SCIENCES ance of six horizontal stripes which, from ventral to dorsal, comprise the me- sodermal, mesectodermal, ventral neuroectodermal, dorsal neuroectodermal, dorsoepidermal, and amnioserosa anlagen, as shown in Ficure 10. The ventral border of the horizontal stripe of nuclei that synthesize NK-2 mRNA is created by repression of the NK-2 gene by snail initially and then by sim, while the dorsal border of the stripe is created by a different, uniden- tified species of repressor mediated by dpp.5! Thus, the ventral and dorsal borders of the NK-2-positive stripe of nuclei are created independently by different species of repressors. The width of the NK-2-positive stripe of nuclei and the position of the stripe on the ventral-dorsal axis of the embryo are not fixed, but can be shifted by the combined effects of proteins that induce and repress the NK-2 gene. Rao, Vaessin, Jan, and Jan®? have shown previously that the position of the neur", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-x3sv-umtb~kzde", "00000000-0000-0000-223F-942840D7321B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Unpublished draft of introduction to the Nobel speech", "101584910X137", null, "1968", "5 December 1968", "In this draft, Nirenberg emphasizes the rapidity of change in genetic research and is gracious for the contributions of others.  He predicts a new area of research in genetics called molecular evolution, in which effects of synthesized genes on the economy of the cell will be explored.  Nirenberg claims we are at a point of a new biological evolution--a time in which the brain will eventually establish direct communication with the gene.", "Speeches, Drafts (documents)", "Nobel Prize,Genes", "Public Reactions to the Genetic Code, 1961-1968", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Belt l-    Immediate draft Truly remarkable progress has been made in the field of molecular genetics during the last ten or 15 years.  The field should continue to advance rapidly during the foreseeable future. Alternative paragraph: The field of molecular genetics is advancing at an incredibly rapid rate and it seems probable that the field will continue to advance perhaps even more rapidly during the foreseeable future.  The new knowledge has had relatively little effect upon man thus far. The field of molecular genetics has advanced with remarkable rapidity and should continue to do so during the foreseeable future.  The immediate effect of the new knowledge is to open up new areas for exploration so I think it probable that the field will continue to advance quite rapidly in the foreseeable future. During the 1930s physicists realized that the release of nuclear energy was theoretically certain, but they doubted whether it would be of practical interest.  Because of this uncertainty, physicists did little to inform society of what might come. When the power became reality, society was unprepared intellectually and institutionally to deal with it.  The scientist therefore must 12/5/68 The field of molecular genetics has advanced with remarkable rapidity, due to the efforts of investigators in virtually every field of science.  One may speculate about probable future developments and about how the new knowledge will affect the eternal questions of who we are and where we are going.  The phenomenon of transformation has been studied in considerable detail and it is clear that cells can be transformed genetically by DNA prepared from natural sources.  The new information can be inherited by the descendants of the recipient cells.  We know that the machinery of the cell will accept and follow instructions that are written in the appropriate molecular language.  The language is now deciphered, so in theory, at least, one can compose new genetic messages of known information content.  Simple genetic messages can be synthesized chemically, largely due to the pioneering studies of Gobind Khorana and his colleagues. It seems probable that mechanisms of storing and recalling genetic information evolved a billion or more years ago, perhaps during the transition from a cellular to the cellular form of life.  Later, as single cells evolved into more complex multicellular organisms, the nervous system evolved.  The genes obviously contain the information that ultimately gives rise to the brain.  At this time a new biological cycle is evolving, for it should be possible within the near future to synthesize genetic messages and then to use them to program cells.  We know that the gene ultimately continues the information that is responsible for the formation of brain.  It seems likely that man eventually will be able to instruct his own cells, and ultimately, influence his own biological evolution.  One can predict that a new area of research will emerge during the next 25 years, that of molecular evolution, in which the effects of synthetic genes upon the economy of the cell will be explored in a systematic fashion. It seems likely that men eventually will be able to instruct his own cells, and ultimately, influence his own biological evolution. We are at this point in time the final stages of a new biological cycle is evolving, a cycle in which the brain, which ultimately is derived from the gene, will, within a relatively short time, establish direct communication with the gene.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mvfk.kbka.wpr2", "00000000-0000-0000-A5FB-7D10ED33937C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Unpublished introduction to comparison between genetic and neural codes", "101584910X138", null, "1967", "10 October 1967", "In this transcribed audio recording from one of Nirenberg's requests to his lab, he discusses the evolution of genetic and neural codes.  He suggests that while the genetic codes could have arisen only once or could have been selected from a number of precursors, the neural codes almost certainly were selected from a large population of precursors.  This is significant because if the genetic code arose de novo it might influence the course of its subsequent evolution.", "Speeches, Drafts (documents)", "Genetic Code", "Transition to Neurobiology, 1965-1969", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Comparison Genetic & Neural Codes 10/10/67 Belt #1 Evolution The genetic code probably evolved in conjunction with the transition from a cellular to cellular kind of organization; that is, during the evolution of single cells. The code probably became fixed at a relatively early date as suggested by Hinegarden, et al., because soon after much information had been acquired, further modification of the code probably was restricted to those which would not prevent the information which had been acquired, from being expressed.  The earliest fossil bacteria, estimated 3,000 x 10^6 years old; however, the fossil record first becomes abundant approximately 600 x 10^6 years ago.  Virtually all of the invertebrate phyla and the first vertebrates had evolved 500 x l0^6 years ago. The genetic code evolved as the original cells evolved.  Fossil bacteria, one to three billion years of age, have been reported; however, the fossil record first becomes abundant about 600 million years ago.  Neurons must have originated as multicellular forms appeared and became more highly differentiated. Therefore, the genetic code is older than neural codes.  Since single-cell organisms, such as bacteria, are highly sophisticated, biochemically, it seems probable that the early neural mechanisms employed extremely sophisticated enzymatic mechanisms.  The mechanisms, almost surely, were based on mechanisms that are operative in single cell organisms or simple multicellular organisms; however, problems involving selective expression of genetic information and basic mechanisms required for differentiation were available.  Basic mechanisms involving cell-cell contact undoubtedly had evolved.  The cells probably were mobile.  Hormones probably had evolved in simultaneous attempts to integrate the activities of multicellular organisms.  Probably directional cell migration and the formation of highly specific cell interactions were possible. One, possibly major, difference between the evolution of the genetic code and the evolution of neural codes should be mentioned though.  One must distinguish between the origin and the evolution of each kind of code.  If the genetic code arose only once then the nature of the de novo code may greatly restrict the course of its subsequent evolution.  Alternatively, the genetic code may have been selected from a large population of precursor codes. Therefore, the genetic code may have evolved from only one precursor rather than from the population of precursors, and so the influence of possible non-random origin may still be apparent.  In contrast, neural codes almost certainly were selected from a large population of precursors.  Basic biochemical mechanisms, upon which the neural codes were based, almost surely evolved at a very early stage by a process of selection, that is, from a statistical event -- not from a single event. Biochemistry in general and certainly the biochemistry of the genetic language demonstrates that molecular complexity is achieved by combining relatively few kinds of molecules in different sequences.  The rather obvious examples are nucleic acids and proteins.  Polysaccharides may also be cited.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2rut.xwbk-wzdk", "00000000-0000-0000-F15D-CB79F993296E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Unpublished conclusion intended for Nobel speech", "101584910X139", null, "1969", "1 March 1969", "In this draft version of the conclusion to his Nobel speech, Marshall Nirenberg explores the relationship between genetic and neurological subjects that foreshadow his professional transition in the coming years.  He suggests the genetic code probably evolved earlier than neural codes and was frozen at a relatively early date because \"soon after a sufficient amount of information had been selected and stored in nucleic acids, alternatives of the code probably were limited to those that would not radically alter the retrieval of information that had been requested.\"", "Speeches, Drafts (documents)", "Nobel Prize,Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "3/1/69 One must distinguish between the origin and the evolution of the genetic code.  If the code arose as the result of an extremely rare combination of events, then the nature of the original code might have influenced the evolution of the code.  Alternatively, the genetic code may have been selected from a large population of precursor codes. The genetic code may have originated in conjunction with the first primitive cells, perhaps 1-3 x 10^9 years ago.  As discussed previously, the code probably was frozen at a relatively early date, because soon after a sufficient amount of information had been selected and stored in nucleic acids, alterations of the code probably were limited to those that would not radically alter the retrieval of information that had been required. Neurons may have originated as single cells evolved into more complex multicellular forms of life.  Therefore, the genetic code probably evolved earlier than neural codes, perhaps when cellular chemistry was quite primitive.  Since the most primitive single-cell organisms that we are aware of are highly advanced biochemically, neural mechanisms almost surely were based upon sophisticated enzyme mechanisms.  Also, the neural coding mechanism of today undoubtedly were selected from a large population of precursor codes.  It is likely that the molecular logic of the neural codes became fixed relatively early in evolution, however, additional mechanisms may have been acquired during the course of evolution. We are now witnessing the evolution of a new biological cycle.  It is clear that living organisms process information systematically in at least two major channels; intra- and intercellular, corresponding to the genetic and neuron-hormone mechanisms, respectively. Speculations One must distinguish between the origin and the evolution of the code.  If the code originated due to a single extremely rare event, then the original nature of the code may have restricted the course of its subsequent evolution.  Alternatively, if many kinds of precursor codes originated, the genetic code may have been selected from a population of precursor codes.  In either case, the code probably originated as the first primitive cells evolved, perhaps l-3 x 10^9 years ago.  As discussed previously the code probably became fixed at a relatively early date, because after some information had been acquired, further evolution of the code probably was restricted to changes that would not prevent the information that had been acquired from being expressed. Living organisms process", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8y75.87qt.hwi5", "00000000-0000-0000-C799-16F68306E4DB", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Unpublished transcript of the Columbia University Symposium on the Relationship between Biological and Physical Sciences", "101584910X140", "101584910X141", "1966", "[ca. 1966]", "Professors Devons and Rabi provide introductions to Nirenberg's speech.  Rabi jokes that Nirenberg's \"translation\" efforts sound more like physics or linguistics than biology, and on that note Nirenberg emphasizes the collaborative nature of the project.  The determination of the sequence of triplets allowed Nirenberg and others to synthesize 63 of the 64 possible triplets by the time of the symposium.  According to Nirenberg, remaining problems included an explanation for why the code looks like it does, an answer to the question of meaning within the code, and a precise explanation for the relationship between differences in codons and biological roles.", "Speeches, Transcripts", "Genetic Code,DNA,RNA,Codon", "Translating the Code of Life and the Nobel Prize, 1962-1968", "20", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "nppannciereete NS serEt CHE <A MTA SS ; i or & hg oa ? Good evening, ladies and gentlemen and welcome once again for Prof.Devons: rs seupaninshtats vineaee a brief interval to this meeting of the Symposium on the Relationship of Biological and Physical Sciences. This Symposium is, as you know, supported by the NYState Founda- tion for Science & Technology. This meeting we have this evening is a delayed pleasure - it's been our ninth meeting Unfortunately Dr. Nirenberg was ill on that occasion and he had to disappoint to you. I am glad to say, as you can see for yourself , he is here and well this evening and will talk to us tonight, and I am going to ask Professor I. I. Rabi , University Professor of Columbia University to introduce our speaker this evening. Prof Rabi: Ladies and Genglemen , Dr. Nirenberg deserves a very much better introducer than he is getting.tonight. Because I am no kind of biologist - I call.myself a physicist - my only connection with biology ina practical way was to plant a few trees some 50 years ago , in Brooklyn of course, (laughter) but I am very glad to introduce Dr. Nirenberg . In the first place his subject doesn't sound very biological to me on the translation of the genetic code - either physics or linguistics ~ now Dr. N. was born in New York and when he was a helpless little boy was taken down to Florida, where he grew up and proved that we get things from Florida, good things from, Florida apart from grapefruit and suntans. He was graduated from the University of Florida in 1948 and then got an M.A. there and finally worked his way north to get a doctor's degree from the University of Michigan. I will not recount muhh further in his career or the various things which he did because £xex actually I couldn't put the proper feeling -2- into the topics for which he became justly famous. He's a young man, by my standards, and the year 1965 was an extra- ordinary year for him - probably the opening guns for a still more extraordinary career following - but I note in this biography given me that he became an Honorary Doctor of Science in the University of Michigan , Yale University, and the University of Chicago - all in one year and received a National Medal of Science from the President of the United States in that very same year, in addition to becoming a fellow of the American Academy of Arts & Sciences. What 1966 will bring for him I don't know - it has probably brought a great deal already but it's not on the record which I have. I happened to be present when the discussions were taking place for the nomination for this distinguished National Medal of Science and all agreed that man had wery great talent indeed and I join with you in looking forward to his talk which would be as I said on the Translation of the Genetic Code. (Applause Dr. Nirenberg: Thank you Dr. Rabi. Having been born in Brooklyn I also want to-thank--you-for. having. planted some trees in Brooklyn . I am~gilad-really to have this opportunity to tell you about the genetic..lang ; ot perhaps 2 to @ billion years continuous dialogue between cells and their descendants has taken place. /sach generation passes to the next a library of information which specifies in detail the way to make many kinds of   » < Sat s _ - A - . Y ‘ ye 3 ( ee ELLE , ee of/life on this .planectasenaegiict use viresalty, ‘the same planguage pe he eo . THe, atthough recent ty severat = a humber of alayects have 3 bean! found-thet-#1 41 desertbe-tater.— May we have the first slide please - This - Very-good - The last slide was a good ihtroduction anyway to introduce some of the collaboratores who have worked on this problem in the last - oh 3 years I guess - 4 years This in a very real sense has been a collaborative project and I would like to call attention to these - to the collab- orators. However, this is the last slide and I think if we could have the first slide (laughter ) we will show it again xakex at the end. Good - this is an illustration from Watson's recent very fine book which diagramatically illus- trates a double strand of DNA and an enzyme RNA preliminaries (?) which cahalyzes the synthesis of message RNA as shown here. The point that I wish to make here is that only one strand of the DNA is copied by RNA prelimnates - is copies with a given polarity not known really what signals or what words specify the beginning and the end of the message RNA synthesis But there must be a code word of some sort which says Start and another one that says Stop. These remain to be defined. Next slide please This shows also diagramattcally a protein synthesis DNA shown here and the different cross hatches represent various segments of DNA corresponding to - each corresponding to a prote&n possibly or several proteins - messenger RNA ribozomes dia- grammatically attached to the messenger RNA and reading begins and as soon as one moves down another one attaches until the message is virtually covered with ribozomes. -4- The reading takes place by means of SRNA, transfer RNA which carries specific amino acids and recognize particular code words on the ribozomes. Thus, the code word is recog- nized not by the amino acid per se, but by an adaptor mole- cule , adapter SRNA. Now, the rationale coeeeky for our previous work on the code was to use synthetic messages - compared with polynucleic type sporelegs ? they were randomly ordered sequences messages, and in this way characteristics of the code could be determined. And also base compositions of code words. But not base sequences. The situation up to one - well, two - years ago was very much like that of an anagram. We knew the letters of the code , but not the order, of the letters within each word. Now, an approach towards determining the sequences of the code words, are shown on the next slide. This illustrates diagrammatically again the code on recog- nition process. This is a ribozome of E coli small sub-unit and large sub-unit . There are two binding sites for SRNA at least per ribozime, shown diagrammatically here, and this shows an amino SRNA presumably three bases in the trans- fer RNA molecule recognized and three bases in the message RNA which binds to the small sub-unit and amino acid as shown here. Now one of the binding sites presumably is for the peptftal SRNA which is shown here. The other for the incoming amino S-less RNA. Three enzymes plus GTP are required for the transfer, then, of the growing peptide chain to the next amino acid. A shift in some way occurs so that the next three bases would be read. Thus protein synthesis starts at a given place and proceeds, reads three bases sequentially and reads -5- with a given polarity. Next slide please .. Well, actually in several laboratories, Schweets labo- ratory, Lippman's laboratory, Rockefeller, KG's , it is shown that if one added a synthetic message RNA, such as polyuradylic acid , to ribozomes, that phenalalamino SRNA bound to the ribozomes prior to peptide bonds synthesis, my colleague Dr. Philip Lieder, and I wondered how small a message would direct the binding of the SRNA to the ribozome and it turned out that only three bases were needed. That is, the triplet itself would direct the binding of the appropriate aminoSRNA to ribozomes . This provided a rather simple route towards determing the sequence of RNA code words. Now our major problem was to devise some techniques for synthesising triplets. At the time we started the work with the triplets a - oh perhaps 20 to 25 of the 64 possible permutations of bases were reported, had been either prepared by enzymatic digestion of RNA or by chemical synthesis, by some of the very elegant techniques devised by Karana and his associates . Two techniques were developed in our laboratory the first by Philip Lieder, Maxine Singer and Richard Rimakome employed polynucleic type phosphoralays which requires a doublet primer illustrated here, and adds nucleic psydiphosphates to this to make triplets,tetramers, pentamers and soforth. A second methdd developed by Merton Bernfield in our laboratory which employed an observation that had been made by Heppel some years before. The pancreatic RNA-ase which is normally thought to be degradative enzyme will catalyze a transister - ification from a two-prime, three-prime cyclic nuclear dite -6- to a five-prime hydroxol to form triplets and higher homolons. Thus, this technique added units to the left end of an accepter and the polynucleic type phosphoral aids added units to the right end of an accepter. With these two techniques it became rather simple to compare various triplets needed. The next slide please .. Now this just shows diagrammatically an oligonucleitide and I wish to illustrate only that ... a given segence , say, triade, may exist in three chemically distinct forms, depend- ing on its geography. in a molecule, that is, a five-prime terminal code-on, internal code-on, and“three-pr ime terminal code-on. All of our evidence todate indicates then that the biologic characteristics of the code-on recognition may in some cases .. many cases .. be influenced by the particular position of the code-on. Thus, each of the 64 possible triplets may exist in three different structural forms. Next slide please .... This shows the influence of various substitutions on the hydroxyl groups - terminal hydroxyls of the sugar. This is a concentration of oligonucleitide plotted against the binding aphenymekaikxxe aphenylalaminecern into ribosols. A simple triplet of tri-U has an activity such as this - if one adds a phosphate to the a eree hydroxylake the sugar the template activity is greatly enhanced, Shown here. A phosphate attached Hmex# to the three-prime terminus lowers Rotman template activity., Recently Fritz Roten in our laboratory prepared some attalogs of tri-U also mmkaxz with a metal group attached to the five-prime phosphate , shown here, and -7- this is, although this should be CH, it's a metal group also 3 attached to the three-prime phosphate. Three-prime terminal phosphate. And this greatly reduces template activity. A triplet with a two-prime three-prime cyclic phosphate has very little template activity. Next slide please We think it possible that terminal hydroxyls of sugars may be modified and that in some cases, possibly these may regulate the template activity of the code-on. The... certainly a substitution at the five-prime terminus may be important because this may serve as a signal which specifies the attachment and/or detachment of the ribozome to the message. Recently, Matra and Herwits , also Stent, have shown in vitro, at least, that messenger RNA contains a triphosphate attached to the five-prime terminal hydroxl. And, although it's not clear what the £Hnsakxanxmk physio- logical funcation of the triphosphate is, it's highly possible that this may in some way specify the phasing, the initiation of reading of the message . It would also select the first word to be read, phase the reading and perhaps affect the susceptibility to m=xnHgeksagenks exonucleasis. Internal code- ons may be also important , may be modified in various ways two-prime hydroxyl, or the base could be modified ... I think that code-on neighbors may in some cases play a role also in ... may affect the code-on recognition process. I would also like to call attention to the possible difference hetween internal initiation and termination vs terminal initiation and termination. The mes... often messages ... messenger RNA seems to contain the infor- mation from more than one purtsing ? if one starts reading -8- at/left end forthe terminal initiation one reads in and then comes to a word that says Stop and an unknown mechansim for starting an internal position. And .. that so that internal initiation and termination may, it's not known, but it's possible that it may be different from a terminal initiation or termination.mechanism. Next slide please ... On this slide is plotted oliganucleitide concentration and binding of aminoacelesito ribozomes. I wimply want to illustrate several points that became obvious very early in thiswork. First, that synonym code-ons corresponded to various aminoaceles RNAs , for example, phenylalemine SRNA responded to both to UUU and UUC. And UUC was slightly more active than UUU. With light CSRNA responded to AAA and AAG there was a quite marked difference in template activity of each synonym. The degeneracy that was observed was a very logical degeneracy because the C=U in the third position and in this case, A=G in the third position. Next slide please... On this slide is summarized virtually all of our work to date. We have synthesized 63 of the 64 possible triplet se- quences and the sequences of RNA code-ons are shown. The underlining represents previous base composition assignemtns which were done with the protein synthesizing system and randomly ordered pinucleitides. And we've observed a very close correspondence between the earlier work with randomly orderéd polynucleitides and base sequence studies. I should point out the types of synonymi that are observed. For example, glutannic acid corresponds to code-on ... GAA and GAG. Aspartic acid .. GAU GAC Uc AG type of degeneracy. Another type of -9- degeneracy is illustrated in this column where the third posi- tion may be occupied by either UC A or G, completely variable. Another type of degeneracy is illustrated by methianate, that is a G may be present in the third position, may be recognized but not in A. I think that the logical degeneracy that has been found has its many consequences, one of the more obvious is that one may have a great deal of silent mutation, that is in one of the , one of the code words or groups of synonym code .. code-ons one may have mutation and ... which may convert any base in the third position to any other base without resulting in amino acid replacement in per ? Another obvious conclusion is that amino acids which are very Similar chemically such as aspartic acid, and glutanic acid , both dicarboxcylic acids, have chemically similar, quite related code-on. It may reflect the evolu tion of the code , it's not clear, but certainly one consequence would be that when error does occur, usually two bases are read correctly , one incorrectly. And very often a chemically- may be related amino acid xs substituted in protein when an error does occur. Thus the general picture of the code is quite a conservative one .. one that minimizes error most often. There's one underlying question though which has not yet been answered, and that is, why ... why does one see this type of code ... why shouldn't phenylalimine, for example, be corresponding to GCU and GCC instead of alimine. This question has not been answered and I think that there may ~10 well be a chemical meaning to this. It is not obvious yet Alternatively, it ... simple chance alone may acoount for this. I tend to think, however, this is ... is a personal preference --. that there will be an underlying meaning will be found. Next slide please.... Could we have that last slide ... just a second ... I wanted to point out the ... previous slide ... I wanted to point out that some code words may ... appear to serve special functions. For example, the recent work of Brenner, Garen, Zinder and others indicate that UAA and UAG may specify the end of the message, although the precise mechanism for punctu- ation is not known. UUG and CUG AUG and some cases GUG May correspond to the initiation .. may specify the start Our recent studies, and those also of CLark and Marker in England , have indicated that these code-ons in terminal positions at least, are recognized by enformulesionate and this may serve as an initiator of protein synthesis. I'll say more of special function words and .. in a few minutes. Let me have the next slide please ... This simply summarizes the various patterns of synonym code-ons that are observed. You would see in the third posi- tion , and so forth. The enformulathiamine SRNA the initiator Next slide please ... Some possible special function code-ons are listed on this slide. Sanger in England first observed the necoli that one of the two speciss, or at least a part of methianine SRNA could accept a formula moiety. -1l1- The amino group of the methianine after the methianine was linked to the SRNA could be formulated. And the work of Kapetchy and his colleagues in Watson's laboratories and others have suggested, Zinder's Lab also, have suggested that this may specify initiation . And as I indicated before, U UG AUG CUG and to some extent also GUG are recognized by the informulinine SRNA. UAA UAG may serve as a terminator, we think it likely also that AGU AGC AGA also AGG may serve as special function words. The functions , however, have not thus far been found. This is, I think, the situation now is extremely interesting because the tools are to hand to try to decipher the mechanism of special function words and also the various functions that they may play in protein synthesis. Next slide please ... Dr. Hatfield in our laboratory has recently prepared some radioactive triplets and have looked at the binding of the triplet to the ribozome. In this box is plotted the binding of the triplet to the ribo- zome; in this the binding phenylalimine SRNA to the ribo- zome. As you see in the presence of the appropriate triplet phenylalimine SRNA binds to ribozomes times plotted against binding. With the radioactive triplet Dr. Hatfield asked the question \"Is SRNA necessary for the binding of the code word to the ribozome?\" And he finds that it is . Because very little triplet binds in the absence of SRNA, in the presence the triplet binds at approximately the same rate as phenyl- alimine SRNA and approximately the same amount of triplet binds as phenylalimine SRNA. Thus, the complex may well be a one-to-one complex on the ribozome. -13- I should also point out that major lucine SRNA binds to ribozome very weakly in response to triplets, and possibly this type of recognition corresponds to only recognition of two out of the three phases. Next slide please ... This shows an experiment with some of Holly's SRNA plotting concentration of alimine SRNA against binding to ribozomes. And the dotted line represents hundred per cent binding, that is, all of the available SRNA in the reaction. This fraction of SRNA, which Holly sent to us, was estimated by their laboratory to be greater than 95% pure. And yet this SRNA fraction recognized quite well three, at least three of the alimine code-ons ... GCU GCC and GCA. It did not respond, or responded to a relatively slight extent to GCG. E-coli SRNA , unfractionated SRNA, responded quite well to GCG , as a matter of fact, this was the best code-on --. alimine code-on found as compared to the coli, rather the yeast, very poor pattern . The GCA GCU and ccc very little response to GCC ... GCC was an excellent code-on for alimine with yeast SRNA. Now since this SRNA was of high purity the results strongly suggest that one molecule of SRNA can recognize alternately at least three of the four alimine synonyms. Further, since Holly's laboratory has recently reported the sequence of alimine SRNA the data afford some insight into the mechanism of code-on recognition. Next slide please ... This is the sequence of alimine SRNA as reported by Holly's laboratory . The amino acid is linked to the terminal denizine and this is shown in only one of the possible conformations that has been suggested by Holly's laboratory. I should point -12- This provides very simple and quite sensitive technique for detecting code-on recognition by SRNA which is not isolated with amino acids. If Some special function words are not recog- nized by activating enzymes, not asolated, this would provide a relatively simple route towards detecting such recognition. Next slide please ... On this slide is summarized the work that has gone on in our laboratory with purified SRNA fractions. This work was done in collaboration with B. P. Docter at Walter Reed and also Donald Kellogg in our labora- tory. Tyrazine SRNA both peaks of VERAR PS cognize both UAC and UAU and this shows the CU pattern in the third position. A minor peak availing recognizes GUC and GUU . The major vailing peak, however, recognizes the GU A GUG and to a smaller extent GUG. All of this work, with one exception, was done with E-coli SRNA fractions. The E-coli fractions, licine 1 and 2 recognize both licine code-ons. Recently carbon, however, KaK is reported, just at the Federation meetings a few weeks ago, that in mammalian liver one species of licine SRNA pref- erentially recognizes AAG. The other preferentially recog- nizes AAA. Lucine peak 1A C recognizes CUG , another peak UUG. The small methianine peak SRNA peak recognizes AUG preferen- tially, whereas, as I said before, the major peak of methianine SRNA will accept a formile group and can recognize U UGC UG and AUG. Triptathane SRNA peak 2 recognizes UGG CGG and to a smaller aegkeeX extent AGG. Thus, one sees this pattern in the first position and’ $88h alimine SRNA recognizes GCU GCC and GCA. The pattern is repeated in the third position. Now this ex- periment was done with ... this SRNA was obtained from Dr. Robert Holly with a highly purified fractions of yeast SRNA and I'll say more about this later. -14- that there are several possible single-stranded regions of interest. This sequence is GToseudoUC has been found in virtually every SRNA that has been examined. Another inter- esting sequence is the CGGs surrounded by two dihydrauriditic acids. And the third is the IGC region which is right in the middle of a molecule. Next slide please ... The two sequences of interest are thowe shown here. The CGG between dihydroU's and the IGC . Now if phase-pairing --. if these really are the SRNA anti-code-ons,that recognize the alimine code-ons that are shown here, recognition would have to be by parallel. pairing between C and G , and the G would then have to recognize U C and A. If, however, phase pairing were Watts and Krick hydrogen-bonding anti-parallel phase pairing C would phase pair with thé G and G with the c and the inner scene in this position would base pair wxk alternately with U C or A but not G, which is the pattern which is observed. Krick has recently proposed a detailed mechanism xak which would permit hydrogen bonding between intercine and U C or A. Next slide please ... And this mechanism simply employs a \"wobble\" \"\"\" A MOVE- ment of either the SRNA or the messenger RNA on the ribozome . in the end position. And I think all of the experimental results are in accord with this type of recognition mechanism as shown here. The basis in the SRNA anti-code-on is shown in this column , the basis in the messenger code-on is shown in this column. The patterns that are found are listed here. Thus in innercine in SRNA in an end position could recognize -15- by alternate base pairing U C or A aG, an end position of SRNA could recorgnize alternately C or U and A could recognize UC G and aU could recognize?¥iternate pairing Aor G. We would also predict that a ribothymadilic acid SRNA would pair also with A or G , perhaps the interaction with A would be stronger than a urydilic acid residue , that a pseudoU in SRNA might recognize alternately A G or U and we have noticed this pattern with SRNA rather often. And possib1$}€Rat a dihydrouridylic acid would not base pair so that the interac- tion with the triplet .. messenger triplet would be/weak interaction but it's entirely possible that a U or aC in a terminal position would not greatly inhibit the interaction so this may be permissible. Also, that a metal group on a two-prime hydroxyl deribose might result in a weaker interac-— tion and by permitting greater degree of motion on the ribo- zome might permit a greater ambiguity. ... a lower specificity. Next slide please .... Now the results with the innercine strongly .. and also the other trace spaces ... rather strongly suggests that .. actually a great deal of work suggests that SRNA is modified enzymatically after it is released from DNA templates. And it seems probable .. that there ... in the cell there exists a whole spectrum of intermediates. One gets a successive modification after modification because the level of trace spaces in SRNA is quite high. Now the consequences of this are rather simple to visualize. If an A, an admine, for example in SRNA is de-aminated and converted to an innercine the A would normally recognize the uradylic acid residue in the message and the conversion would result in a recognition of -16- aUcCor A. Similarly, if a G were converted to an I when we get this type of inter-conversion , or a C were de-aminated we would get this type of conversion. The ... it!s too early really to say ... I think it likely that this type of inter- conversion occurs and that it has very possibly great biologic Meaning. Next slide please There .... really a great deal of work has been done recently which suggests that ... really a great deal of work has been done ... which shows that in many cases that it is possible to modify the specificity of code-on recogni- tion. I Think that this is biologically a very very impor- tant point and will have truly profound biologic consequences. Gorini and Gilbert and Davies at Harvard have shown that streptomycin will bind to a protein on the 30 s ribozome the small sub-unit and all the available evidence suggests that the binding of streptomycin to ribozome may in some way distort the geography of the code-on recognition site, to that a greater ambiguaty, a lower specificity may occur and this may be at least one reason - it may not be the only reason - one reason which may account for the action of streptomycin on bacteria. Simply the greater degree of error occurs in protein synthesis. There may be other mechanisms, other effects of streptomycin on the cells as well. Id like to actually list just a few ..pf the recent ... relatively recent examples of modification of specificity of code-on recognition that are being studied in various labora- tories. Streptomycin effect is certainly one, Next slide please ... -17- v Well, before I come to this slide ... I should mention also the ... some work that ... some very regent work that we've been doing in collaboration with the Siwilkets at Princeton . They observed that upon infection of bacterial cells of C-coli with a virus T2-phage that within one minute after infection a protein was synthesized by the bacteria which modified kam a pre-existing lucine SRNA component . This modification resulted in a shifted movement of the SRNA on mack columns so it could be purified. We have tested the code=on recognition of this modified ... this SRNA modifica- tion which is due to viral infection. And find that it recog- nizes only poly UG but it doesn't recognize any triplet. And we have tested all of the UG triplets . Now together with the modification of the SRNA one finds the cessation of post-protein synthesis and we don't understand the mechanism of this ... mechanism of turning off the host protein synthesis but we think it likely that .. that the enzyme modifies the lucine SRNA component and this lucine SRNA may in some way interfere with the initiation of host protein synthesis, but not a phage synthesis. There's a very subtle way of subverting the metabolism of the cell so that viral proteins may be synthesize@ in large amount. And currently this problem is being worked on . And here also is some recent study that has been done in our laboratory by Richard Marshall and Tom Kaske which we have compared the specificity of code-on recognition in am- phibian .. with amphibian SRNA .. zenith eslavos , liver and guinea pig liver SRNA with that of E-coli SRNA . E-coli -18- SRNA does not recognize AGG and recognizes CGG with biargine SRNA only to a very slight extent. Whereas xn both} Smphibian and mammalian liver we see a very strong response of arginine SRNA to AGG also to CGG. The alimine SRNA I mentioned previously with the yeast .. contrast /Pfle yeast and coli patterns shown here. In both amphibian liver and guinea pig liver GCC is a very active code-on whereas in the amphibian liver GCG is a very ... has no activity for alimine SRNA as con- trasted to coli-SRNA. auayEecognized by isolucine SRNA in higher forms although we have not detected it in coli. In all species tested AAA is recognized, whereas AAG has only slight activity in coli but is a very active code-on in higher forms. Seriun SRNA UCG xx recognition is variable, AGU and AGC recognition is variable also as shown here. 3NE ACG is a var iabley$soghati Phere. Now we have found no differences in the code-on recognition of SRNAs corresponding to aspartic acid, cystine, glutannic acid, histamine, phenyl- alimine, proline, tyrazine and also valine which should be on this slide. Thus it seems clear that the same sequences correspond to quibalominic acids throughbut .. from bacteria to mammalian liver. However, very subtle differences in relative responses of synonym code-ons are observed. I think that such differences may, in some cases, play biologic roles. We wonder whether such differences may be involved in some types of differentia- tion ... may affect the rate selectively of protein synthesis in some cases. These are problems I think for the future that will be worked on within the next year or so and .. we are very interested in determining the outcome of questions such -19- as these. Thank you very much. Applause Prof.Devons: Well as Dr. Rabi said \"it's not befitting for a physicist £o make comments of appreciation of these things.\" All I can say is that Dr. Nirenberg has indicated some of the rather breath- taking speed with which this subject is developing. I am going to ask Professor Zubay , professor zoology here to say a few more professional words of appreciation than I can offer. Prof. Zubay: Prof.Zubay - I was hoping Dr. Nirenberg would talk for another 5 minutes because I was working on some concluding remarks - However, let me say that I found that this lecture was somewhat more technical perhaps than most of us have been hearing on these Monday nights . I am sure that many of you were snowed by it - as a molecular biologist I assure you everything he said was correct (laughter) or at least 90% correct. I found it very exciting and I am sure you noticed how totally immersed and how exciting he finds his own work. This great crusade of his began in .. around 1961 when he made the very exciting announcement in Mostow - I was t", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-d2vf_yuu8_9czw", "00000000-0000-0000-B552-1C2BC9A017C4", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "On the Translation of the Genetic Code", "101584910X141", "101584910X140", "1968", "January 1968", "Draft of speech given at Columbia University for the \"Symposium on the Relationship between Biological and Physical Sciences.\"  This draft contains many of Dr. Nirenberg's handwritten notes.  Emphasizing the remarkable similarity in codewords used by bacteria, amphibian, and mammalian processes and the fact that most, if not all, forms of life on the planet use almost the same genetic language, Nirenberg suggests that developments in genetic research also point to a physio-chemical or molecular basis for processes \"governing such fundamentally biological phenomena as cell metabolism and replication.\"  Nirenberg discusses the development of synthetic triplets, problems associated with degeneracy, base pairing explanations, and the possible role of protein synthesis in cell differentiation.", "Speeches, Drafts (documents)", "Genetic Code,DNA,RNA,Codon", "Translating the Code of Life and the Nobel Prize, 1962-1968", "24", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "On the Translation of the Genetic Code F 2 MARSHALL I NIRENBERG National Heart Institute - National InstituteS of Health /+ ae poe vod dige to take tare oppurtun hy vo. relate some thig. Cale - 2? No wed Or Know Lease of the genetic language. | For some two to four’ v a billion years some such Language has probabd.y provided the ba sis for a continuous dialogue between ceils and their descend- , cv naamscogern UE at fr fa ants. Fossil records aff bacteria. about 3 billion years etd~- y ‘ave been reported (ey Baghoorn’Schopé) the first vertebrate appeared approximately 500 million years ago; ana amphibians and mammals about 350 ana 180 million years ago, respectively. seat The presence of bacteria 3 billion years ago nay-indicate the presence of an operational code at that tinep Almost surely “ne code nas Zunctioned for more than 500 million years. The oy remarkable similarity in codetwords used in bacterial, amphi- bian and mammalian replicative processes suggests that most, if not all, forms of life on this planet use almost the same genetic language, and that this language has been used, pos- sibly wich few major changes, for at least 500 miliion years. 7 a s it is by virtue of this language that pach generation is e to pass to the next generation a library of information t- ab which specifies in detail how to make the many kinds of protein catalyst) that the cells will need for theizx development. And on Le . ai. thous t now seems clear that all, or aimost all.forms of 4 r-' ife on this planet use virtualiy the same language, ecently a number of dialects\" have been found. I shall describe this Geer. Ipea 1 {tae : 4 + *4 = The elucidation of the genetic code has been “he subject / 7 ins of much intensive work, particularly in the past four or five years, and I wouid sixe to stress -atuthe-oursety, that this work, ane-particularly-the work with which I have been associ- o . tet . a e ated ‘nas been, in a very real sense a collaborative project. ? . Zé : 2 <enini—this will—become--evident as I proceed. Se ee re wat  cpaprnmeancts TTT pasa wnceinmrerne = BY F< recald briefly - as must have been dgne fre-. nf AAn Av     Wasson coneye ‘of protein: Ae & 1)> ‘Here ig. show de wy lg Yee nM of - pfoby mA beep ves nied Pol” an a ail a, f Na ef TE ; ‘schematically the aobte- -soranded Sir vist -egastes wit be i “* oe foot v     enzyme,- RNA~polymerase ,+ cataiyses the synthesis of messen er yn A. . ; ae 4 ; Bf RNa, osing the DNA as a template. Only one strand of twe DNA wo is copiea by Sse RNA-polymerase; amd the copying process is se- quential) and erere-are signals, whose exact nature is unkown, ok amb Witeeh specify she begir naing and the end of ‘the messeag#Z-RN ae e eRe i ‘ gytithesic. | She next diagram (Fig. 2), shows. schematicallyy the process of protein synthesis. In the DNA shown here, the dir- ferent cross-hatchings represent various segments of DNA, each “eg corresponding tc a specific protein,’ or group of eroteins. Ri- = bosomes are shown,schematicaliy, attached to the messenger=ANA —   1 o bist of collaborators at Bethesda. ee rs Fae basic features of the JOwick- Po. Sem ie ett we TL wacle reading, or translation, begins} amr as soon as one ribo- some moves down the MeSGeRSOLARNA, another becomes attached un-       cil the messes alae i is EN covered with [a gomes Bren, ee n LF MND in pgly rater Ce | Tae aettar rea ding Yaleowplice” @ ean e% RN . (soluble-- L. maA}} which ee speciiic amino¥acids and recognizeg parti- t cad BA Dy cular m¢RNA code words\\\\en_the-vibesemes. Thus the codenwere, ¢ é “OF codo..f is recognized not by the amino- acidg, per-se, but by   Aire ete Am a ME OS IN I coer coleaiese-the $HRNAD-    _y+-—--Fig..- illustrates, again diagramatically but in more cetai 4 ae ty the codon recognition process as exemplified by that most inten-     & Ce be . sively sfudied organism, oti... fi Tt f f. : th oe pe, AR “gt, Cron i Pee MA tg al. 4 Tae _gibosome’ of Beeehi cbiptisexyewo su sublunits: the larger. 305 aad tae smailex 30$- The messempewRNA iies on the smaller the Cart & {tens ‘perc of the ribosome, @ and presumably [three bases)in 1 the ACh ot ot ek ARE te ge MomeerpeteeRNA molecule te—-eeden!) are > recognized by{thred, Basel 2   can then bind . ahecrae Pomel | fas) : fat one of two possible binding sites on the Larger ribosome’ sub- unit, a@-particular amino-acid, (aa). One of these binding sites . ae 70s . is for the peptidai S+RNA, so the STRNA which is at tached to the growing (protein) Pee reneae. molecule; and the other) for the incoming amino-acid s}RNA,. hus the ree _ enzymes (Sae—two-s-RNA ls Ghost a, i ~ ore Bae oh plustict Ph which supplies tne acti- 2 vacion energy, are cequired for the transfer of the growing poiy- e. pepcide chain to the next (incoming) amino~a eid S+RNA complex, Wnen this is accomplished the SrRNA required for the previous &; YALE howe’ amino“acid is discarded, and a shift in-seme-way occurs so that the next codon (triplet of bases) on the m#RNA can be recognized 7 fT by 4 new stRNA. In this way the protein synthesis starts at a tebe are ran Py we . ae a RE cmt Ph ARS ye ee 4 aoc wore - . given place (on the m+RNA) , reads groupings of three bases se~ quentiaity and with a given polarity. Fo = f “ : \\\\ oe\" ols Co i ‘ “ in an accual living cell, even “thre~smettest bacterial’ ‘cell, trey, me Cty ae copumowetsoe~byochemical ai processes axe simultaneous ly, in-process. all part of the cell metabolism. The Synthesis of even a single Bes a € protein is quice an elaborate process involving, Ninter-alia, the cru.csfer of a long DNA message to an m+RNA molecule which Yas cypneaily suffic tent nucleotides (about 1,500) to code seme ': he cee Pe fe Pope ea aminc~acids for the-protein. polypeptide chains, Moreover, in an actual celi these 1,500 nucleotides will not be arranged at \" a zn any cae sequence, refie cing ene ta ‘that there is a hog ean oad tact, jaan on eS qe ut “ ’ aeons Ly, Cog pit Et So away great number o£ different sequences o£;amino-acids ‘(of -whieh-20. i, hes va tovfvaclid, oo . . different—varieties.-are ) whith constitute different proteins.. Nonetheless,oy a gveat' variety of biochemical and genetic LK investigations, especia ity with bacteria and viruses, a=g@eeat N “many features of She protein synthesis, f includi ing { an particular|y el i ry sea@n. information sbout the code, nas been obtainedY ine work f snall Me cescribag is, however, characterized by the use of much Simpier, in vitro systems, where the essentially chemical feacures QD of some of the basic stens in the whole process are studied. Th , the success of these methods, « and the concurrenc. of, vesults £23 fiem-with those from in vivo experiments, where—beth-are- avai. 5 SRR cemenmeemicinetiine ape wiil I hope demonstrate how a physio-chemical or mole- cular basis can be found for the base processes governing such fundamentally biological phenomena as cell metabolism and repli- cation. The basis for our earlier work on the DNA-RNA code was the use of synthetic messages,’ (in place, thec is, of actual m+RNA) which were randoml % oriented sequences of the four code letters, UJ CA Lae . aga cdl } ; cyfoes e), _ACengne) Gtpanine) , the four bases AGS Z ow of m#RNA. In this racteristic of the code could be     “ha   - ee t ~ Cerexminedey inp router. the. base compositions Of dae code- *. to.y 1 Pk x. * Le fit 4 Tek i fw. a words, Dut not t Sequente-pi-the-bases ia-the. in the 4 words. Thus the orobien/ up to two or three years ago was like Fi that of an anagram: we Knew the letters comprising the codewords but ndé¥ the order of the Peteers within each word. hs i Et~hes beennweke established’ i in several laboratories that tole ct-one~edded’ 4 syntheti c messenrger=RNA, in particular polyuri- Ce - Af dah dylic acid™ @ sy nthetic RNA with entirely U bases) ¥’to a suit-~ adie mixture of ribosomes, StRNA's >» enzymes, ATH, G GTP and amino- a acids Ses the poly-~U vweebd selectively bind® phenylalanine s+RNAY/ (l.e., the particular st RNA associated with the incorporation of + amino-acid ohenylalanine in protein),. to sae ribosomes. My coi- league Philip weaer and f then speculated sow small a message (c~ che RNA type) would direct the binding of s+RNA to the ribo- Some, Experiment showed thac only three bases were needed, chat is, very small molecules comprising only the triplet itsel¢ would direct the binding of of the appropriate amino-acid StRNA to tne ribosomes. This provided a,rather Simple route towards the determination of the sequence of letters in the RNA code-° words. Our main problem was to devise Suitable techniques for Syn- thesizing triplets. At the time we started our work with such triplets, methods had been reported for making some 20 or 25 of the 64 (=4) triplets which can be constructed from the four d nucleotides U, Cc, Ay G. These had been prepared by enzymatic breakdown of RNA, ox by chemical Synthesis, in the latter case using some of the very elegant techniques devised by Khorana and his associates. Iwo general techniques were developed in our laboratory, the first by Leder, Singer and Brimacombe, and the second by “erton Bernfieid. The first employed polynucleotide phosphory- L s e ey _ OA Tee ca = A \" 4 \\\\ te A get Pa ve . 1ase £522 enzyme Ach Ahr jul prcetailiis, tt padi, >, ? of . / ° | a a ead coe ; _s en de 4 : [ry Log. un pomGewl3- Single nucleotides to ditnucleotides to make Fig. ? 7 trimers , tetramers, pentamers, ete. The second method em- t eh, a] ployed the enzyme pancreatic RNA-ase, w nough normally pa tt ry pale a breakdown or degradative enzyme, will also catalyze an ex- change reaction between polynucleotides and car be used to make . . “ . ee Ls . tripiezs with well-defined sequences. Using the methods of ‘ ee Khorana and these two enzymatic tecaniques, it was possible to Synthnesize amost all of the 64 reap tees In connection with the use of thie small polynucicocice or oligonucleotide\" molecules such as the trinucleotides, it is important to. point out BReme that any given sequence of nucleo- tides can exist, when incorporated in actual m¢RNA in three chemicaiiy distinct forms, depending on the location of the se- quence in the wacle messenger molecule. The chemical forms re- sate to the three positions (a) as an internal codon (trinucieo- tide) ox as one of the other of the terminal groups - so called t w 3'-terminal codon and 5'-terminal codon. This is illustrated hy n Fig. . e Fig. fie ft. - All of the evidence te-date suggests that the biological char- a&cceriscics oF codon recognition may in some, perhaps in many, a wh AE cases be influenced by the particular position of the codon in che m#RNA (or equivalently in the DNA). Thus each of the 64 triplets referred to above thay exist in three effect ively dif- ferent structural forms. tae significance of these ‘'secondary\" chemical features is of en) indicated by exzeriments / in vitro, witn the o.igonucleotides,   The heiical RXA (or DNA) has a definite sense or direction - with a cefinite \"beginning\" and a definice \"snaing', 3! and 5! refer to features of the chemical structure at these espective cexyminalis. = eS Ma ay _ t a ? * and specifically by studying the influence of various (phos- phoYiacing) Suosctitutions on either the 3' or 5' terminal hy- droxyi groups of the sugar in the trinucleotides. Thus Fig. Fig. Shows the binding of phenylalanine s+RNA to ribosomes as a function of the concentration of the trinucleotide. A simple ¥ we triplet, UUU, has an activity shown by (a). I one adds a ob. pnosphate to the 5! hydroxyl group te the Sugar the activity is greatly inex “eased, i. e., the binding or template effectiveness r of the trinucleotide is greatly enhanced;”\"(b) . A phosphate at- tached to the 3' terminal lowers the tempiate effectiveness, (c}). Recently, Fritz Rotman prepared some analogues of UUU Pettey ClpELG. hase with a methyl group attached to the 5! phosphate, and also, wich & methyl group attached at both terminals, i.e. both 5' and 3! phosphate. The methyl group at the 3' phosphate terminal great- iy reduced the template effectiveness. A triplet with 2'; 3! cyclic phosphate shows very little template activity. it seems possible that'significant terminal variations of this sore may occur in different biological circumstances, and that ota? rane Poa chesefmay possibiy regulate the template activicy of the codons. For example, the terminal hydroxyls of the sugars (ribose) may   The bincing of che StRNA to the ribosome is determined by tech- L i Gioactive tracer is incorsorated in the SPRNA, ivity associated finaily with the ribosome of this binding. It is in thet the term |. enoces the effectiveness o2 che binding. pbk oo 5 fe he be modixied in such a manner. Certainly a substitution at the 5'-terminus may be important because this could furnish a Ssig- nal which specifies the attachment and/or the detachment of the ribosome from the message, (m+RNA or substitute}. Recently Mitra and Hurwitz, and also Stent, have shown that, in vitro at least, méessenges-RNA contains a triphosphate attached to the terminal nycroxyi; and aithough it is not clear what physiological func- tion this triphosphate serves, it is highly plausible that it may in some way specify the initiation of reading the message. It couid aiso determine the first (three letter) word to be % reac, phase the reading, and, perhaps affect the susceptibility “GO enzymes thac could attack the termini of the messengex-RNA. ancernal codons may also be modified by these secondary chemical changes; the 2' hydroxyl or the base could be modi- fied and such cnanges may be relevant to the punctuation of the faa he bt mie, £ a message. it-a@iso- cannot be excluded that the codon recognition process is in some instances affected by the particular neigh- bors of that codon on the message. Cus. at Lt-should also. be pointed.out that there could ‘poseibty be a difference between internal initiation and termination (i.e. initiation or csermination of polypeptide sequence (5 rotein) by & codon interne..y locazec in the message) and serminc1 initia- ction and termination (the same Process effected by terminal co- Gons). Consicer the situatio where. ‘the meseencves<\"0/. appears Tes) \\\\ LO to contain the information for the assembly of more than one protein, (or more than one polypeptide chain of « protein). If one starts to read (from the left in Fig. ) the codon for Fig. the terminal initiation, one then reads in the message unti.z one veaches the word that says stop\") and chen trere-wiii-be an unknown mecaanism See starting the second message at an in- 2 terval position. It seems quite piausible, although not known, that chese terminal and internal initiation and termination me Q 3 % ry a ” rH Nn Q ould be different --possibly different codons. Anotner feature of codon recognition concerns tne degener- acy of the code, or the existence of synonyms, i.e. different codons which code the same amino~Acid in the polypeptide se- quence. With che appropriate oligonucleotides, one can examine, t.. in vitro, the effectiveness of different synonym messages in &£ binding the particular amino- acidg : s+RNA! s to the ribosomes. The results of such are illustrated in Fig. . For example, a Fig. phenylalanine SP RNA responded to both the oLigonucleotides UUU and UUC, but UUC was slightly more active than UUU. Sinlarly fy Lysine+ £s¢RNA xesconced to both AAA and AAG but hese-cthere-ts Tu 2a = - - a * A ek at ane rence in the templace activity betweem the tua we gauke vo kede two synonymg, Tne first of these degeneracies, that between the wna a bh o t -- : te (smaller) pyrimicine bases C and U when they occur* as, third let- ter of the cocon,is universal throughout the code. The second Potes. af wept hoe sf? A Cex tp itae deg cneracy, @& the (large) purine bases A and G in f coir \"+88 occurs in all but two or three words (c.f. Fig. ) we turn now from these refinements end cetailed feacures oi the triplec-binding method to the actual results obtained rocecure. Shmee the triplets have a weil-defined se- ee. val quence or nucleotide es 7 there are 64 possible ~qmh t tripl ets} asa) . ‘fe we have synthesized 63 of these and determined the amino-acids which they code The results are summarised ) in Fig. “ “* 2 APs : Pe a Se aR FR Fig. 2 ERS “4 The astezisks indicate base compositions oz codons which < . A o - o 7 * a # 77 . were cetermineda by directing protein synthesis an Eecoli- extracts . . hbo as with syntnetic randomly-ordered polynucleotides. jt is clear. caat—there is~a-very..close/ sexsespendenee with the results of “HE earlier work. it is interesting to notice the types of syno- nyms which occur (some of which have aiready been mentioned).   jail - “3 qed ; vf poe cua uutene yet Sies_glutamic acid eetincopcndtentn | ‘the codons” CAA and GAGF- an ex- Se sre meent ey ermine Te mn ie “ CC is tay vet se ee &, af wd ampie a ‘A=G degeneracy in the third Place. bikewise Lspar ahd coclenso, woth. acid and; ea C }-eorrespondina-te U=C degeneracy in the third piace, Another. ctype of degeneracy is illustrated by Threonine sirconine | 4 hird bas) ct wnich is coded sy AC and any os the four U, C, A,.Gi place, —Besakos ine, on the other hand, is one of the rare cases (irypt Ops> may be another) in which GReec—ee 0 cord piace ce- 12 Oe LD ee Le Lol racy AUG gexicsenfee but AUA codes for Asoleucine. yen Mead 03 (oO “his degeneracy of the code can have many consequences, One of the more obvious is the possibility of a great deal of wat k a Sage ohh tae oy OF polio. tah me \"silent\" mutation, that is fon one of the code-words, or grouss wets ‘ of synonymous coce-words, there may be CONVCY Saga .0f~a~—bage--j.n- oy the—third-positioa to anotner base without resulting in an amino-~ . . . - . a, acic rep.acement. Another obvious conclusion is that amino i) cics which are very similar chemically, such as the dicarboxylic acids ( aspartic acid and glutamic acig, have closely related co- 2 Gons. This may reflect the evolution of the code, but whether or not this is so, one consequence would certainly be that when an error in repiication does occur, usually the first two bases are read correctly and the third one incorrectly. And very of- cen the result of an error in reading will be the substitution pe nitenn : q, in a protein of a chemically reiated aminoacid, thus the general picture of the code igs that it is quite conservative-- in the sense that it usually minimizes error or the consequences of er- ror. The various patterns df Synonym codons are summarized in Fig. - <(N-formyimethionine S#RNA shown here is the initiator), ta] ig +S. g in addition to the 0 odons for the specific amino“acids, ‘dere-as~as.has-been-mentioned earliex, some code-words Sneek. b's “ppear to serve special functions (\"punctuation\" etc.). For ex- ampie, the recenz work oi Brenner, Garen and Zinder, and of others, ae indicaces chat UAA and UAG may indicate the end of a message - although the precise mechanism for punctuation is unknown. 1 ULG, CUG, AUG and in some cases GUG may specify the initiation bh © 4 fo ry * of a message. Our recent scudies, and also those o and Marxer in England, have indicated that these codons - at 1east when in terminal positions ~ are recognized by formylmethi- onine and this may serve as an initiator of protein synthesis. Some possidie szecial function codons are listed in Fig. . 4 nh At beg ‘) fos! Sanger ,first * Observed imiengli that one of toe two. saN ra et a og yee w a * yr 8 “Pocmyt Stoup: x WS that is tne -amino “group~—of bee - methionine, after the methionine-   a wo ' eS oa + LU ie COROT gy was Tinked-to-the-s+RNA-ecoutd-be fomaylated. The work o£ Capec- chi and colleagues, and of Zinder, kawe suggested that this may mentioned HI specify initiation of Message translation. And as already, UUG, AUG, CUG and to some extent GUG are recognized by Kom formylmechiensae WPRNA; also that UAA and UAG may serve as ter- minators. It also appears likely that the words AG F wistin ending» he ~~ o JU, ©, A or G may also serve aS speciai function words; but s€= 9.5 Aa these functions have not sé-- -farc been found. The present situa- at pote tion in this field is e most interesting one, in that.the neces- sary tools for deciphering che special functiozx WOLGS _G52~£6 sccm, wed, and ic should soon be possible to understand more about the mechanism of these special words and the sole they play in pro- tein synthesis, be oi E=-would aike-to turn now-to- a variation of the triplet- Ear cg el Be binding methoc,- whi ch throws. further \\\\etgisteont the coding mechan- ism. D. Hatfield has recently prepared some radioactive trip- Lets / Cin the earlier experiments it was the StRNA which con- tained the racicactive tracer), “and has studied the binding of these tripiets to the ribosomes in the presence of the amino- acid S4RNA. Fig. shows both the binding of the triplet and of the S+RNA (here phenylalanine S#RNA) to the ribosome. Fig. Asean 5eseen, in the presence of the appropriate triplet polynucleotide phenyiaianine s#RNA binds to the ribosome; in the absence of the s}RNA very little triplet binds to the ribo- some. Because of this, in the presence of the s4RNA both the triplet polynucleotide and the phenylalanine s+RNA bind to the ribosome 4c approximately the same rate. Thus the compiex on cne ribosome may well be a one-to-one association of triplet and XNA. this technique provides a very simple and quite sensitive method for detecting codon recognition by s7RNA which is not t hea a } acylated with amino-acids. Thus some special function words may not be recongized by activating enzymes, s+RNA's, which are   e wo x . n > : ™ : : Owes . 4 Bue . ; po tee : | ek wd 5 if : 2 we OTE OS NO et ee vg is “f ok ey Joe fhe alt ee : e not acylaced, and this method would provide a relatively simsle route towards detecting such recognition. We have also made investigations (in collaboration with B. P. Docter and Waicer Reed) with purifies s+RNA fvactions, i.e., media containing essentially only 4 singie type of s7RNA, de- ah Tees ra , ; rived from Esecoli fractions. We find that ycosine-sfRNA re- cognizes both UAC ana UAU, which again exemplifies the C=U de- generacy in oJ the third place. (There are two types of Tyrosine= ° t da S#RNA, difi rh er in ; both types recognize UAC and UAU.) Si milarly Vaiine/s?RNA recognizes both GUA and GUG (G=A degeneracy) but the GUG to a much lesser extent than GUA. The Ercoli fraction leucine-1-s-RNA and Leucine-2-s?RNA both xecognize the leucine codons (UUA, UUG, CUU, CUC, CUA, CUG). Recently, however, J. A. Carbon has reported that in mammal Liver one species of Leucine=s~-RNA preferentially recognizes AAG, and the other pre- ferentially recognizes AAA. There are also types of Leucine-s#RNA which recognize CUG, and others which recognize UUG. The major variant of methionine~s-RNA which, as mentioned previously, wili accept a formyl group recognizes UUG and CUG, Joe Tay out a less prominent methionine+s+ -RNA recognizes AUG preferern- tially. Uikewise there is a Mryptovhan s#RNA which recognizes UGG, CGG and co a smaller extent AGG. The pattern here is clear: a close relationship between U, C and A in the first place of 16 tne coding criplet. R. nolley, working with purified fractions cecal Z C2OD of yeast s?RNA, found “alan ine=s#RNA recognized “@é,GCC and CCA -- again the group U, C, or A but now in the third place of sane coding tviplec. It should also be pointed out srominent binds to ribosomes very weakly in response to the ucleotice triplets: it is possible that this tyve of weak re- cognition involves only two of the three nucleotide basis in the * This work with pure fcactions = such és +e élanine~s+RNA prepared from yeast, can afford some further insigh<c into the mechanism of codon recognition. This is especially so in this case since Yolley and ais collaborators have recently reported ¥ a the sequence of bases in the alanine-s#RNA. Hi Fig. ts: shown” af a bot the variation -of binding of- alanine}s+RNA to ribosomes wash” con-~ centration of the s+RNA. TT F H gS. she Gocced Line represents 100% binding, i.e. ali the available Th S+RNA is bound to ribosome.’ This fraction of S-RNA which Holley f 5 = * . . @ vars ts — Suppiied to us, was estimated to be greater than 95% pure; and yet this s+KNA recognized quite well at least three of the alanine codons -- GCL, GCC, and GCA. It ditd=enct respond = ~wex omy very 4 a # ae ol fy ch Siigntiyy cc GOG. (On the other hand, with unf ractionated Eecoli. StRKNA, alanina-s+ \"NA responded quite well to 227 -- f “pT rh ~~ nt aA on vA an ~ 5 * : a aT\" WES tne best alanin “stRNA COGoON,-ana tae response to aU ed this , GCC ana GCA was relatively weak.) Since the yeast extracted StRNA <vaction was of high-purity, the results strongly suggest thac a singla molecule of S#RNA can recognize alternatively at least three of che four alanine synonyms. The wnole sequence of the nucleotides in this alanine S+RNA are shown in Fig. Fig. yr ' bp s at bos me alanine amino-acid im Linked to the terminel adenosine, and are ; tnais is shown in the diagram in only,of the suggested possible conformations. There ave several single-stranded regions of at ™ The ye the S+RNA, of possible interest. There-is-a sequencer G, T,¥U, c (¥U is an isomer of U) fowhich—-seqtrenee has been found in vir- tually every s4RNA that has been examined. Another interesting Loon fp ; . . tans : Sequence is the C, G, Gf surrounded by two dihydrouridyliec acids. A tnird is the iGC region (ISinosine) right in the middle of cae S+RNA molecule. These latter two regions of interest are Shown in more detail in Fig. Fig. the . ~ . If these-ews triplets CGG and IGC were really the S{RNA anti= codons, that is, the nucleotide groups which recognized the nu- cleotide-triplct cco. 5 for alanine, recognition would be by paral- z oe Lae - of . lei pairing between C and U; and the G woulc thsén have to recognize   Ni £ SAL Coe . - pe Mae, ROD pe 2 re oe coe of “ Soe. pete. weal U, C ana A. Ii, however, base pairing weve’ according to the | Watson-Crick hycroge ondbonding, or anti-parallel scheme, C would pair with G, G with C and the inosine I in this position would base-pair with one of U, C or A, but not G. This latter is the pattern observed for the alanine code; and Crick has re- cently proposec a detailed mechanism which wouid permit hydrogen’ bonding between Z and U or C or A. Tnis mechanism, by which I recognizes U, C or A in the cE RE atts antifcodon - codon pairing, termed the \"wobble\"sby Crick, in- ae volves a movement, at the end position of the triplet, of either che sfRNA or the messemgex-RNA on the ribosome. All the experi fe mental results are, I believe, in accord with this type of re- &, cognition mechanism. The table shows the base~Sequences in the Table t., s tRNA anti}godon and the corresponding base-sequences in the messenger=RNA codon. Thus Thosine in an end position in s#RNA 7 oticr TACs tort - ' Can recognize by alternate base pairing U, C or A; 4G in the ad es eles ora és, end position of s#RNA could’ similarly recognize “aleermately. C ox Yo and A coulca recognize U, C or G and @ U could recognize Ada ae by alternate pairing A or G. we would also predict on this mo- del that a riocthymidylic acid ass PRNA would pair also A and G, (perheass the interaction with A would be stronger than for a . e3 ‘ ent ° tee . 7 uricylic acia in S#RNA) 5 that ayvyuwU in StRNA might recognize aim mo . — Oh Ae sof Z We ta Tw Oe hee Cy cemeace’ A, Gor U - a pattern that has been noticed rather a wi St; RNA. ess pod f ae “Up. fm’ Another possibility is @ dihydrouridylic acid would not. wo» * J Q bia ct © 4 Dd 0 se pair whtn-.che.expeeted-eomplementary’4), so that the sf . . > iacevaction witn the messenger would be a week interaction: ~ ? buc it is also quite possible that a U or C in a terminal po- sition wouid noc greatly inhibit the interac A metal group on a 2'-nydroxyl ceoxyribose (sugar) mighc also result in 4 weaker interaction, and furthermore, by permitting a greater freedom of motion on the ribosome, such a modification might resuit in greater ambiguity, i.e.) lower specificity of These resuits with infrequently occurring (or “trace'y bases, and particularly those with Inosine, cohen St rongly sug- gest that s#RNA may be modified enzymatically, _ after it is xre- Leased from the DNA template (where it is assembied in the cell). Since the level of \"trace\" bases is quite high in an actual cell, it seems Likely chat there exists a whole spectrum of interme- diates, stRNA's in various stages of successive modification. the consequences of this are f£ “r. easy to visualize. For ex- “A =a ° ~ . s oN ° a a a > ample, if an acenine(A) in stRNA is de-aminalid and so converted > + » which would normally recognize the Gri- oy dylic acid base in the message, would now be revlaced by some- into an inosine(i), the thing (the I) which can recognize U, C or A. Simila: intercon- versions would result from the desamination of a C or the conver- Sion of G to i. It is possible, althoucn perhaps rather pre- Mature LO speculate, that this type of interconversion plays an umportant tiologiecal role. There nascextaialy-beem a great deal 02, Work [recen em ve Cuarhce b K Lure vida of ¢ whey Ate a, _Wietekt sugzests vnat, £t ,is possible in actual cells, fesome 7 yea ON ee “ wey—te modify che specificity of codon. recognition and—this is—eertai. ~taveprofound biological   consequences. An example of this is the effect of the anti- biotic screptomycin, tt—has—been—shown,—by Davis, Gil", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-n2sy.bcvz-yhhh", "00000000-0000-0000-C17A-C22192C88A06", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Frances Bacus to Marshall W. Nirenberg", "101584910X142", null, "1971", "3 December 1971", "Marshall Nirenberg's fame brought him many different types of requests from the public.  In this letter, journalist Frances Bacus requests any information that Nirenberg might have on the genetic basis of racial characteristics, or stereotypes.", "Letters (correspondence)", "Genetic Code,Continental Population Groups", "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "December 3, 1971 Dear Mr. Nirenberg: I am writing an article for ADAM magazine titled \"Your Race and Your Intellect\". Have you done any research in your department that would have a bearing on this subject genetically or otherwise? For instance, are Scotsmen really thrifty, Indians loyal, Frenchmen sexy? Could you tell me where I could get information that would show race to be a factor in crime rate, intelligence, personality, alcoholism, drug addiction, or any other life pattern that would be of interest to ADAM magazine readers? Thank you for your interest. Sincerely, Frances Bacus", "Bacus, Frances", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ztga-ka49.kjpp", "00000000-0000-0000-052A-FB69C50EA5DA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "An Enzymatic Spectrophotometric Method for the Determination of Phenylalanine in Blood", "101584910X143", null, "1960", "March 1960", "This article describes a simple enzymatic method for the quantitative detection of phenylalanine in blood by using snake venom.  Useful as a test for diagnosis of the disease phenylketonuria and evaluating the effectiveness of a diet designed to be low in phenylalanine, the method is rapid and precise.", "Articles", "Phenylalanine,Enzymes,Spectrophotometry", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "6", "pages", "Text", "English", "Reproduced with permission of Mosby, Inc..", "Copyright may apply", null, null, "Reprinted from THe JourNAL of LABORATORY AND CLINICAL MbeIcing, St. Louis. Vol. 55, No, 3, Pages 491-496, March, 1960. (Printed in the U. S. A.) (Copyright © 1960 by The C. V. Mosby Company) AN ENZYMATIC SPECTROPHOTOMETRIG METHOD FOR THE DETERMINATION OF PHENYLALANINE IN BLOOD Bert N. La Du, M.D., Pu.D., anp Parrrcia J. Micuarn, B.A, Betuerspa, Mp.   HERE is a need tor a simple rapid method to measure phenylalanine in serum or plasma, not only as an aid in the diagnosis of phenylketonuria, but also in the evaluation of the effectiveness of a diet low in phenylalanine in the treatment of patients with this condition. The method most commonly used to measure phenylalanine in plasma is that of Udenfriend and Cooper,: in which plasma is deproteinized and treated with Streptococcus faecalis to decarboxylate i-phenylalanine to phenylethylamine. The latter is then determined colorimet- rically after reaction with methyl orange. Although this method has been used successfully by many workers,” it requires relatively large amounts of plasma and takes several hours to perform each set of analyses. Furthermore, extremely careful technique is necessary to avoid an appreciable and variable blank from methyl orange. Another method for the determination of serum phenylalanine is that of Berry,” by paper chromatography. This method, although simple, has limitations in quantitative analysis inherent in any method which depends upon estimation of the intensity of colored spots on paper chromatograms.   Scr, CHCOOH Sc. C—COOH Sp 9s Snake venom TL-amino acid oxidase     L-phenylalanine phenylpyruvie acid H SN beg ee « 2 \"I , oO NZ B SN 0 0 K Dek —b=0 H   Borate-enol complex of phenylpyruvic acid Arsenate borate   From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U. S. Public Health Service, Bethesda, Md. Received for publication June 22, 1959, 491 492 LA DU AND MICHAEL J. Lab. & Clin. Med. A simple rapid method is described whieh permits the analysis of plasma L-phenylalanine in 0.1 ml. of serum without deproteinization. The method uti- lizes L-amino acid oxidase from snake venom to oxidize L-phenylalanine to phenylpyruvic acid. In the presence of arsenate and borate ions the resulting a-keto acid is rapidly converted to an enol-borate complex which has a high ab- sorption in ultraviolet light. Catalase is added to protect the a-keto acid from peroxide formed in the oxidative deamination by L-amino acid oxidase. Enol-borate complexes have been employed by Knox and Pitt® to determine p-hydroxyphenylpyruvie acid oxidase activity in mammalian liver preparations. More recently these complexes have been used to measure the activity of several enzymes whieh either form or break down the aromatic a-keto acids related to phenylalanine, tyrosine, tryptophan, and histidine.” METHOD Reagents.— 1. Phosphate buffer: 0.2M sodium phosphate buffer, pH 6.5. 2. Arsenate-phosphate buffer: 2.0M sodium arsenate was dissolved in the 0.2M phosphate buffer, and the final pH was readjusted to 6.5 with dilute HCl. 3. Borate-arsenate reagent: 1.0M borate, dissolved in 2.0M arsenate, pIl 6.5, (61.8 Gm. borie acid + 624 Gm. sodium arsenate [Na,HAsO,-7H.0]) was adjusted to pH 6.5 with HCl and made up to 1 L. 4, Snake venom L-amino acid oxidase (the venom of Crotalus adamanteus*): a suspen- sion of dried venom in water was made which contained 10 mg. per milliliter. This was ecen- trifuged, and the clear supernatant so.ution was removed and used. The enzyme solution is kept at 0 to 5° ©, and remains active with little loss of activity for several days. The dry venom is stored in the refrigerator and maintains high enzyme activity for several months. The activity of the L-amino acid oxidase preparation can be assayed by using 0.1 ml. of the standard phenylalanine solution in place of serum in the directions given below. 5. Catalase: crystalline beef liver catalase,t diluted 1:5 with 0.2M phosphate buffer, pH 6.5. The enzyme solution is kept at 0 to 5° C. and can be used for several weeks. 6. Standard phenylalanine solution: L-phenylalanine, 1 «M per milliliter (0.1 ml. con- tains 16.5 ag). 7, Blood serum: Blood is drawn and allowed to clot. (Some samples of heparinized plasma develop turbidity during analysis which makes them unsuitable for this determination.) Procedwure.— Three 1.2 ml. quartz Beckman cuvettes with a 1 em. light path are used for the determinations, The additions to each of the cuvettes are as follows: O By Ey PO, buffer, 0.2M, pH 6.51: 0.39 0.39 0.39 Arsenate-phosphate, pH 6.5: 0.5 — = 1M borate in 2M arsenate: _ 0.5 0.5 Catalase, 1:5 dilution: 0.01 0.01 0.01 Venom, 10 mg. per milliliter: 0.10 0.10 0.10 (Serum added later.) The contents are mixed and the duplicate experimental cuvettes, E, and FE., are read against the control cuvette, C,, at 308 mu, in the Beckman spectrophotometer.§ If the addi- *From the Ross Allen Reptile Institute. Silver Springs, Fla. 7From the Worthington Biochemical Corporation, Freehold, N. J. tIf more than 0.1 ml. of serum is to be added, the volume of phosphate buffer must be reduced proportionately. §Model DU with ultraviolet attachment. Nahas > MEASUREMENT OF PHENYLALANINE IN PLASMA AND BLOOD 493 umber tions have been made correctly, this reading should be approximately zero. Then 0.1 ml. of serum is added to cuvettes C,, E,, and E,, mixed, and readings are taken at 1 or 2 minute intervals for 10 minutes at 308 mu, to be certain that the enzyme activity is adequate, and that the reaction is essentially complete, as indicated by finding no further change in the optical density reading. Final readings are made at 308, 330, and 350 my at this time. Calculations.— Readings are taken at 330 and 350 mu, as well as at 308 mu, to correct for the ab- sorption of the keto acids derived from tyrosine and tryptophan, since the latter 2 amino acids are also oxidized by the venom L-amino acid oxidase. ‘The correction applied is based upon the relative absorption of the enol-borate complexes of these keto acids at 308, 330, and 350 mz: RELATIVE OPTICAL DENSITY READINGS AT DIFFERENT WAVELENGTHS (mp) KETO ACIDS OF 308 330° 350 X = tyrosine x 0.60X 0.06X Y¥ = phenylalanine Y 0.10Y 0 Z = tryptophan 0.852, 1.41Z Z Let the reading at 308 mp — A; 330 mu = B; 350 my = C Then: A= X + ¥ + 0,852 B= 0.6X + 0.1Y + 1417 C = 0.06X + Z Solving these equations gives: X = 2.38B — 0.238A — 3.16C (1) Y = L234 ~ 226R + 2.15C (2) Z = 1.19C - 0.14B + 0.014A = C -— 0,06x (3) The absorption at 308 my caused by phenylpyruvie acid is given by solving for Y in equation (2): Y 0.031 * 10 = gg phenylalanine per milliliter of serum (if 0.1 ml. of serum were analyzed). One microgram phenylalanine under these conditions of 1.1 ml., ete, at 308 mu reads 0.031 O.D. units. x By the same means, solving for X in equation (1) and dividing: $045 X10 = ug tyrosine per milliliter of plasma (1 wg tyrosine under these conditions reads 0.045 at 308 Z m#); and, similarly, using equation (3): 0.033 * 10 = ug tryptophan per milliliter of serum (1 wg of tryptophan reads 0.033 at 350 mz). RESULTS AND DISCUSSION Spectficity—The L-amino acid oxidase of snake venom catalyzes the oxida- tion of a number of amino acids, However, only 4 of these are present in ap- preciable quantities in serum which yields a-keto acids with highly absorbing enol-borate complexes; these are phenylalanine, tyrosine, tryptophan, and his- tidine. Fortunately, histidine is not oxidized under the experimental conditions described above, and the relative contribution of each of the other 3 amino acids can easily be determined from the absorption of the combined complexes at 3 different wavelengths. Therefore, this method permits the simultaneous deter- mination of phenylalanine, tyrosine, and tryptophan in serum. If the value of phenylalanine alone is desired, the correction for absorption eaused by the keto acids of tyrosine and tryptophan would appear to be a disadvantage of the method. This is partly compensated for by the commercial availability of the 494 LA DU AND MICHAEL J. Lab. & Clip, Med. enzyme, its stability, and its high activity. Furthermore, the ease with which both phenylalanine and tyrosine can be measured is of special interest in some studies on phenylketonuria; for example, in the detection of the heterozygous carrier of this trait by the ratio between the levels of phenylalanine and tyro- sine in the blood after the administration of phenylalanine as a tolerance test.® The wide range of substrates which can be used with this enzyme has made it posstble to extend this method to measure a number of other aromatie amino acid analogues and antimetabolites, such ag p-fluorophenylalanine, m-tyrosine, monoiodotyrosinc, mononitrotyrosine, and 8-2-thienylalanine. These compounds are also rapidly oxidized by the L-amino acid oxidase of snake venom, and the resulting keto acid products ean be measured as enol-borate complexes.” 1.000           o PHENYLALANINE ALONE o @ RECOVERY FROM PLASMA     «800 +600 «400 -200 OPTICAL DENSITY, 308 m.      t l I J 10 20 30 MICROGRAMS OF PHENYLALANINE Fig. 1.—Graph of the optical density at 308 mz versus concentration of phenylalanine when determined in Standard solutions and when added to normal plasma. The ‘linearity between optical density and phenylalanine concentration is demonstrated. Reproducibility and Accuracy.—The method described is well suited to measuring the elevated blood level of phenylalanine found in untreated phenyl- ketonuric¢ individuals and to following the effectiveness of a diet low in phenylal- anine in lowering the serum level in these individuals. The specificity of the absorption spectrum of the phenylpyruvie acid enol-borate complex and the ease and rapidity of the analysis permit a large number of determinations to be carried out within a short period of time. Fig. 1 demonstrates the linear relationship between phenylalanine concentration and optical density change between 2 and 30 ng analyzed alone and the reeovery of phenylalanine added to normal plasma in this range. These results are in agreement with the theo- retic values expected. However, the precision of the analytic method is lower in analyses of phenylalanine in normal serum than when the values are elevated as in serum of an individual with phenylketonuria. In normal serum the con- tributions of tyrosine and tryptophan to the reading at 308 my are appreciable. Even though duplicate pairs of analyses in a series of normal serum samples analyzed on suceessive days rarely disagreed by more than 7 per cent, larger aliquots of serum would be desirable if the method were to be used to measure, with high accuracy, the endogenous phenylalanine, tyrosine, and tryptophan levels. Attempts to carry out the analyses directly on 0.2 or 0.3 ml. of serum have bec only partly successful, since a turbidity often occurs. For this reason, None MEASUREMENT OF PHENYLALANINE IN PLASMA AND BLOOD 495 several methods were tested to deproteinize the sample before analysis. Preliminary experiments have been made using perchlorie acid precipitation followed by neutralization and the removal of KC10,, as described by Segal, Blair, and Wyngaarden,! before cnzymatie assay of blood pyruvate. This procedure for deproteinization appears to be satisfactory in eliminating the turbidity problem, and the solution which results can be used directly in the enzymatic assay. Phenylalanine Levels in Normal and Phenylketonuric Individuals —-The serum phenylalanine levels of 30 adult blood bank donors in a nonfasting con- dition were found to have an average value of 1.55 mg. per cent (range, 0.84 to 2.64 mg. per cent). It is probable that the level in normal subjects in a fast- ing condition is somewhat lower than these values. In a smaller number of analyses in laboratory workers in a fasting condition, the group average is slightly lower than the above value. The level of phenylalanine in a group of 10 phenylketonuric individuals on a regular diet was found to have an average value of 36.8 mg. per cent (range, 26.2 to 59.1 mg. per cent). These values in normal and in phenylketonurie people are in good agreement with those in the literature? 1*™ obtained by other methods. The wide difference in the levels of normal and phenylkctonurie individuals makes the analysis of serum phenylalanine a valuable diagnostic procedure. SUMMARY A simple enzymatic spectrophotometric method has been described for the quantitative determination of phenylalanine in serum. The method is based upon the measurement of the absorption of the enol-borate complex of pheny]- pyruvic acid generated enzymatically from phenylalanine by L-amino aeid oxi- dase of snake venom. For elevated serum levels of phenylalanine the method is rapid, specific, and precise. It should be useful as a confirmatory test in the diagnosis of suspected phenylketonuria and in the evaluation of the effectiveness of a dict low in phenylalanine. The values obtained by this method agree well with those in the literature obtained by other techniques. Tyrosine and tryp- tophan are also determined by this method, and a suitable modification of the method is deseribed which should allow an accurate estimation of these amino acids and phenylalanine in normal serum. REFERENCES 1. Udenfriend, 8. and Cooper, J. R.: Assay of L-Phenylalanine as Phenylethylamine After Enzymatic Decarboxylation; Application to Isotopie Studies, J. Biol, Chem. 203: 953, 1958. . Meister, A., Udenfriend, 8., and Bessman, 8. P.: Diminished Phenylketonuria in Phenyi- pyruvic Oligophrenia After Administration of 1-Glutamine, 1-Glutamate or L- Asparagine, J. Clin. Invest. 35: 619, 1956. 3. Hsia, D. Y.-Y., Driscoll, K. W., Troll, W., and Knox, W. E.: Detection by Phenylalanine Tolerance Tests of Heterozygous Carriers of Phenylketonuria, Nature, London 178: 1239, 1956. 4. Hsia, D. Y.-Y., and Paine, R. S.: Phenylketonuria: Detection of the Heterozygous Carrier, J. Ment. Deficiency Res. 1: 53, 1957, . Hsia, D. Y.-Y.:  Phenylketonuria: The Phenylalanine-Tyrosine Ratio in the Detection of the Heterozygous Carrier, J. Ment. Deficiency Res, 2: 8, 1958. tw oO 496 LA DU AND MICHAEL J. Lab. & Clin, Med. March, 1960 . Knox, W. E., and Messinger, E, C.: The Detection in the Heterozygote of the Meta- bolic Effect of the Recessive Gene for Phenylketonuria, Am. J. Human Genet. 10: 53, 1958. . Berry, H. K.: Paper Chromatographic Method for Estimation of Phenylalanine, Proc. Soc. Exper. Biol. & Med. 95: 71, 1957. . Knox, W. E., and Pitt, B. M.: Enzymatic Catalysis of the Keto-Enol Tautomerization of Phenylpyruvie Acids, J. Biol. Chem. 225: 675, 1957. 9, Lin, E. C. C., Pitt, B. M., Civen, M., and Knox, W. L.: The Assay of Aromati¢ Amino Acid Transaminations and Keto Acid Oxidation by the Enol Borate-Tautomerasc Method, J. Biol. Chem. 233: 668, 1958. . La Du, B. N., and Michael, P. J.: A New Assay for Analogues and Antimetabolites of Tyrosine and Phenylalanine. In preparation. . Segal, S., Blair, A. E., and Wyngaarden, J. B.: An Enzymatic Spectrophotometric Method for the Determination of Pyruvie Acid in Blood, J. Las. & CLIN. Merb. 48: 137, 1956. . Hsia, D. Y.-Y., and Driscoll, K. W.: Detection of the Heterozygous Carriers of Phenyl- ketonuria, Lancet 2: 1337, 1956. . Jervis, G. A., Block, R. J., Bolling, D., and Kanze, E.: Chemical and Metabolie Studies on Phenylalanine. II. The Phenylalanine Content of the Blood and Spinal Fluid in Phenylpyruvie Oligophrenia, J. Biol. Chem. 134; 105, 1940. . Bickel, H., Gerrard, J., and Hickmans, EK. M.: The Influence of Phenylalanine Intake on the Chemistry and Behavior of a Phenylketonuric Child, Acta paediat. 43: 64, 1954.", "La Du, Bert N. ; Michael, Patricia J.", null, "Journal of Laboratory and Clinical Medicine", "C.V. Mosby Company", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8exi~2ddy-sna8", "00000000-0000-0000-4F3D-843F15335A4C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on deciphering the amino acid codes", "101584910X144", null, "1964", "[ca. 1964]", "In these handwritten notes from a laboratory notebook, Marshall Nirenberg sketches some of the possible combinations of nucleotides in RNA codes and their corresponding amino acids, revealing the process of elimination and sequencing difficulties associated with early coding efforts.", "Laboratory notes", "Codon", "Translating the Code of Life and the Nobel Prize, 1962-1968", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Gan Aaa ual UuXu Aue Cun       U kre WS Se A, Q) Gye 64,8) Who, va A 4. ay aD dros ras c A . arr P~ PUBS LR     New \\\\G. A u ie - ' ah. A> a (_ 2 asa 2 | \\\\ “ ‘ . e a UES Ley | ¢) Be ey me WAY ida l i AO4 Lee CAC LS AUK 1 a cau de Re? Pak WAR Sehun ACO Len MMI Nepatne i, AML -  Uewhedic ad Ce ot 6 a ~ C uk   UX 6 6 ae kA UG EC GH Ac UAG gf { ke) —t Pad : dor | Lee Typomr Cheers | be Ree | Aap tem ie m4 OK OA A - CQ Ly no Duhon. eg, Due NIA , Apne There , bat NH. , Czy NIIy (Are, ARC MMe Oephe pce", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8r59.mf95_8rgh", "00000000-0000-0000-ADAE-E35C75E59861", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "64 triplets and complementary triplets", "101584910X145", null, "1965", "[1965]", "This handwritten table lists the 64 possible triplets and their complements known by 1965.", "Tables (documents), Laboratory notes", "Codon", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "VAX 6Y Top dets Bo crrp hen be AY c. ot C uY Cc AM AAG AA UY AC uu Ur A Pe ULE ru AG /yEO INn@Y (Nor uu ccT AoA LiCé 3 G/* CAG 2AU GING cok eu A CuK u 66 C66 CGK4 CE coe CC AN ce & 5 Ge 6ClG acu GCC CEC CEA LES c {Ce CEG CEY Cee ECC EC K & O68 n ec CCG cc 4 eco EGER & hy Ba & ce cA CA & ati CAT GU ¢ a er tate 4 AC /¥CG 44 ACC uj & Cc ty & : i* Uo 12 UX UWE Huy Une ARS. BAA KING if UA UMHG f+ UAL AAC Aad A tle ru ot AM Ade AY AUC AG WAY o> AAG > Lau CUS CHU Cuc EAC ee a ee EING mob gAaE Uc 6 UC uy 4 AE C A& i ns < rm 1GY 64S GU & GUC , CAC CaN CAS i TUG U6G UBY U6C ACG ACN AOE", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-y9yk~xhuv~ba9p", "00000000-0000-0000-19C8-968715CE05CC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on triplets", "101584910X146", null, "1964", "[ca. 1964]", "These handwritten notes include amino acids coded by two or more different polymers, various single and double code words, sketches of strands, and sequencing tables.", "Laboratory notes", "Amino Acids,Codon", "Translating the Code of Life and the Nobel Prize, 1962-1968", "4", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "anhyas s RNA Por lent & Any ua t “Coe Cc Len UG © D Uc ¢ UP A (4s) Ba - Nh Ly G me s Ogee Ny. Ang UA x CA c HK, UG Mu ce CI t 2) z AG | A Paks Anon _ S ime EOD t AC A 6c ? Q = Abe uc ('\\\\ ae. _A Ac A ao L 6c l?l ¥ Le un    sk ae [at sRNB CAA ' a | Mea ecy PAE ot GRA | 64a . | 4c ! UG G6 u [. Ae one. Ne | CENA AU uc Q ung © | a. Us mee oo 6 | . 7 AWS un fe er Wc / Lor) we AAC ARG Thee ee Acic Wai Ps oa Uru n> a UAL uscd HAS a CAC LUG cee GUG CUA [cE CER ACK. he Re WE Muu te au UE ot Ac6“% pue ey Rear he ag SCO 7 ach fee RS het 5E U~G rare Ure Cu Kyo C+4 Aan On. Se4, Gur ‘ . AAG G6K GAR ecg UGA UAA ACé 6an Olu GeEk GAC a Cag bn OAD Aa’ A wA Tyee AMA oye ; A A a xe AA& Uirerne AUG URGE?) eX cc nee Make UNG op AG A ABC? AY CE ays U6G AA & — Yue UULY Acc. A U6G YUYUGS Uo. COFY Op Ni BRAT Lg AU, or CU Cok” { ' - AAU Pdi Cee ani, fe AC. Bek OR tice? COA f NA, ) s CAC CAC Coc Cle > yp & a> fa 2} s* sf 7 PC PR ‘se fe ?", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kjjd.g4j4~yr8n", "00000000-0000-0000-8CBA-29FA636AF83F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Proposed Training", "101584910X147", null, "1961", "[ca. 1961]", "This NIH proposal provides an outline of Marshall Nirenberg and Heinrich Matthaei's findings from their earlier studies of cell-free E. coli, TMV, and messenger RNA.  Future plans include the study of RNA coding units, which includes determining the coding ratio, the specificity, and universality of the code at NIH.  Funds for professional development are also requested.", "Proposals", "Amino Acids,Poly U,Proteins", "Translating the Code of Life and the Nobel Prize, 1962-1968", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "(a) Characteristics of RNA coding units A cell-free E. coli amino acid incorporating system dependent upon the addition of messenger or template RNA has been obtained (Nirenberg and Matthaei, Proc. Nat. Acad. Sci., 47, 1588 (1960)).  Both naturally occurring and synthetic polyribonucleotides have been found to direct protein synthesis in the system.  The purpose of this investigation will be to compare the characteristics of naturally occurring and synthetic polyribonucleotide coding units.  These characteristics will include (a) the coding ratio, i.e., the number of nucleotides comprising one coding unit (b) specificity of coding units, i.e., whether two or more coding units direct the incorporation of one amino acid into protein, and (d) the universality of coding units, i.e., whether a coding unit corresponding to an amino acid is the same in different species. Polyuridylic acid serves as template RNA by directing the cell-free synthesis of polyphenylalanine (Nirenberg and Matthaei, Proc. Nat. Acad. Sci., 47, 1588 (1960)).  Also, randomly mixed polynucleotide direct the incorporation of other amino acids into protein.  Thus, the nucleotide composition of RNA coding units corresponding to 15 amino acids has been determined (Martin, Matthaei, Jones and Nirenberg, Biochem. Biophys. Res. Comm. (in press)). The addition of tobacco mosaic virus RNA to the cell-free E. coli system greatly stimulated incorporation of amino acids into protein.  The soluble C14-protein formed during the course of the reaction has many properties characteristic of tobacco mosaic virus protein.  For example, it is antigenic and is precipitated by tobacco mosaic virus antibody; it may be purified by DEAE column chromatography in the same manner as tobacco mosaic virus protein; a peptide can be isolated from the middle of the molecule after digestion with trypsin which contains C14-amino acid in the proper place (Tsugita, Frankel-Conrat and Nirenberg, unpublished results).  However, the product of the reaction did not combine with tobacco mosaic virus RNA to form infective virus.  The product appears to be similar, but not identical to tobacco mosaic virus protein.  Attempts will be made to compare the finger-prints of the product of the reaction and authentic tobacco mosaic virus protein and thus determine the precise differences between them.  These studies should show whether coding units contained in tobacco mosaic virus RNA can direct the synthesis of similar proteins in both tobacco plants and E. coli.  In other words, a direct comparison may be obtained between the function of RNA coding units in two different species. Soluble RNA has been shown to be an intermediate polyphenylalanine synthesis (Nirenberg, Matthaei and Jones, Proc. Nat. Acad. Sci. (in press)).  Since soluble RNA appears to be a cofactor which functions as an \"adapter\" carrying an amino acid to its proper place on template RNA, a variant soluble RNA base-pairing with a different code letter of template RNA would substitute one amino acid for another during protein synthesis.  It is possible that in species other than E. coli, polyuridylic acid may be either meaningless or may serve as a template for a different amino acid.  Soluble RNA will be purified from different species and will be charged enzymatically with different C14-amino acids.  The C14-amino acyl soluble RNAs will be used in a purified E. coli amino acid incorporating system and synthetic polyribonucleotides will be added to direct amino acid incorporation.  It is hoped that data obtained from these experiments will show whether soluble RNA from different species can recognize the same RNA coding unit.  These data, as well as the experiments mentioned previously, may indicate whether RNA coding units in different species are the same. Randomly mixed polyurydilic-guanylic acid directs the incorporation of a number of amino acids including leucine and valine.  The amounts of leucine and valine incorporated into protein were equal and were proportional to the guanylic acid content of each polymer.  Thus, the number of coding units prevailing in leucine per polymer were the same.  However, omission of C12-valine did not affect the incorporation of C14-leucine, which demonstrated that the coding units for valine and leucine are different.  Degeneracy in this system was demonstrated by the finding that both polyuridylic-guanylic acid and polyuridylic-cytidylic acid stimulated the incorporation of leucine into protein. The specificity and degeneracy of RNA coding units in tobacco mosaic virus RNA will be investigate in a similar manner.  Thus, the information concerning the code which has been obtained using synthetic polynucleotides may be directly compared with the coding characteristics of a viral RNA. (b and c) This type of training in enzymology and biochemical genetics will aid Dr. Byrnes in his future research.  Upon completion of his fellowship, Dr. Byrnes should be exceptionally well qualified to do independent research and to teach. Facilities Available The Laboratory of Molecular Biology in the Institute of Arthritis and Metabolic Diseases, National Institutes of Health, will contain all of the equipment necessary for this type of work.  This laboratory is staffed by people trained in different disciplines, such as crystallography, physical chemistry, biophysics, organic chemistry, biochemistry and genetics.  Interaction and exchange of information between such people should be valuable and Dr. Byrnes should obtain training of unusually broad nature.  Frequent inter-disciplinary seminars help to maintain a stimulating intellectual and research environment. Funds Requested An annual stipend for Dr. Byrnes of $6,500.  It will not be necessary to receive the institutional allowance in partial payment of costs incurred by the National Institutes of Health in pursuance of Dr. Byrnes' research and training.  Part of these funds, however, may be an aid to Dr. Byrnes in defraying tuition expenses of elective courses given at the National Institutes of Health if Dr. Byrnes wishes to participate in them.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rfcz_w4jh_s58u", "00000000-0000-0000-3F83-BFCCF16F0398", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Draft of speech given at the International Congress of Biochemistry in Moscow", "101584910X148", null, "1961", "[ca. August 1961]", "The International Congress of Biochemistry in Moscow was held in August 1961.  In this typed draft of the classic speech that pushed Nirenberg into the scientific spotlight, he announces how poly-U amino acids stimulate the incorporation of phenylalanine into a polypeptide chain.  Handwritten corrections are included.", "Speeches, Drafts (documents)", "RNA, Messenger ; Amino Acids,Proteins", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Moscow Talk Original MS We have obtained a stable, cell-free E. coli system which incorporates C14 amino acids into protein at a rapid rate, which has many characteristics of protein.  Conditions have been found which demonstrate a novel characteristic of this system; that is, a requirement for messenger or template RNA even in the presence of excess soluble RNA.  Naturally occurring RNA such as C coli messenger RNA and TMV RNA and [ . . .  ] synthetic polynucleotide, were active in this system. First slide, please.  Each reaction mixture contains these constitutents in micromoles per milliliter.  The enzyme extract consisted of E. coli ribosomes and 100,000 x g supernatant solution.  Complete details are presented in Biochemical and Biophysical Research Communications 4 (1961). In slide No. 2, counts per minute per milligram protein is plotted against time and minutes.  In the absence of added DNAase, valine was rapidly incorporated into protein. At the end of 90 minutes [END PAGE ONE] [BEGIN PAGE TWO] The synthetic polynucleotide's polymaleic[?] acid.  Addition of microgram[?] qualities of Pol 4 reaction mixtures result in a 500 to 1000-fold elimination in incorporation of C14-phemyloline into a protein which strongly resembles polyphemyoline[?].  We've tested many other synthetic polynucletudes.  Only poly 4 stimulates phenylene [ . . . ]. Randomly mixed polymers of [ . . . ] and were completed [ . . . ].  A solution of ply U and Poly A, which lose pains[?] to form all and triple stranded felicies[?], had no content[?], whatsoever, suggesting that single -- strandedness is a necessary requisite for adults[?]. The incorporation is also extremely specific, We've tested every C14 amino acid individually and poly 4 stimulates the incorporation only of phenylene[?]. [END PAGE TWO] [BEGIN PAGE THREE] The mechanism of the incorporation process is under current investigation.  Attempts are also being made to determine other letters of the code.  In summary, a stable, cell-free system has been obtained from E. coli in which incorporation of amino acids into protein was both [ . . . ] and 100,000 kg [ . . . ] solution were [ . . . ] for [ . . . ].  Phenylene[?] incorporation also[?] required puromycin, chloramphenicol and Rnase.  The product of the reaction was partially characterizing[?] and it appears to be polyphemyoline[?].  These results are in press and will be published shortly in the Power[?] of the Nat [ . . . ].  Poly-U appears to function in this system as a synthetic template, or messenger RNA.  One or more [ . . . ] [ . . . ] [ . . . ], then, appear if [ . . . ] code for polyphemyoline[?]", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hn2m.f5fq_xxif", "00000000-0000-0000-0826-6B9CD163CC8E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "S[ynthetic] RNA codewords", "101584910X149", null, "1963", "[ca. 1963]", "During the period from 1962-1966, Marshall Nirenberg and his research team were devoted to translating the code by determining the various combinations of nucleotides in the RNA codons.  These handwritten notes provide a record of some of the possible nucleotide combinations that were worked out on paper.", "Laboratory notes", "Codon", "Translating the Code of Life and the Nobel Prize, 1962-1968", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "a *% es [ to~ BQ : Se i 3S Eo . PSA Pe ARG AGE , 666GU & | [a 2 Y 6 AA Ky AG K 6 K 6 © au > 43     BRU AAC ~AUA UUs UU uu UR yu UuQ& @ue uu (eg) Wee “eS uufa}", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-866j_75cb.t4fv", "00000000-0000-0000-A99C-AD65E63A31BC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Telegram from Sten Friberg, Karolinska Institute to Marshall W. Nirenberg", "101584910X15", null, "1968", "16 October 1968", "Telegram from the Karolinska Institute notifying Marshall Nirenberg of his receipt of the Nobel Prize.", "Telegrams", "Nobel Prize", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Reproduced with permission of Sten Friberg, III.", "Copyright may apply", null, null, "Dr Marshall Nirenberg NIH Bethesda MD Karoka Institutet has decided to award the Nobel Prize in Physiology in Medicine for 1968 jointly to yourself to Robert Holley and to Gobind Khrana for your discoveries concerning the interpretation of the gnetic code and its function in protein synthesis Fribg Rector", "Karolinska Institutet ; Friberg, Sten", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kqiw_hk4k_8547", "00000000-0000-0000-843A-52FF46A7B9CB", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Popular Comparison - Molecular Commentary of Cell as Man's Communication in the Future", "101584910X150", null, "1967", "24 October 1967", "In this typed draft of a speech, including handwritten corrections and additions, Nirenberg characterizes RNA as a \"robot.\"  The code is the \"language of civilization\"--man has grown to understand that language has built libraries with information, but has only just begun to translate the text.  Nirenberg warns that the texts available contain precise instructions for construction of microscopic machines that are more advanced than the chemical and industrial technology of man, and therefore require a great deal more work before the meaning of it all will be revealed.", "Speeches, Drafts (documents)", "Communication", "Translating the Code of Life and the Nobel Prize, 1962-1968", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "10/24/67 Belt #2 Man recently has begun to communicate with another world, a world with a very ancient and an extraordinarily organized civilization, quite different from the civilization that he is accustomed to.  The customs, strategies, and basic principles of the society are, for the most part, largely unknown.  This civilization is a very ancient one, probably one to three billion years old. It is a highly organized, complex civilization.  Every individual uses essentially the same language.  Man now understands this language; he has found libraries which contain a truly vast amount of information, and has begun, but only barely, to translate the text.  Millions of texts are available which contain the precise instructions for the construction of very complicated and remarkably ingenious microscopic machines which are required to build a wide variety of materials which are used by each individual.  Each text is rewritten in a slightly different form by a scribe and the new text then is taken by robots who follow each instruction blindly; and with the aid of ancillary machinery, fabricate machines which are needed to create the things that are needed by every individual.  In short, the texts describe a chemistry and an associated industrial technology which is quite different and far more advanced than that of man's. Man now understands the language of the civilization, has written quite elementary messages in the form that robots understand, and in such texts has communicated directly with the robots.  The robots read and faithfully carry out the instructions. Man has obtained many millions of texts from this civilization but, thus far, has translated only a handful of them.  He understands and can use some of the machinery of the civilizations.  He has not yet had time to inventory the immense libraries at his disposal, much less translate the texts.    He understands the construction of only a few machines but has obtained hundreds of kinds of machines and can use, to some extent, many of the machines.  For example, man can use a machine which rewrite the texts in the form that the robots understand.  The major problem is sorting out the machinery, the texts, and, of course, learning how to use the machines.  However, man is rapidly learning how to write texts with the relatively primitive tools which are at his disposal.  He probably will be able to write meaningful texts within five years.  It probably will be much easier to do this than to separate one text from another.  It is difficult to separate one text from another in the libraries that are available because the instructions for millions of machines may be encoded on a single tape.  Therefore, the problem of separating instructions for one machine or the other seems extremely difficult.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bi6r_8hbe_f96j", "00000000-0000-0000-FEFE-EA33817B2F04", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on the future of science", "101584910X151", "101584910X121", "1966", "[1966]", "Nirenberg's notes read \"What may be expected in the future? One hesitates to predict future events, but one must.  As an eminent virologist, Salvatore Luria, has stated 'the impact of science on human affairs imposes on its practitioners an inescapable responsibility.\"  Nirenberg expressed the meaning of this responsibility in the now famous Science editorial, \"Will Society Be Prepared?\"", "Notes", null, "Transition to Neurobiology, 1965-1969", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ubzb~d698.yp2y", "00000000-0000-0000-FE6D-B7C73D114C3B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Draft of acceptance speech given upon receiving the Gairdner Award", "101584910X152", null, "1967", "1967", "In the draft for his acceptance speech for the award granted by the Gairdner Foundation, a non-profit corporation devoted to the recognition of outstanding achievement in the field of biomedical research, Nirenberg asserts that a driving force behind science \"is that the individual strives for truth and [publicly] accepts full responsibility for his work.\"", "Speeches, Drafts (documents)", "Awards and Prizes", "Translating the Code of Life and the Nobel Prize, 1962-1968", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Dr., Mr. Gairdner, Honored Guests, Ladies and Gentlemen. I deeply appreciate the honor that the Gairdner Foundation has conferred upon me.  Previous recipients of the Gairdner Award, by their accomplishments have contributed much to medical science.  To be included in this group is a recognition which I shall always remember, and for which I am grateful to you. In ancient times in the Orient, an artist always used an assumed name to sign the pictures that he painted.  The reason was that no on other than the artist would know who created the picture, and so people then could never directly criticize or reward the artist for his work.  The artist then would always be free -- he could never be punished directly, and he could never be corrupted by praise. Certainly the concepts of freedom and creation for the sake of creation are ideals to cherish.  But contrast this philosophy with the fundamental philosophy of science.  Certainly one of the most magnificent aspects of science is the devotion of the practitioners of science to truth.  The essential factor in this process is that each individual accepts personal responsibility for the correctness of his work.  Every scientist labors long and hard to be sure that his findings and his interpretation of facts are correct.  For his colleagues judge his scientific ability on the basis of what he creates or what he strives to create. The basic values of the scientific community are simple and good.   The very heart of the philosophy though is that the individual strives for truth and publicly accepts full responsibility for his work. The individual then is freely available to everyone in the scientific community and can be punished by criticism or rewarded by praise.  So constructive criticism plays an essential role in the scientific community and praise, which is far less frequent than criticism is also needed for balance. As for the possibility that the scientist can be corrupted by praise, I can only say that I do not know anyone who has been adversely affected by praise.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6s4e.krkm~z6dj", "00000000-0000-0000-8F36-8F731AAFE51B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Draft of speech to the American College of Physicians and Surgeons", "101584910X153", null, "1967", "[ca. 1967]", "The top right corner of document reads: \"American College Physicians and Surgeons San Francisco\"  In this handwritten draft of a speech given at the ACPS circa 1967, Nirenberg covers the topics of protein synthesis, approaches to defining nucleotide sequences of codons, and the accuracy, logic, and plasticity of the code.", "Speeches, Drafts (documents)", "Codon", "Translating the Code of Life and the Nobel Prize, 1962-1968", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "American College Physicians and Surgeons San Francisco 1. Two experimental approaches were devised to translate the codes. We were able to down that Protein synthesis is dependent upon messenger RNA and that add-free protein synthesis[?] could be directed. By synthetic RNA[?] [END PAGE ONE] [BEGIN PAGE TWO] These approaches were used to define nucleotide sequences of codes[?].  The results have shown that the code is a logical, flexible code.  The translation can be attained by modifying part of the translation apparatus, such as TRNA, coordinating [?] enzymes [ . . . ] and [ . . . ] Accuracy is achieved by arranging the language so that frequent errors either have similar meetings or server special functions. So examples of the Rogue and plasticity of the code will be shown in the next few slides.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-equr_j27w-ybka", "00000000-0000-0000-ABB8-2B889C51B3D0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Preliminary notes for speech given at the Lasker Awards luncheon", "101584910X154", "101584910X155", "1968", "[ca. 21 November 1968]", "Marshall Nirenberg won the Lasker Award for medical research in 1968 for his contributions to deciphering the genetic code.  In these handwritten notes for his acceptance speech, Nirenberg asserts the new knowledge in molecular genetics \"will have a marked effect\" on the \"eternal questions of who we are and where we are going,\" but not immediately as more basic information is needed.  He predicts a new area of research in genetic research will emerge in the last part of the twentieth century, including work on molecular evolution and genetic surgery.", "Speeches, Drafts (documents)", "Genes", "Translating the Code of Life and the Nobel Prize, 1962-1968", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "1. The field of molecular genetics has advanced with incredible rapidity during the last 10 to 15 years.  You may wonder whether the new knowledge will have any effect upon the eternal question of who we are and where we are going. I think it will have a marked effect, but no immediately 2. Much more basic information must first be obtained. The initial objective is to understand the chemistry of the normal cell.  This leads naturally to an understanding of the aberrant cell, and is obviously a great importance to medicine. Once can predict that a new era of research will emerge [END PAGE ONE] [BEGIN PAGE TWO] 3. during the next 25 years, -- that of molecular evolution in which the effects of synthetic genes upon the economy of the cell will be explored. We know that the machinery of the cell will accept and follow any instructions written is the appropriate molecular language.  The language has been deciphered 4. and it seems probable that most if not all forms of life on this planet use the same language with minor variations.  Genetic surgery is a reality, both microorganisms and with mammalian cells.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-683j_p92z.fbvh", "00000000-0000-0000-76AE-3AC546D584D2", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Lasker Lunch Remarks", "101584910X155", "101584910X154", "1968", "21 November 1968", "These remarks are transcribed and are an extension of the views presented in Marshall Nirenberg's \"Preliminary notes for speech given at the Lasker Awards luncheon.\"  Nirenberg adds, \"The major point is that it should be possible in the future to synthesize genetic messages and use them to program cells.  It seems likely that man eventually be able to instruct his own cells, and ultimately, influence his own evolution.\"", "Speeches, Notes", "Genes", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "\\\\ LASKER LUNCH REMARKS-NOV.21, 1968 THE FIELD OF MOLECULAR GENETICS HAS ADVANCED WITH INCREDIBLE RAPIDITY DURING THE LAST 10 TO 15 YEARS. YOU MAY WONDER WHETHER THE NEW KNOWLEDGE WILL HAVE ANY EFFECT UPON THE ETERNAL QUESTIONS OF WHO WE ARE AND WHERE WE ARE GOING. I THINK IT WILL HAVE A MARKED EFFECT, BUT NOT AN IMMEDIATE ONE, MUCH MORE BASIC INFORMATION MUST FIRST BE OBTAINED. THE INITIAL OBJECTIVE IS TO UNDERSTAND THE CHEMISTRY OF A NORMAL CELL, THIS LEADS NATURALLY TO AN UNDER- STANDING OF THE ABERRANT CELL, AND IS OBVIOUSLY OF GREAT IMPORTANCE TO MEDICINE, ONE CAN PREDICT THAT A NEW AREA OF RESEARCH WILL EMERGE DURING THE NEXT 25 YEARS--THAT OF MOLECULAR EVOLUTION IN WHICH THE EFFECTS OF SYNTHETIC GENES UPON THE ECONOMY OF THE CELL WILL BE EXPLORED. 2 Qin KNOW THAT THE MACHINERY OF THE CELL WILL ACCEPT AND FOLLOW ANY INSTRUCTIONS WRITTEN IN THE APPROPRIATE MOLEC- ULAR LANGUAGE, THE LANGUAGE HAS BEEN DECIPHERED AND IT SEEMS PROBABLE THAT MOST, IF NOT ALL, FORMS OF LIFE ON THIS PLANET USE THE SAME LANGUAGE WITH MINOR VARIATION, GENETIC SURGERY IS A REALITY, BOTH WITH MICROORGANISMS AND WITH MAMMALIAN CELLS, DNA FROM ONE STRAIN OF CELLS CAN BE USED TO PROGRAM A DIFFERENT CELL STRAIN, AND THE PROGRAM IS INHERITED BY THE DESCENDANTS OF THE RECIPIENTS, SIMPLE GENETIC MESSAGES CAN BE SYNTHESIZED CHEMICALLY LARGELY DUE TO THE PIONEERING STUDIES OF DR, KHORANA, THE MAJOR POINT IS THAT IT SHOULD BE POSSIBLE IN THE FUTURE TO SYNTHESIZE GENETIC MESSAGES AND USE THEM TO PROGRAM CELLS, IT SEEMS LIKELY THAT MAN EVENTUALLY WILL BE ABLE TO INSTRUCT HIS OWN CELLS, AND ULTIMATELY, INFLUENCE HIS OWN BIOLOGICAL EVOLUTION,", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3yhs~pxz3~pm2c", "00000000-0000-0000-4443-5449D547A4FC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Draft of \"Genetic Versus Neural Information Processing Systems\"", "101584910X156", "101584910X180,101584910X181", "1969", "27 March 1969", "This draft outlines Marshall Nirenberg's \"speculations\" as to the analogous relationship between the genetic code and the neural code.   This talk was Nirenberg's most complete account of his considerations of the neural code and its relationship to the genetic code.  When Nirenberg was asked to participate in a symposium on mental retardation in 1969, he insisted that his speech not be published as it was overly speculative and therefore did not meet the standards of his other scientific work.  This draft of this speech compares the genetic code that directs protein synthesis with the neural code that directs neural processes.  Topics covered in the talk include information storage and retrieval, questions of origins and evolution, and molecular-level mechanisms.", "Speeches, Drafts (documents)", "Genetic Code", "Transition to Neurobiology, 1965-1969", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "gl iS | becca Ye ria a Dee I, or PTs 2, <a 3 for | (, mA, a “ Be a Se pee 7p LO of | banat ws Sear. Cee cel . dfa Adri f arn PO ’ p~< DN, | & oki qT Kr hee. (go [same y ~ 3,700 A’ f ie t~ Yeo AR (pA SS 00 x0\" AP of Qe Hess fe Aad oA ts / ayy! ee ky ae a ate ke, he. vfs ec ak 30 Le Pf tf bee, f ¢ ‘Fee Xa. [ee he S Ns Ti, 4, Cato F a folly a % (epee. ek A", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dk4z_u34w.5p5c", "00000000-0000-0000-5EDB-B9B4F3E7CD97", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Bibliography of publications written by Dr. Nirenberg", "101584910X158", null, "2000", "19 January 2000", "This bibliography was a part of Marshall Nirenberg's curriculum vitae.  It includes more than 175 publications from the period 1956-1999.", "Bibliographies", null, "Biographical Information", "16", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "10. ll. 12. BIBLIOGRAPHY Marshall Nirenberg Nirenberg, M.W. and Hogg, J.F.: On the mode of hexose uptake by ascites tumor cells. J. Am. Chem. Soc., 78 6210- 6211 (1956). Nirenberg, M.W. and Hogg, J.F.: Inhibition of anaerobic glycolysis in Ehrlich ascites tumor cells by 2-deoxy-D-glucose. Cancer Research, 18: 518-521 (1958). Nirenberg, M.W. and Hogg J.F.: Hexose transport in ascites tumor cells. J. Am. Chem. Soc., 80: 4407-4412 (1958). Nirenberg, M.W.: An enzymic defect in ascites-tumor cells. Biochem. Biophys. Acta, 30: 203-204 (1958). Nirenberg, M.W.: A biochemical characteristic of ascites tumor cells. J. Biol. Chem., 234: 3088-3093 (1959). Nirenberg, M.W. and Jakoby, W.B.: Enzymatic utilization of g- hydroxybutyric acid. J. Biol. Chem., 235: 954-960 (1960). Nirenberg, M.W.: The induction of two enzymes by one inducer; a test case for shared genetic information. Fed. Proc., 19: 42 (1960). Nirenberg, M.W. and Jakoby, W.B.: On the sites of attachment and reaction of aldehyde dehydrogenases. Proc. Natl. Acad. Sci. USA, 46: 206-212 (1960). Nirenberg, M.W. and Jakoby, W.B.: Constraints in the determination of active-centre topography. Nature, 188: 747-748 (1960). Nirenberg, M.W. and Matthaei, H.: The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic template RNA. in Biological Structure and Function at the Molecular Level, Engelhardt, V.A. (Ed.), The MacMillan Co., New York, Proc. Fifth Int. Cong. Biochem., Vol. I, Moscow, 1961, pp. 184-189. Matthaei, H. and Nirenberg, M.W.: The dependence of cell-free protein synthesis in E. coli upon RNA prepared from ribosomes. Biochem. Biophys. Res. Commun., 4: 404-408 (1961). Matthaei, J.H. and Nirenberg, M.W.: Characteristics and stabilization of DNAase sensitive protein synthesis in E. coli extracts. Proc. Natl. Acad. Sci. USA, 47: 1580-1588 (1961).   1/19/00 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Nirenberg, M.W. and Matthaei, J.H.: The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. USA, 47: 1588-1602 (1961). Martin, R.C., Matthaei, J.H., Jones, O.W. and Nirenberg, M.W.: Ribonucleotide composition of the genetic code. Biochem. Biophys. Res. Commun., 6: 410-414 (1961/62). Matthaei, J.H., Jones, O.W., Martin, R.G. and Nirenberg, M.W.: Characteristics and composition of RNA coding units. Proc. Natl. Acad. Sci. USA, 48: 666-677 (1962). Tsugita, A., Fraenkel-Conrat, H., Nirenberg, M.W. and Matthaei, J.H.: Demonstration of the messenger role of viral RNA. Proc. Natl. Acad. Sci. USA, 48: 846-853 (1962). Barondes, S.H. and Nirenberg, M.W.: Fate of synthetic polynucleotide directing cell-free protein synthesis. I. Characteristics of degradation. Science, 138: 810-813 (1962). Barondes, S.H. and Nirenberg, M.W.: Fate of synthetic polynucleotide directing cell-free protein synthesis. II. Association with ribosomes. Science, 138: 813-817 (1962). Nirenberg, M.W., Matthaei, J.H. and Jones, O.W.: An intermediate in the biosynthesis of polyphenylalanine directed by synthetic template RNA. Proc. Natl. Acad. Sci. USA, 48: 104- 109 (1962). Jones, O.W., Jr. and Nirenberg, M.W.: Qualitative survey of RNA codewords. Proc. Natl. Acad. Sci. USA, 48: 2115-2123 (1962). Nirenberg, M.W., Matthaei, J.H., Jones, O.W., Martin, R.G. and Barondes, S.H.: Approximation of genetic code via cell-free protein synthesis directed by template RNA. Fed. Proc. Symp., 22: 55-61 (1963). Nirenberg, M.W.: Cell-free protein synthesis directed by messenger RNA. in Methods in Enzymology, Eds. S.P. Colowick and N.A. Kaplan (1963), Academic Press, New York, Vol. 6, pp. 17- 23. Nirenberg, M.W. and Jones, O.W., Jr.: The current status of the RNA code in Symposium on Informational Macromolecules, H. Vogel (Ed.), 1963, Academic Press, New York, New York, pp. 451-465. Singer, M.F., Jones, O.W. and Nirenberg, M.W.: The effect of secondary structure on the template activity of polyribonucleotides. Proc. Natl. Acad. Sci. USA, 49: 392-399 (1963). 1/19/00 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. Nirenberg, M.W., Jones, O.W., Leder, P., Clark, B.F.C., Sly, W.S. and Pestka, S.: On the coding of genetic information. Cold Spring Harbor Symp. on Quant. Biol., 28: 549-557 (1963). Leder, P., Clark, B.F.C., Sly, W.S., Pestka, S. and Nirenberg, M.W.: Cell-free peptide synthesis dependent upon synthetic oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA, 50: 1135- 1143 (1963). Nirenberg, M.W.: The Genetic Code: II. Scientific American, 208: 80-94 (1963). Nirenberg, M.W.: Nucleic acids in relation to the coding of genetic information in Brain Function, Vol. II: RNA and Brain Function; Memory and Learning. Mary A.B. Brazier (Ed.). UCLA Forum held in 1962, Univ. of Calif. Press, Berkeley and Los Angeles, pp. 5-28, (1964). Bryne, R., Levin, J.C., Bladen, H.A. and Nirenberg, M.W.: The in vitro formation of a DNA-ribosome complex. Proc. Natl. Acad. Sci. USA, 52: 140-148 (1964). Eiserling, F., Levin, J.G., Byrne, R., Karlsson, U., Nirenberg, M.W. and Sj6 strand, F.S.: Polyribosomes and DNA-dependent amino acid incorporation in Escherichia coli extracts. J. Mol. Biol., 10: 536-540 (1964). Nirenberg, M.W. and Leder, P.: RNA codewords and protein synthesis. I. The effect of trinucleotides upon the binding of SRNA to ribosomes. Science, 145: 1399-1407 (1964). Leder, P. and Nirenberg, M.W.: RNA codewords and protein synthesis. II. Nucleotide sequence of a valine RNA codeword. Proc. Natl. Acad. Sci. USA, 52: 420-427 (1964). Leder, P. and Nirenberg, M.W.: RNA codewords and protein synthesis. III. On the nucleotide sequence of a cysteine and a leucine RNA codeword. Proc. Natl. Acad. Sci., 52: 1521-1529 . (1964). Nirenberg, M.W.: Protein synthesis and the RNA code. in Harvey Lectures, Series 59, pp. 155-185 (1964). Aach, H.G., Funatsu, G., Nirenberg, M.W. and Fraenkel-Conrat, H.: Further attempts to characterize products of TMV-RNA- directed protein synthesis. Biochemistry, 3: 1362-1366 (1964). Bernfield, M.R. and Nirenberg, M.W.: RNA codewords and protein synthesis. IV. The nucleotide sequences of multiple codewords for phenylalanine, serine, leucine and proline. Science, 147: 479-484 (1965). 1/19/00 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. Pestka, S., Marshall, R. and Nirenberg, M.W.: RNA codewords and protein synthesis. V. Effect of streptomycin on the formation of ribosome sRNA complexes. Proc. Natl. Acad. Sci., 53: 639-646 (1965). Bladen, H.A., Byrne, R., Levin, J.G. and Nirenberg, M.W.: An electron microscopic study of a DNA-ribosome complex formed in vitro. J. Mol. Biol., 11: 78-83 (1965). Millar, D.B.S., III, Cukier, R. and Nirenberg, M.W.: Interaction of E. coli ribosomal RNA with synthetic polynucleotides. Sedimentation properties, and thermal stability as measured by fluorescence polarization. Biochemistry, 4: 976-985 (1965). Trupin, J.S., Rottman, F.M., Brimacombe, R.L.C., Leder, P., Bernfield, M.R.and Nirenberg, M.W.: RNA codewords and protein synthesis. VI. On the nucleotide sequence of degenerate codeword sets for isoleucine, tryosine, asparagine, and lysine. Proc. Natl. Acad. Sci. USA, 53: 807-811 (1965). Nirenberg, M., Leder, P., Bernfield, M., Brimacombe, R., Trupin, J., Rottman, F. and O'Neal, C.: RNA codewords and protein synthesis. VII. On the general nature of the RNA code. Proc. Natl. Acad. Sci., 53: 1161-1168 (1965). Brimacombe, R., Trupin, J., Nirenberg, M., Leder, P., Bernfield, M. and Jaouni, T.: RNA codewords and protein synthesis. VIII. Nucleotide sequences of synonym codons for arginine, valine, cysteine, and alanine. Proc. Natl. Acad. Sci. USA, 54: 954-960 (1965). Kellogg, D.A., Doctor, B.P., Loebel, J.E. and Nirenberg, M.W.: RNA codons and protein synthesis. IX. Synonym codon recognition by multiple species of valine-, alanine-, and methionine-sRNA. Proc. Natl. Acad. Sci., 55: 912-919 (1966). Trupin, J., Dickerman, H., Nirenberg, M. and Weissbach, H.: Formylation of amino acid analogues of methionine sRNA. Biochem. Biophys. Res. Common., 24: 50~55 (1966). Jones, O.W., Jr. and Nirenberg, M.W.: Degeneracy in the amino acid code. Biochem. Biophys. Acta., 119: 400-406 (1966). Grunberger, D., O'Neal, C. and Nirenberg, M.: Stimulation of amino acid incorporation into protein by polyuridylic-8- azaguanylic acid. Biochem. Biophys. Acta., 119: 581-585 (1966). Anderson, W.F. and Nirenberg, M.: The genetic code and protein synthesis. in Handbook of Biochem. and Biophys., H.C. Damm, P.kK. Besch, A.J. Goldwyn (Eds.), Cleveland and New York, World Publishing Company, pp. 96-108 (1966). 1/19/00 48. 49. 50. 51. 52. 53. 54A. 54B. 55. 56. 57. 58. Pestka, S. and Nirenberg, M.: Regulatory mechanisms and protein synthesis recognition. X. Codon recognition of 30 S$ ribosomes. J. Mol. Biol., 21: 145-171 (1966). Rottman, F. and Nirenberg, M.: RNA codons and protein synthesis. XI. Template activity of modified RNA codons. J. Mol. Biol., 21: 555-570 (1966). Nirenberg, M., Caskey, T., Marshall, R., Brimacombe, R., Kellogg, D., Doctor, B., Hatfield, D., Levin, J., Rottman, F., Pestka, S., Wilcos, M. and Anderson, F.: The RNA code and protein synthesis. Cold Spring Harbor Symp. on Quant. Biol., 31: 11-24 (1966). Pestka, S. and Nirenberg, M.: Codeword recognition of 30 S$ ribosomes. Cold Spring Harbor Symp. on Quant. Biol., 31: 641-656 (1966). Marshall, R.E., Caskey, C.T. and Nirenberg, M.: RNA codewords and protein synthesis. XII. Fine structure of RNA codewords recognized by bacterial, amphibian, and mammalian transfer RNA. Science, 155: 820-826 (1967). Nirenberg, M.: The genetic code in The Neurosciences, Quarton, G.C., Melnechuk, T. and Schmitt, F.O. (Eds.), New York, The Rockefeller University Press, pp. 143-152 (1967). Nirenberg, M.: Man's power to shape his own biologic destiny - will society be prepared to use it wisely? Quarterly Bulletin of Research Corporation, Spring 1967. (Part reproduced as an editorial in Science). Nirenberg, M.: Will society be prepared? Science, 157: 633 (1967). Nirenberg, M.W., Caskey, C.T. and Levin, J.G.: RNA codon recognition. Seventh International Congress of Biochemistry, (Japan) Suppl. 1, p. 1068 (1967). Kano-Sueoka, T., Nirenberg, M. and Sueoka, N.: Effect of bacteriophage infection upon the specificity of leucine transfer RNA for RNA codewords. J. Mol. Biol., 35, 1-12 (1968). Levin, J.G. and Nirenberg, M.: RNA codons and protein synthesis. XIII. RNA codon recognition by deacylated tRNA and aminoacyl tRNA. J. Mol. Biol., 34: 467-480 (1968). Caskey, C.T., Beaudet, A. and Nirenberg, M.: RNA codons and protein synthesis. 15. Dissimilar responses of mammalian and bacterial transfer RNA fractions to mRNA codons. J. Mol. Biol., 37: 99-118 (1968). 1/19/00 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. Wilcox, M. and Nirenberg, M.: Transfer RNA as a cofactor coupling amino acid synthesis with that of protein. Proc. Natl. Acad. Sci. USA, 61: 229-236 (1968). Caskey, C.T., Tompkins, R., Scolnick, E., Caryk, T. and Nirenberg, M.: Sequential translation of trinucleotide codons for the initiation and termination of protein synthesis. Science, 162: 135-138 (1968). Scolnick, E., Tompkins, R., Caskey, T. and Nirenberg, M.: Release factors differing in specificity for terminator codons. Proc. Natl. Acad. Sci. USA, 61: 768-774 (1968). Nirenberg, M.: Genetic Memory. J. Am. Med. Assn., 206: 1973- 1977 (1968). Nirenberg, M.: The Genetic Code in Les Prix Nobel en 1968. Nobel Foundation, Stockholm, P.A. Norstedt and S6 ner, pp. 221- 241, (1969). Nirenberg, M.W.: On the translation of the genetic code in Biology and Physical Sciences, Devons, S. (Ed.), N.Y., Columbia University Press, 1969, pp. 57-73, (1969). Marshall, R. and Nirenberg, M.: RNA codons recognized by transfer RNA from amphibian embryos and adults. Developmental Biology, 19: 1-11 (1969). Gartland, W.J., Ishida, T., Sueoka, N. and Nirenberg, M.W.: Coding properties of two conformations of tryptophanyl-tRNA in Escherichia coli. J. Mol. Biol., 44: 403-413 (1969). Nelson, P., Ruffner, W. and Nirenberg, M.: Neuronal tumor cells with excitable membranes grown in vitro. Proc. Natl. Acad. Sci. USA, 64: 1004-1010 (1969). Nirenberg, Von M.: Der genetische code (Nobel-Vortrag). Angew. Chemie, 81: 1017-1027 (1969). Seeds, N.W., Gilman, A.G., Amano, T. and Nirenberg, M.W.: Regulation of axon formation by clonal lines of a neuronal tumor. Proc. Natl. Acad. Sci. USA, 66: 160-167 (1970). Nirenberg, M.: The flow of information from gene to protein in Aspects of Protein Biosynthesis, Part A., Anfinsen, C.B., Jr., (Ed.), New York, Academic Press, Inc., pp. 215-246, 1970. Blume, A., Gilbert, F., Wilson, S., Farber, J., Rosenberg, R. and Nirenberg, M.: Regulation of acetylcholinesterase in neuroblastoma cells. Proc. Natl. Acad. Sci. USA, 67: 786-792 (1970). 1/19/00 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. Smrt, J., Kemper, W., Caskey, T. and Nirenberg, M.: Template activity of modified terminator codons. J. Biol. Chem., 245: 2753-2757 (1970). Kan, J., Nirenberg, M.W. and Sueoka, N.: Coding specificity of Escherichia coli leucine transfer ribonucleic acids and effect of bacteriophage T2 infection. J. Mol. Biol., 52: 179-193 (1970). Hatfield, D. and Nirenberg, M.: Binding of radioactive oligonucleotides to ribosomes. Biochem., 10: 4318-4323 (1971). Minna, J., Nelson, P., Peacock, J., Glazer, D. and Nirenberg, M.: Genes for neuronal properties expressed in neuroblastoma x L cell hybrids. Proc. Natl. Acad. Sci. USA, 68: 234-239 (1971). Gilman, A.G. and Nirenberg, M.: Effect of catecholamines on the adenosine 3':5'-cyclic monophosphate concentrations of clonal satellite cells of neurons. Proc. Natl. Acad. Sci. USA, 68: 2165-2168 (1971). Gilman, A.G. and Nirenberg, M.: Regulation of adenosine 3':5'- cyclic monophosphate metabolism in cultured neuroblastoma cells. Nature, 234: 356-358 (1971). Amano, T., Richelson, E. and Nirenberg, M.: Neurotransmitter synthesis by neuroblastoma clones. Proc. Natl. Acad. Sci. USA, 69: 258-263 (1972). Peacock, J., Minna, J., Nelson, P. and Nirenberg, M.: Use of aminopterin in selecting electrically active neuroblastoma cells. Experimental Cell Research, 73: 367-377 (1972). Minna, J., Glazer, D. and Nirenberg, M.: Genetic dissection of neural properties using somatic cell hybrids. Nature New Biology, 235: 225-231 (1972). Wilson, S.H., Schrier, B.K., Farber, J.L., Thompson, E.J., Rosenberg, R.N., Blume, A.J. and Nirenberg, M.W.: Markers for gene expression in cultured cell from the nervous system. J. Biol. Chem., 247: 3159-3169 (1972). Vogel, Z., Sytkowsky, A.J. and Nirenberg, M.W.: Acetylcholine receptors of muscle grown in vitro. Proc. Natl. Acad. Sci. USA, 69: 3180-3184. Thompson, E.J., Wilson, S.H., Schuette, W.H., Whitehouse, W.C. a nd Nirenberg, M.W.: Measurement of the rate and velocity of movement by single heart cells in culture. Am. J. of Cardiology, 32: 162-166 (1973). 1/19/00 84. 85. 86. 87. 88A. 88B. 88C. 89. 90. 91. 92. 93. Catterall, W.A. and Nirenberg, M.: Sodium uptake associated with activation of action potential ionophores of cultured neuroblastoma muscle cells. Proc. Natl. Acad. Sci. USA, 70: 3759-3763 (1973). Sytkowsky, A.J., Vogel, Z. and Nirenberg, M.W.: Development of acetylcholine receptor clusters on cultured muscle cells. Proc. Natl. Acad. Sci. USA, 70: 270-274 (1973). Greene, L.A., Sytkowsky, A.J., Vogel, Z. and Nirenberg, M.W.: a-Bungarotoxin used as a probe for acetylcholine receptors of cultured neurons. Nature, 243: 163-166 (1973). Chalazonitis, A., Greene, L.A. and Nirenberg, M.: Electrophysiological characteristics of chick embryo sympathetic neurons in dissociated cell culture. Brain Research, 68: 235- 252 (1974). Schrier, B.K., Wilson, S.H., Nirenberg, M.: Cultured cell systems and methods for neurobiology in Methods in Enzymology, Vol. 32, Part B, pp. 765-783, Fleischer, S. and Packer, L. (Eds.). Academic Press, New York (1974). Hamprecht, B., Amano, T. and Nirenberg, M.: Choline acetyltransferase. Assay 2. Ibid. pp. 783-785 (1974). Richelson, E. and Nirenberg, M. Tyrosine hydroxylase assay. Ibid. pp. 785-788 (1974). Klee, W.A. and Nirenberg, M.: A neuroblastoma x glioma hybrid cell line with morphine receptors. Proc. Natl. Acad. Sci. USA, 71: 3474-3477 (1974). Breakefield, X.0. and Nirenberg, M.: Selection for neuroblastoma cells that synthesize certain transmitters. Proc. Natl. Acad. Sci. USA, 71: 2530-2533 (1974). Klee, W.A., Sharma, S.K. and Nirenberg, M.: Opiate receptors as regulators of adenylate cyclase. Life Sci., 16: 1869-1874 (1975), in The Opiate Narcotics., Neurochemical Mechanisms in Analgesia and Dependence. Goldstein, A. et al. (Eds.), Pergamon Press, New York, pp. 117-122 (1975). Thompson, E.J., Griffith, J.M., Schoenberg, D.G. and Nirenberg, M.W.: An improved method for extracellular recording of action potentials from single cultured neuroblastoma cells. Med. Biol. Eng., 13, 104-106 (1975). Sharma, S.K., Nirenberg, M. and Klee, W.A.: Morphine receptors as regulators of adenylate cyclase activity. Proc. Natl. Acad. Sci. USA, 72: 590-594 (1975). 1/19/00 94, 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. Sharma, S.K., Klee, W.A. and Nirenberg, M.: Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc. Natl. Acad. Sci. USA, 72: 3092-3096 (1975). Matsuzawa, H. and Nirenberg, M.: Receptor-mediated shifts in cGMP and cAMP levels in neuroblastoma cells. Proc. Natl. Acad. Sci. USA, 72: 3472-3476 (1975). Nirenberg, M.W.: Coding of neural information by neuroblastoma cells. in Talwar, G.P. (Ed.): Regulation of Growth and Differentiated Function in Eukaryote Cells. New York, Raven Press, pp. 537-539 (1975). Greene, L.A., Shain, W., Chalazonitis, A., Breakefield, X., Minna, J., Coon, H.G. and Nirenberg, M.: Neuronal properties of hybrid neuroblastoma x sympathetic ganglion cells. Proc. Natl. Acad. Sci. USA, 72: 4923-4927 (1975). Nelson, P., Christian, C. and Nirenberg, M.: Synapse formation between clonal neuroblastoma x glioma hybrid cells and striated muscle cells. Proc. Natl. Acad. Sci. USA, 73: 123-127 (1976). Vogel, Z. and Nirenberg, M.: Localization of acetylcholine receptors during synaptogenesis in retina. Proc. Natl. Acad. Sci. USA, 73: 1806-1810 (1976). Vogel, Z. Daniels, M.P. and Nirenberg, M.: Synapse and acetylcholine receptor synthesis by neurons dissociated from retina. Proc. Natl. Acad. Sci. USA, 73: 2370-2374 (1976). Lampert, A., Nirenberg, M. and Klee, W.A.: Tolerance and dependence evoked by an endogenous opiate peptide. Proc. Natl. Acad. Sci. USA, 73: 3165-3167 (1976). Klee, W.A. and Nirenberg, M.: Mode of action of endogenous Opiate peptides. Nature, 263: 609-612 (1976). De Mello, F.G., Bachrach, U. and Nirenberg, M.: Ornithine and glutamic acid decarboxylase activities in the developing chick retina. J. Neurochem., 27: 847-851 (1976). Puro, D.G. and Nirenberg, M.: On the specificity of synapse formation. Proc. Natl. Acad. Sci. USA, 73: 3544-3548 (1976). Klee, W.A., Lampert, A. and Nirenberg, M.: Dual regulation of adenylate cyclase by endogenous opiate peptides in Kosterlitz, H. (Ed.): Opiates and Endogenous Opioid Peptides. Amsterdam, Elsevier/North-Holland Biomedical Press, 1976, pp. 153-159. Goldstein, A., Cox, B.M., Klee, W.A. and Nirenberg, M.: Endorphin from pituitary inhibits cyclic AMP formation in homogenates of neuroblastoma x glioma hybrid cells. Nature, 265: 362-363 (1977). 1/19/00 107. 108. 109. 110. lil. 112. 113. 114. 115. 116. 117. 118. Chalazonitis, A., Minna, J.D. and Nirenberg, M.: Expression and properties of acetylcholine receptors in several clones of mouse neuroblastoma x L cell somatic hybrids. Exp. Cell Res., 105: 269-280 (1977). Christian, C.N., Nelson, P.G., Peacock, J. and Nirenberg, M.: Synapse formation between two clonal cell lines. Science, 196: 995-998 (1977). Agarwal, N.S., Hruby, V.J., Katz, R., Klee, W. and Nirenberg, M.: Synthesis of leucine enkephalin derivatives: Structure- function studies. Biochem. Biophys. Res. Commun., 76: 129-135 (1977). Giagnoni, G., Sabol, S.L. and Nirenberg, M.: Synthesis of Opiate peptides by a clonal pituitary tumor cell line. Proc. Natl. Acad. Sci. USA, 74: 2259-2263 (1977). Sharma, S.K., Klee, W.A. and Nirenberg, M.: Opiate-dependent modulation of adenylate cyclase. Proc. Natl. Acad. Sci. USA, 74: 3365-3369 (1977). Puro, D.G., De Mello, F.G. and Nirenberg, M.: Synapse turnover: the formation and termination of transient synapses. Proc. Natl. Acad. Sci. USA, 74: 4977-4981 (1977). Sugiyama, H., Daniels, M.P. and Nirenberg, M.: Muscarinic acetylcholine receptors of the developing retina. Proc. Natl. Acad. Sci. USA, 74: 5524-5528 (1977). McGee, R., Simpson, P., Christian, C., Mata, M., Nelson, P. and Nirenberg, M.: Regulation of acetylcholine release from neuroblastoma x glioma hybrid cells. Proc. Natl. Acad. Sci. USA, 75: 1314-1318 (1978). Nathanson, N.M., Klein, W.L. and Nirenberg, M.: Regulation of adenylate cyclase activity mediated by muscarinic acetylcholine receptors. Proc. Natl. Acad. Sci. USA, 75: 1788-1791 (1978). Ruffolo, R.R., Jr., Eisenbarth, G.S., Thompson, J.M. and Nirenberg, M.: Synapse turnover: A mechanism for acquiring synaptic specificity. Proc. Natl. Acad. Sci. USA, 75: 2281-2285 (1978). MacDermot, J. and Nirenberg, M.: Turnover of opiate receptors in neuroblastoma x glioma hybrid cells. FEBS Lett., 90: 345-347 (1978). Nirenberg, M.: Studies on synapse formation and opiate dependence. National Cancer Institute Monograph, 48: 339-342 (1978). 1/19/00 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. Nelson, P.G., Christian, C.N., Daniels, M.P., Henkart, M., Bullock, P., Mullinax, D. and Nirenberg, M.: Formation of synapses between cells of a neuroblastoma x glioma hybrid clone and mouse myotubes. Brain Research, 147: 245-259 (1978). Christian, C.N., Nelson, P.G., Bullock, P., Mullinax, D. and Nirenberg, M.: Pharmacologic responses of cells of a neuroblastoma x glioma hybrid clone and modulation of synapses between hybrid cells and mouse myotubes. Brain Research, 147: 261-276 (1978). Eisenbarth, G.S., Ruffolo, R.R., Walsh, F.S. and Nirenberg, M.: Lactose sensitive lectin of chick retina and spinal cord. Biochem. Biophys. Res. Commun., 83: 1246-1252 (1978). Burgermeister, W., Kline, W.L., Nirenberg, M. and Witkop, B.: Comparative binding studies with cholinergic ligands and histrionicotoxin at muscarinic receptors of neural cell lines. J. Mol. Pharm., 14: 751-767 (1978). Sabol, S.L. and Nirenberg, M.: Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by a-adrenergic receptors, I. Inhibition of adenylate cyclase mediated by a- receptors. J. Biol. Chem., 254: 1913-1920 (1979). Sabol, S.L. and Nirenberg, M.: Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by a-adrenergic receptors, II. Long-lived increase of adenylate cyclase activity mediated by a-receptors. J. Biol. Chem., 254: 1921- 1926 (1979). MacDermot, J., Higashida, H., Wilson, S.P. Matsuzawa, H., Minna, J. and Nirenberg, M.: Adenylate cyclase and acetylcholine release regulated by separate serotonin receptors of somatic cell hybrids. Proc. Natl. Acad. Sci. USA, 76: 1135-1139 (1979). Eisenbarth, G.S., Walsh, F.S. and Nirenberg, M.: Monoclonal antibody to a plasma membrane antigen of neurons. Proc. Natl. Acad. Sci. USA, 76: 4913-4917 (1979). Klein, W.L., Nathanson, N. and Nirenberg, M.: Muscarinic acetylcholine receptors regulation by accelerated rate of receptor loss. Biochem. Biophys. Res. Commun., 90: 506-512 (1979). Wilkening, D. and Nirenberg, M.: Lipid requirement for long- lived morphine-dependent activation of adenylate cyclase of neuroblastoma x glioma hybrid cells. J. Neurochem., 34: 321-326 (1980). 1/19/00 129. 130. 131. 132A 132B 133. 134. 135. 136. Nirenberg, M., Wilson, S., Higashida, H., Thompson, J., Eisenbarth, G., Walsh, F., Rotter, A., Kenimer, J. and Sabol, S.: Synapse plasticity in Cellules, Nerveuses, Transmetteurs et Comportement, Edited by Rita Levi-Montalcini, Pontificiae Academiae Scientarvm Scripta Varia, 45: 123-127 (1980). McGee, R., Jr., Smith, C., Christian, C., Mata, M., Nelson, P. and Nirenberg, M.: A new capillary tube system for measuring the uptake and release of materials from cultured cells. Anal. Biochem., 101: 320-326 (1980). Wilkening, D., Sabol, S.L. and Nirenberg, M.: Control of opiate receptor-adenylate cyclase interactions by calcium ions and guanosine-5 -triphosphate. Brain Research, 189: 459-466 (1980). Trisler, G.D ., Schneider, M.D. and Nirenberg, M.: A topographic gradient of molecules in retina can be used to identify neuron position. Proc. Natl. Acad. Sci. USA, 78: 2145-2149 (1981). This paper also has been reproduced in the following: Trisler, G. David, Michael D. Schneider and Marshall Nirenberg: A topographic gradient of molecules in retina can be used to identify neuron position in Patterson, P.H. and Purves, D. (Ed.): Readings In Developmental Neurobiology. Cold Spring Harbor Laboratory, 1982, pp. 513-517. Trisler, G.D., Schneider, M.D. and Nirenberg, M.: Topographic gradient of cell-membrane molecules in avian neural retina detected with monoclonal antibody in Ocular Size and Shape: Regulation During Development, Hilfer, S.R. and Sheffield, J.B. (Eds.), Springer-Verlag, N.Y., p. 141-161 (1981). De Blas, A.L., Busis, N.A. and Nirenberg, M.: Monoclonal antibodies to synaptosomal membrane molecules in Monoclonal Antibodies Against Neuronal Antigens, McKay, R., Raff, M.C. and Reichardt, L.F. (Ed.), Cold Spring Harbor Reports in the Neurosciences Vol. 2, Cold Spring Harbor Laboratory, N.Y. (1981), pp. 181-191. Trisler, G.D., Schneider, M.D., Moskal, J.R. and Nirenberg, M.: A gradient of molecules in avian retina with dorsoventral polarity in Monoclonal Antibodies Against Neuronal Antigens, McKay, R., Raff, M.C. and Reichardt, L.F. (Ed.), Cold Spring Harbor Reports in the Neurosciences Vol. 2, Cold Spring Harbor Laboratory, N.Y. (1981), pp. 231-257. Kenimer, J. and Nirenberg, M.: Desensitization of adenylate cyclase to prostaglandin E, or 2-Chloroadenosine. Mol. Pharm., 20: 585-591 (1981) 1/19/00 137. 138. 139. 140. 141. 142. 143. 144. 145. Hirata, F., Del Carmine, R., Nelson, C.A., Axelrod, J., Schiffmann, E., Warabi, A., De Blas, A.L., Nirenberg, M., Manganiello, V., Vaughan, M., Kumagai, S., Green, I., Decker, J.L., and Steinberg, A.D.: Presence of autoantibody for phospholipase inhibitory protein, lipmodulin, in patients with rheumatic diseases. Proc. Natl. Acad. Sci. USA, 78: 3190-3194 (1981). Nirenberg, M. Synapse Plasticity in Proceedings of the Symposium On Trends In Bioassay Methodology: In Vivo, In Vitro And Mathematical Approaches, NIH Publication No. 82-2382, 1981, pp 201-207. Trisler, G.D., M.D. Schneider, J.R. Moskal and M. Nirenberg: Molecules that define a dorsal-ventral axis of retina can be used to identify cell position in Clayton, R.M. and Truman, D.E.S. (Ed.): Stability and Switching in Cellular Differentiation. Plenum Press, New York, 1982, pp. 123-127. Trisler, G.D., Schneider, M.D., Moskal, J.R., and Nirenberg, M. Molecules that Define a Dorsal-Ventral Axis of Retina can be Used to Identify Cell Position. in Problems of Normal and Genetically Abnormal Retinas. (Eds) Clayton, R.M., Haywood, J., Reading, H.W., and Wright, A. Academic Press, 61-63 (1982). Thompson, Jeffrey M., Eisenbarth, George S., Ruffolo, Jr., Robert R., and Nirenberg, M. Synapse Selection Based on Differences In Synapse Turnover International Journal of Developmental Neuroscience, 1: 25-30 (1983). Nirenberg, M., Wilson, S., Higashida, H., Rotter, A., Krueger, K., Busis, N., Ray, R., Kenimer, K., Adler, M. and Fukui, H. Synapse Formation by Neuroblastoma Hybrid Cells in Molecular Neurobiology. The 48th Cold Spring Harbor Symposium on Quantitative Biology, XLVIII: 707-715 (1983). Nirenberg, M., Wilson, S., Higbashida, H.,Rotter, A., Krueger, K., Busis, N., Ray, R., Kenimer, J.G., and Adler, M.: Modulation of Synapse Formation by Cyclic Adenosine Monophosphate. Science 222, 794-799. (1983). Nirenberg, M., Wilson, S., Higashida, H., Rotter, A., Krueger, K., Busis, N., Ray, R., Kenimer, J.G. and Adler, M. Modulation of Synapse Formation by Cyclic Adenosine Monophosphate. (1984) in Biotechnology and Biological Frontiers. (Abelson, Philip H., ed.) pp 442-453, American Association for the Advancement of Science, Wash., D.C. This article was published originally in Science 222, 794-799. (1983) Trisler, D., Grunwald, G., Moskal, J., Darveniza, P., and Nirenberg, M.: Molecules That Identify Cell Types Or Position In The Retina in Neuroimmunology, P. Behan and F. Spreafico eds., 89-97, Raven Press 11965984) - 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. DeBlas, A., Adler, M., Shih, M., Chiang, P., Cantoni, G., and Nirenberg, M.: Novel Inhibitors of CDP-Choline Synthesis, Action Potential Calcium Channels, and Stimuls-Secretion Coupling. Proc. Natl. Acad. Sci. 81: 4353-4357 (1984). Busis, N., Daniels, M.P., Bauer, H.C., Pudimat, P.A., Sonderegger, P., Schaffner, A.E. and Nirenberg, M.: Three Cholinergic Neuroblastoma Hybrid Cell Lines that Form Few Synapses on Myotubes are Deficient in Acetylcholine Receptor Aggregation Molecules and Large Dense Core Vesicles. Brain Research. 324: 201-210. (1984). Fredman, P., Magnani, J.L., Grunwald, G.B., Trisler, D., Nirenberg, M. and Ginsburg, V. Developmental Regulation of Brains and Retinas in Cellular and Pathological Aspects of Glycoconjugate Metabolism (H. Dreyfus, Ed.), Plenum Press, N.Y., October, 1984. Strauss, W.L. and Nirenberg, M.: Inhibition of Choline Acetyltrasferase by Monoclonal Antibodies. Journal of Neuroscience 5: 175-180 (1985). Grunwald, G.B., Gierschik, P., Nirenberg, M., and Spiegel, A.: Detection of a-Transducin in Retinal Rods but not Cones. Science 231: 856-859, 1986. Higashida, H., Streaty, R.A., Klee, W., and Nirenberg, M.: Bradykinin Activated Transmembrane Signals are Coupled via N, or N, to Production of Inositol 1,4,5-Trisphosphate, a Second Messenger in NG108-15 Neuroblastoma-Glioma Hybrid cells. P", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6a9c_6ufr_e2dp", "00000000-0000-0000-2B9C-5550F5103EF3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Synapse Plasticity", "101584910X160", null, "1980", "1980", "This article discribes Marshall Nirenberg's work at NIH with the biochemical genetics team.  Using model systems for the study of synaptogenesis, the experiments described in the article use clonal lines of hybrid cells from chick embryo retina and spinal cords to demonstrate the different impacts of developmental conditions on survival of synapses and growth of new cells.", "Articles", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "6", "pages", "Text", "English", "Reproduced with permission of the Pontifical Academy of Sciences.", "Copyright may apply", null, null, "PONTIFICIAE ACADEMIAE SCIENTIARVM SCRIPTA VARIA 45   M. NIRENBERG, S. WILSON, H. HIGASHIDA, J. THOMPSON, G. EISENBARTH, F. WALSH, A. ROTTER, J. KENIMER ann S. SABOL SYNAPSE PLASTICITY Abstract of the Proceedings of the Study Week on: NERVE CELLS, TRANSMITTERS AND BEHAVIOUR   ACADEMIA SCIENTIARVM EX AEDIBUS ACADEMICIS IN CIVITATE VATICANA MCMLXXX SYNAPSE PLASTICITY M. NIRENBERG, S$. WILSON, H. HIGASHIDA, J. THOMPSON, G. EISENBARTH, F. WALSH, A. ROTTER, J. KENIMER anv S. SABOL Laboratory of Biochemical Genetics National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Md., USA Two model systems were used to study the process of synaptogenesis: clonal cell lines of neural origin and neurons dissociated from embryos and cultured in vitro. Twenty-three neuroblastoma or hybrid cell lines were tested for their ability to synthesize and release acetylcholine and form synapses with rat striated muscle cells and clonal muscle cells. Six cell lines form synapses with muscle cells with high frequency; whereas 17 cell lines were found which have defects in synapse formation. Rates of acetylcholine synthesis were 34-463 pmol acetylcholine/min/mg/homogenate protein; intact cells incubated with [*H]-choline contained 55-1,600 pmol [?H]-acetylcholine/ mg/protein and released 80-6,400 fmol [*H]-acetylcholine/min/mg pro- tein into the medium. Five kinds of decets were detected with cell lines that form few or no synapses: 1) little or no acetylcholine synthesis, 2) large dense core vesicles present but not clear vesicles, 3) clear vesicles present but not large dense core vesicles, 4) little or no action potential Ca** ionophore activity, and 5) a defect in another step required for stimulus-dependent acetylcholine secretion. The ability of hybrid cells to form synapses was found to be regulated. Growth of hybrid cells in the presence of dibutyryl-cAMP increased the concentration of intracellular acetylcholine, the abundance of vesicles, the amount of acetylcholine released from cells in response to excitory stimuli, the efficiency of synaptic communication, and the number of synapses formed. These effects also were obtained with cAMP, but not with cGMP. 124 PONTIFICIAE ACADEMIAE SCIENTIARVM SCRIPTA VARIA - 45   The effects of putative neurotransmitters and hormones on intra- cellular cAMP or cGMP levels, cell membrane potential, and acetylcholine secretion from cells were determined. At least 10 species of receptors were detected on synapse competent cell lines. Activation of receptors for serotonin, PGF2 acetylcholine, bradykinin, neurotensin, or angiotensin resulted in secretion of acetylcholine from cells. Receptor mediated in- creases in cAMP or cGMP levels had no immediate effect on acetylcholine secretion from cells. However, growth of hybrid cells for 24 or more hours in the presence of ligands of receptors that are coupled to the activation of adenylate cyclase and/or exposure of cells to other inhibitors of cyclic nucleotide phosphodiesterase resulted in increase in cAMP levels of cells and mimicked all regulatory effects of dibutyrylcAMP on acetyl- choline storage and release from cells, These results show that cell lines with and without synapse defects can be generated and that receptor mediated reactions that activate adenylate cyclase and elevate cAMP levels of cells regulate the storage and stimulus-dependent secretion of acetyl- choline, thereby regulating synapse formation and the flow of informa- tion across synapses. Synapses were turned on or off slowly over a period of days, which suggests that cAMP, directly or indirectly, regulates the acquisition of components that are required for synaptic activity. We previously showed that activation of NG108-15 opiate receptors or muscarinic acetylcholine receptors results in inhibition of adenylate cyclase and that exposure of cells to morphine or carbamylcholine reduces cAMP levels of cells initially, but gradually, over a period of 24-48 hours, cAMP levels increase and return to the control value due to a compen- satory, long-lived increase in the specific activity of adenylate cyclase. Subsequent studies have shown that NG108-15 cells possess presynaptic a2-receptors and that exposure of cells to 1 uM norepinephrine similarly reduces cAMP levels initially, and that cAMP levels slowly return to the control value over a 10 hour period as the specific activity of adenylate cyclase increases. The cells then are dependent on norepinephrine to inhibit the elevated adenylate cyclase activity. Withdrawal of norepi- nephrine or blockade of the a-receptors results in a 4-9 fold increase in intracellular cAMP. Approximately 8 hours are required for the elevated enzyme activity to return to the control value. Cyclic AMP levels are elevated during the withdrawal period, and cells are supersensitive to ligands for other species of receptors, such as PGE, that activate adenylate cyclase. The demonstration that 3 species of receptors which mediate inhibition of adenylate cyclase also evoke persistent increase in adenylate cyclase activity suggests that the phenomenon is a general one, and that NERVE CELLS, TRANSMITTERS AND BEHAVIOUR 125   other species of receptors that inhibit adenylate cyclase also may act as dual regulators of the enzyme. The hypothesis that activation of adenylate cyclase may lead, conver- sely, to a reduction in adenylate cyclase activity also was tested. Incuba- tion of NG108-15 cells with PGE: for 12 hours resulted in 60-80% decreases in basal adenylate cyclase specific activity and NaF-, Gpp,NH)p-, 2-Cl-adenosine, and PGE:-stimulated activities. Basal and PGE:stimulated adenylate cyclase activities of cells exposed to PGE: decayed exponentially with half-lives of 6 hours. On withdrawal of PGE:, adenylate cyclase activity slowly increased and returned to the control value over a period of 24 hours; cyclohexamide inhibited the increase in adenylate cyclase activity > 90%. These results show that activation of adenylate cyclase leads to a loss of enzyme activity and that the recovery of enzyme activity to the control value requires protein synthesis and approximately 24 hours of incubation. These long-lived, receptor-mediated effects on adenylate cyclase activity, acetylcholine storage, stimulus-secretion coupling, and the de- monstration that synapses can be turned on or off by regulating acetyl- choline release, have properties that resemble those expected for simple forms of learning and memory, such as habituation, tolerance, dependence and sensitization, but whether synapse plasticity in a cultured cell system is related to behavioral phenomena is not known. Cultured neurons dissociated from chick embryo retina and spinal cord also were used to study the process of synapse formation. Both intact chick retina and cultured retina cells were shown to have high choline acetyltransferase activity and abundant nicotinic and muscarinic acetylcholine receptors. 'I-Labeled a-bungarotoxin and 3-[2H]-quinucli- dinyl benzilate, which bind with high affinity and specificity to nicotinic or muscarinic acetylcholine receptors, respectively, were used as probes to determine the properties of the receptors, the number of binding sites, and their distribution within the retina during embryonic development. Most of the nicotinic acetylcholine receptors and all of the muscarinic receptors of chick retina were localized in layers within the inner synaptic layer of the retina; 11 layers were distinguished within the inner synaptic layer of chick retina on the basis of muscarinic and nicotinic acetylcholine receptor concentrations and acetylcholinesterase activity. The layers ap- pear in an ordered sequence during development with respect to temporal and positional relationships. These results and those of others show that neurites of the same type sort out from neurites of other types on the basis of species of receptor, transmitter, or enzyme of transmitter me- 126 PONTIFICIAE ACADEMIAE SCIENTIARVM SCRIPTA VARIA - 45   tabolism. A possible mechanism for generating sets of stratified or co- lumnar neurons with similar properties and relating one set to another by cross-linking neurons of the same type to one another via synaptic con- nections was proposed. The specificity of synapse formation by dissociated chick embryo retina neurons was examined by culturing retina cells with inappropriate synaptic partner cells, such as striated muscle cells which possess nicotinic acetyl- choline receptors. The results show that neurons are generated in chick embryo retina that are able to form synapses with striated muscle cells and then lose the ability to form synapses with a half-life of 21 hours. These neurons first appear in chick retina on the sixth day of embryo development, and are most abundant on the eighth day, comprising perhaps 8% of the retina cell population. Almost all myotubes are innervated after coculturing retina and muscle cells for only 2 hours. However, the mismatched synapses between retina neurons and muscle cells are transient and slowly disappear over a petiod of 8 days. Neurons lose the ability to form new synapses by the 16th day, but not the ability to synthesize and secrete acetylcholine. Cultured retina neurons also form synapses in abundance with other retina neurons (approximately 110° synapses/mg of protein), and synapses between retina neurons were found after all synapses between retina neurons and muscle cells had been terminated. Preparations of neurons from spinal cord, which presumably contain motorneurons that normally innervate striated muscle cells, also formed synapses with muscle cells in vitro but the number of synapses remained constant during subsequent culture. Thus, spinal cord neurons either form stable, long-lived synapses with muscle cells or attain a steady state wherein rates of synapse formation and termination are equal. These results show that populations of cholinergic neurons from retina and spinal cord differ in the rate of synthesis of synapses with muscle cells and probably also in the rate of synapse termination, and that populations of synapses can be selected on the basis of differences in synapse turnover rates. The results suggest that part of the specificity of synaptic circuits may be acquired by a process of selection. A factor extracted from chick embryo retina and spinal cord was found to agglutinate rabbit erythrocytes in vitro. The amount of agglutinin activity varies markedly during embryonic development in the spinal cord, rising to a peak on the 10th day of embryonic development and then decreasing 7-fold by the time of hatching. The factor was first detected in 10 day embryo retina and increases in concentration until the 16th day NERVE CELLS, TRANSMITTERS AND BEHAVIOUR 127   in ovo. Despite differences in hemagglutination activity and the patterns of development, both the retina and spinal cord lectins exhibit the same specificity for saccharides. Lactose was the most potent inhibitor of hemagglutination found (half-maximal inhibition with 2 10°M lactose). To identify retina molecules required for synaptogenesis or communica- tion across the synapse, we have used the technique recently introduced by Milstein and coworkers of monospecific antibody synthesis by clonal spleen cell x myeloma hybrid cell lines formed by fusion of mouse myeloma cells with mouse spleen cells immunized against retina cells. Large quantities of monospecific antibodies can be obtained. Hybridoma cell lines were obtained which synthesize relatively homogeneous monospecific antibodies of high titer directed against cell membrane antigens of cells in retina and brain which were not detected with numerous other tissues. These antibodies bind to molecules that are specific markers for certain cells in the nervous system. Hybridoma A2B5 synthesizes an antibody directed against a molecule in plasma membranes of cell bodies of retina neurons which was not detected on axons or dendrites: whereas, other cell lines synthesize antibodies directed against membrane molecules both cell bodies and processes of retina cells. Further characterization of anti- body A2B5 and other monospecific antibodies directed against retina or neuroblastoma cell surfaces are in progress, including evaluation of their effects on neural functions.", "Thompson, Jeffrey M. ; Sabol, Steven L. ; Eisenbarth, George S. ; Nirenberg, Marshall W. ; Higashida, Haruhiro ; Rotter, Andrej ; Wilson, S. ; Kenimer, James G. ; Walsh, Frank (Frank S.), 1953-", null, "Pontificiae Academiae Scientiarvm Scripta Varia", "Pontificia Accademia delle scienze", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5hak~4p89~kxz8", "00000000-0000-0000-F98C-2D7B6249024F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Inhibition of Anaerobic Glycolysis in Ehrlich Ascites Tumor Cells by 2-Deoxy-D-Glucose", "101584910X161", null, "1958", "June 1958", "This investigation was supported in part by American Cancer Society Institutional Grants and material was taken from part of Nirenberg's dissertation at the University of Michigan.  The article reports on the process of anaerobic glycolysis in Ehrlich ascites tumors exposed to glucose.", "Articles", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "4", "pages", "Text", "English", "Reproduced with permission of the American Association for Cancer Research.", "Copyright may apply", null, null, "Inhibition of Anaerobic Glycolysis in Ehrlich Ascites Tumor Cells by 2-Deoxy-D-Glucose” MarsHaLL W({Nmmenserct AND JAMES F {Hose (Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, Mich.) Little is known of the mechanisms by which -deoxy-p-glucose (2-DG) inhibits anaerobic gly- colysis in yeast cells (1, 9, 18, 14) and in brain, diaphragm, and liver slices (15). This compound also inhibits anaerobic glycolysis in various tumors (15), prolongs the survival time of mice bearing the Krebs ascites carcinoma (2), and causes a decreased rate of growth of several tumors in rats or mice (2, 8). The inhibition with yeast (1) is competitive if either glucose or fructose serves as the substrate. 2-DG is phosphorylated by the hexokinase of yeast (1), brain (10), and tumor (15), but it is not catabolized further by these tissues. 2-DG has been claimed to inhibit the transport of monosaccharides through cell membranes (1, 9). During the course of this inves- tigation Wick et al, (12) reported that 2-deoxy-p- glucose-6-phosphate (2-DGP) competitively inhib- its phosphohexoisomerase in mammalian tissues. The observation of a step preceding glycolysis and possibly involved in hexose transfer into as- cites tumor cells (5, 6) prompted the study of 2-DG to characterize further the transport step in hexose utilization. The findings of Wick et al. (12) were confirmed for the ascites tumor, and, in addition, 2-DGP was found to inhibit glycolysis at a point subsequent to the formation of hexose diphos- phate. The possibility remains, however, that 2- DG inhibits hexose transport, since the enzyme inhibitions do not fully explain the inhibitory effects on glycolysis in cells. MATERIALS AND METHODS The Ehrlich ascites tumor (Hauschka, clone 2 {8]) was grown in white Swiss mice. At the 8th day after inoculation with 0.2 ml. of ascites tumor, the tumor cells were collected * This investigation was supported in part by American Cancer Society Institutional Grants Nos. 22-C and 22-D. The material was taken in part from a dissertation submitted to the Horace H. Rackham School of Graduate Studies by Marshall Nirenberg in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the University of Michigan, 1957. + Present address, National Institute of Arthritis and Met- abolic Diseases, National Institutes of Health, Bethesda, Md. Received for publication October 28, 1957. in a heparinized syringe, centrifuged at 400 X g for 5 minutes, and then washed twice with Krebs-Ringer bicarbonate buffer at pH 7.4. Small amounts of contaminating erythrocytes which collected at the bottom of the centrifuge tube were removed each time with a capillary pipette. After suspension to about 33 per cent (v/v) concentration in alkaline, isotonic KCI (4), the cells were homogenized either by application and rapid release of a pressure of 1500 Ib/sq in of Nz in a small stainless steel cylinder at room temperature or by 100 passes in a Brendler homogenizer at 5° C. Anaerobic glycolysis was measured in a Warburg apparatus at 37°C. with an atmosphere of 95 per cent Nz —5 per cent CO, (11). Experiments with intact cells were performed in Krebs-Ringer bicarbonate buffer; those with homogenates in a medium modified from that of LePage (4) (Table 2). The manometric measurements were confirmed by simul- taneously determining the disappearance of hexose from the medium (5, 6). Tumor extracts containing both hexokinase and phosphohexoisomerase were obtained by centrifuging a homogenate at 20,000 X g for 30 minutes at 5°C. A lipide layer rising to the top was discarded. The active enzymes were contained in the aqueous supernate. The activities of hexokinase and phosphohexoisomerase were measured by di- rect spectrophotometric assay (7) (Chart 1). The formation of reduced triphosphopyridine nucleotide (TPNH) was fol- lowed by measuring the absorption at 340 mz in a Model DU Beckman spectrophotometer with a photomultiplier at- tachment. The sample of glucose-6-phosphate dehydrogenase (Sigma) used in the assay was free of hexokinase and phos- phohexoisomerase activity. Doubling the amount of all reac- tants except the tumor enzyme did not increase the rate of the reaction, whereas doubling the amount of the tumor enzyme increased the reaction rate. The tumor extracts ap- parently contained phosphogluconate dehydrogenase, since the ratio, TPNH production/hexose consumption, was 4P- proximately 2. Glucose and fructose were Pfanstiehl reagents. 2-DG was kindly supplied by Dr. Harold Blumenthal. 2-DGP was pre pared by incubating 111 pmoles 2-DG with 2 mg. yeast hexokinase (Sigma), 150 umoles disodium adenosine triphos- phate (ATP) (Schwarz), and 300 umoles MgCl in 2.0 ol. of a 0.2 u phosphate buffer at pH 7.4. After 30 minutes of incubation at 30° C., the solution was placed in a boiling water bath for 2 minutes, and the precipitated protein 4° then removed by centrifugation. The solution was store! at —18°C. Fructose-6-phosphate (F-6-P) (Nutritional Bio- chemical Corporation) was neutralized to pH 7.0 with K2eCOs before use. RESULTS When equimolar concentrations of 2-DG and glucose. were supplied to intact Ehrlich cells. # 1M. W. Nirenberg and J. F. Hogg, unpublished results. 518 NIRENBERG AND Hoaa—Tumor Inhibition by 2-Deoxy-v-Glucose reduced rate of glucolysis was observed. Under similar conditions, 2-DG completely inhibited fruc- tolysis (Table 1). In homogenates (Table 2), when 2-DG alone was used as a substrate, CO. was displaced from the bicarbonate buffer, presumably as a result of the conversion of 2-DG to 2-DGP. The measurement of acid production in homog- enates, then, is not a true measure of inhibition of the entire pathway of glycolysis by this com- pound. When the amount of CO: produced by the conversion of 2-DG to 2-DGP was subtracted from the CO, obtained with substrates plus 2-DG, 0.8 o A OPTICAL DENSITY, 340 ma es es es es 2£ 2 Do me mm t t J ' q t , 2 . at i   MINUTES Cuart 1.—The effect of 2-deoxy-p-glucose-6-phosphate upon Ehrlich ascites tumor phosphohexoisomerase. O, 2.7 umoles F-6-P; (], 2.7 pmoles F-6-P -+ 22.0 pmoles 2-DGP; VY, 22.0 umoles 2-DGP. The medium contained, in 3.0 ml: appropriate substrates, 100 moles tris(hydroxymethyl)- aminomethane buffer (pH 8.0), 5 umoles ATP, 10 ymoles MgCh, 1.2 mmoles triphosphopyridine nucleotide (TPN) (Schwarz), & umoles KF, 0.2 K units of glucose-6-phosphate dehydrogenase, and sufficient tumor extract (0.05-0.10 mi.) to give the desired reaction rate. a large inhibition of glycolysis became apparent. The maximum rate of acid production, however, was still observed with 2-DG present. Therefore, since glucose might prevent phosphorylation of 2-DG, it was possible that no inhibition had occurred in the homogenates. To eliminate this qualification of conclusion, More specific measurements were made. First, 519 the effect of 2-DG upon tumor hexokinase was tested spectrophotometrically. An equimolar con- centration of 2-DG had little effect upon glucose phosphorylation but had a large inhibitory effect with fructose, the change of optical density per minute being 0.11-0.12 for all combinations except fructose + 2-DG, when the rate was 0.074. These results could be explained by the possibility (a) that 2-DG competed more successfully with fruc- TABLE 1 THE EFFECT OF 2-DEOXY-D-GLUCOSE UPON THE RATE OF ANAEROBIC GLYCOLYSIS OF EBRLICH ASCITES TUMOR CELLS Each vessel contained 2.2 ml. of a 3 per cent cell suspension which was incubated for 90 minutes. No endogenous glycolysis was observed. COo/hr/ mg dry wt Per cent Additions per vessel cells inhibi- (umoles) (ul.) tion 11 Glucose 26.8 11 Fructose 21.1 11 Glucose+11 2-DG 19.4 28 11 Fructose+11 2-DG 0 100 TABLE 2 THE EFFECT OF 2-DEOXY-D-GLUCOSE UPON THE RATE OF ANAEROBIC GLYCOLYSIS OF AN EHRLICH ASCITES TUMOR HOMOGENATE Each vessel contained 3.8 ml. of a 3 per cent tumor homoge- nate. CO2 production was measured at 20-minute intervals for 80 minutes. The medium consisted of: 0.0024 M K:HPO,, 0.025 M KHCO,, 0.04 M nicotinamide (Merck), 0.002 M diso- dium adenosine triphosphate (ATP), 0.0004 M diphosphopyri- dine nucleotide (DPN) (Pabst), 0.0005 M potassium fruc- tose-1,6-diphosphate (HDP) (Schwarz), 0.0005 M pyruvate (Schwarz), and 0.01 M MgClo. _ CO/hr/mg Apparent CO2/hr/mg dry wt inhibi- Additions per vessel! dry wt Corrected tion (amoles) (ul.) for 2-DG (per cent) None 8.4 16.7 2-DG 23.8 16.7 Glucose 32,2 16.7 Fructose 31.7 16.7 Glucose+16.7 84.2 10.4 68 2-DG 16.7 Fructose+16.7 29.5 5.7 82 2-DG tose for hexokinase or (b) that 2-DGP inhibited the phosphohexoisomerase reaction required in the assay with fructose. The latter possibility was checked by using F-6-P as substrate in the same system, thereby permitting an assay for phosphohexoisomerase. A high level of 2-DGP completely inhibited the tumor phosphohexoisom- erase (Chart 1), the inhibition being reversed by higher levels of F-6-P (Chart 2). Thus the inhibition of homogenate glycolysis by 2-DG (Ta- ble 2) was verified. The inhibition of tumor phosphohexoisomerase 520 Cancer Research Vol. 18, June, 1958   by 2-DGP could explain the inhibitory action of 2-DG upon glucose utilization by the cells, but it could not explain the even greater inhibition of fructose utilization. Fructolysis via the Emb- den-Meyerhof pathway, in contrast to the coupled system for the spectrophotometric assay, bypasses the phosphohexoisomerase step. This suggested that 2-DGP was also inhibiting one or more re-     Oo 0.97 0.8, } 0.7F A t Eo.et p J Qo a a o J > 0.55 - oO A 2 o 0.4b J = 4 o —0.3F JS 5 Pay Sob j/ ot £ | I | l l J 0 1 3. 4 5 6 MINUTES Cuart 2.—Reversal of 2-deoxy-p-glucose-6-phosphate in- hibition of Ehrlich ascites tumor phosphohexoisomerase by fructose-6-phosphate. O, 2.5 ymoles F-6-P; A, 2.5 ymoles F-6-P + 13.9 pmoles 2-DGP; (1, 20 wmoles F-6-P + 13.9 umoles 2-DGP. See Chart 1 for medium. actions of the Embden-Meyerhof pathway follow- ing the formation of F-6-P. This possibility was tested by adding various combinations of 2-DGP, F-6-P, and fructose-1,6-diphosphate (HDP) to tumor homogenates and determining the rate of anaerobic glycolysis manometrically (Table 3). Since the phosphorylated inhibitor was used, the previous problem of acid production from 2-DG was eliminated here. A small amount of HDP was required in the medium routinely used for homogenate experiments in order to obtain an ade- quate rate of glycolysis (4); therefore a low rate of glycolysis occurred in the vessels containing no added substrate. The 2-DGP inhibited the gly- colysis of F-6-P, thus demonstrating inhibition of a step in the glycolytic pathway after the formation of F-6-P. Although one might expect 2-DGP to inhibit phosphofructokinase, 2-DGP again inhibited glycolysis greatly when additional HDP -was supplied as substrate. Since equal in- hibitions by 2-DGP were obtained with F-6-P or HDP, clearly 2-DGP can inhibit the Embden- Meyerhof pathway at some point after phospho- fructokinase action. DISCUSSION 2-DGP inhibition of HDP utilization and of phosphohexoisomerase clearly is a mode of in- hibition of cellular glycolysis by 2-DG. With in- tact tumor cells, however, fructolvsis was much more sensitive to 2-DG inhibition than glucolysis. Furthermore, fructolysis does not pass through TABLE 8 THE EFFECT OF 2-DEOXY-D-GLUCOSE-6-PHOSPHATE UPON THE RATE OF ANAEROBIC GLYCOLYSIS OF AN EHRLICH ASCITES TUMOR HOMOGENATE See Table 2 for conditions. CO2/hr/mg Additions per vessel dry wt Per cent (umoles) (ul.) inhibition None (1.5 HDP in medium) 11.4 22.0 2-DGP 5.8 49 7.5 F-6-P 22.6 7.5 F-6-P+22.0 2-DGP 7.6 66 7.5 HDP 17.0 7.5 HDP+22.0 2-DGP 5.2 69 the phosphohexoisomerase step. Therefore, the action of 2-DGP cannot fully explain 2-DG in- hibition of cellular glucolysis. Possibly 2-DG in- hibits fructolysis by serving as a competitive sub- strate for hexokinase. The Michaelis-Menten con- stants of brain hexokinase for p-glucose, 2-DG, and p-fructose are 8.0 X 10 m, 2.7 X 10> M, and 1.6 X 10-? m, respectively (10). Therefore, at an equimolar concentration, 2-DG should com- pete effectively for hexokinase with fructose, but not with glucose. Alternatively, 2-DG may com- pete with glucose and fructose for the hexose transfer mechanism, fructolysis being the more inhibited because fructose has the lesser affinity for the binding site (5, 6). Regardless of what may be the additional mode of inhibition by 9-DG, its use as a tool for the study of hexose transport is seriously qualified by its conversion to an effective inhibitor of glycolytic enzymes. SUMMARY Equimolar concentrations of 2-deoxy-p-glucos¢ inhibited anaerobic glycolysis of Ehrlich ascites Nirenserc anp Hoae—Tumor Inhibition by 2-Deoxy-p-Glucose 521   tumor cells when either p-glucose or p-fructose was used as the substrate, glycolysis with the latter being more strongly inhibited. 2-Deoxy- p-glucose did not inhibit anaerobic glycolysis pri- marily at the monosaccharide transport level. In- stead it was converted to 2-deoxy-p-glucose-6- phosphate by tumor hexokinase and the latter compound inhibited both phosphohexoisomerase and a further step in the Embden-Meyerhof path- way subsequent to phosphofructokinase action. The Michaelis-Menten constants of hexokinase suggest that 2-deoxy-p-glucose may also inhibit hexokinase action on p-fructose. REFERENCES 1, Cramer, F. B., and Woopwarp, G. E. 2-Desoxy-p-glucose as an Antagonist of Glucose in Yeast Fermentation. J. Franklin Inst., 263:354-60, 1952. 2. Ey, J. O. 2-Deoxy-p-glucose as an Inhibitor of Cancerous Growth in Animals. J. Franklin Inst., 268:157-60, 1954. 3. Hauscuxa, T. S. Cell Population Studies on Mouse As- cites Tumors. Trans. N. Y. Acad. Sc., 16:64-73, 1953. 4, LePage, G. A. Glycolysis in Tumor Homogenates. J. Biol. Chem., 176: 1009-20, 1948. 5. Nirenperc, M. W., and Hoaa, J. F. On the Mode of Hexose Uptake by Ascites Tumor Cells. J. Am. Chem. Soc., 78:6210, 1956. 10. 11. 12. 18. 14. 1. ————. Hexose Uptake in Ascites Tumor Cells. Fed. Proc., 16: 227, 1957. . Sue, M. W.; Cort, G. T.; and Corr, C. F. A Compara- tive Study of Hexokinase from Yeast and Animal Tissues. J. Biol. Chem., 186:7638-80, 1950. . Soxo.orr, B.; Caameniy, I.; Saetaor, C.; and Bzacs, J. The Effect of a Glucose Analog on the DNA Content in Tumor Tissue. Proc. Am. Assoc. Cancer Research, 2:148, 1956. . Sous, A. Selective Fermentation and Phosphorylation of Sugars by Sauternes Yeast. Biochim. et Biophys. Acta, 20: 62-68, 1956. Sous, A., and Cranz, R. K. Substrate Specificity of Brain Hexokinase. J. Biol. Chem., 210:581-95, 1954. Umearert, W. W.; Burnis, R. H.; and Stavurrer, J. F. Manometric Techniques and Tissue Metabolism. Min- neapolis, Minn.: Burgess Publishing Co., 1949. Wicx, A. N.; Drury, D. R.; Naxapa, H. I.; and Wotrs, J.R. Localization of the Primary Metabolic Block produced by 2-Deoxyglucose. J. Biol. Chem., 224:963-69, 1957. Woopwaxp, G. E. 2-Desoxy-p-glucose as an Inhibitor in the Aerobic Glucose Metabolism of Yeast. J. Franklin Inst., 264:553-55, 1952. Woopwanp, G. E.; Cramer, F. B.; and Hunson, M. T. Carbohydrate Analogs as Antagonists of Glucose in Car- bohydrate Metabolism of Yeast. J. Franklin Inst., 256: 577-87, 1958. Woopwarp, G. E., and Hupson, M. T. The Effect of 2-Desoxy-p-glucose on Glycolysis and Respiration of Tu- mor and Normal Tissues. Cancer Research, 14:599-605, 1954.", "Nirenberg, Marshall W. ; Hogg, James F.", null, "Cancer Research", "American Association for Cancer Research", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xzfi-j9z4_a2qn", "00000000-0000-0000-1476-5DAA4C32EC0C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "An Enzymic Defect in Ascites-Tumor Cells", "101584910X162", null, "1958", "1958", "This article, published a year after Nirenberg received his Ph.D., examines the correlation between ascites tumors and lack of phosphorylase activity, which can also lead to a lack of physical response to epinephrine or glucagons.", "Articles", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "2", "pages", "Text", "English", "Reproduced with permission of Elsevier Science Publishers.", "Copyright may apply", null, null, "An enzymic defect in ascites-tumor cells* Although glycogen phosphorylase is found in almost all mammalian tissues, greatly reduced phos- phorvlase activities recently have been ascribed to ascites tumors! and a solid tumor®. The following hormonally-dependent sequence of reactions been proposed for phosphorylase activation in liver and adrenal cortex3-*. ATP, Mgt* Epinephrine Dephosphophosphorviase Glucagon ---ee-e----errerrrreore > > (Inactive) “ ’ Dephosphosphophory1 snosine-3’, 3’-cyclic tp O8 ceceeeeceececees ephosphosphophorvlase Adenosine-3’, 3/-cyclic phosphate > | kinase (ATP?) Phosphorylase (active) This communication demonstrates a complete absence of phosphorylase activity in eight different types of tumors. The cells were found to contain phosphorylase-activating enzymes but lacked specifically both active and inactive forms of phosphorylase, and therefore, they did not respond physiologically to epinephrine or glucagon. The following ascites tumors were used: Hepatoma, Ehrlich carcinoma, !ymphocytic leuke- mia, plasma cell, mast cell, Krebs-2 carcinoma, and sarcoma-37; also the HeLa carcinoma grown in tissue culture. Freshly harvested tumors contained very low levels of glycogen, approximating 3 «moles glucose equivalents/g protein, as determined analytically by the method of STap1gE, HAUGAARD AND Marsn’, Histochemical examination with HIO,Schiff reagent indicated that the glycogen was confined entirely to normal polymorphonuclear leukocytes which comprised 1~2°%% of the ascites-cell populations. Ascites-tumor cells grown in vivo and the HeLa carcinoma grown in tissue culture contained no detectable glycogen. In contrast, normal mouse-liver epithe- lial cells grown in tissue culture are known to contain glycogen®. The phosphorylase activities of whole, undialyzed tumor and normal mouse-liver homogen- ates were determined by the method of SUTHERLAND AND WosiLait!. Anactive phosphorylase was found in mouse-liver homogenates (1.53 “¢moles phosphate released/mg protein/1o min). Phospha- tase activity was negligible. Greatly decreased phosphorylase activities were found in hepatoma homogenates (<< 0.1 spmole phosphate/mg protein/1o min). Optimal activity was obtained between pH 0-7. Addition of 5’-AMP did not result in increased phosphorylase activity, nor did the use of a wide variety of homogenization techniques. Similar results were obtained with the previously listed tumors. .\\\\ histochemical phosphorylase assay® was applied to the hepatoma, Ehrlich carci- noma and Krebs-2 carcinoma. An active phosphorylase was found in the polymorphonuclear leukocytes; none was present in the tumor cells. The phosphorylase reaction was then measured in the reverse direction, from glycogen to G-1-P, by following the disappearance of added glycogen   * The following abbreviations are used: 5’-AMP, adenylic acid; ATP, adenosine triphosphate; G-1-P, glucose-1-phosphate; G-6-P, glucose-6-phosphate; TCA, trichloroacetic acid; TRIS, tris (hydroxymethyl) aminomethane; DPP kinase, dephosphophosphorylase kinase. 204 PRELIMINARY NOTES VOL. 30 (1958) from the medium. Normal mouse-liver and -muscle homogenates rapidly degraded glycogen; tumor homogenates did not degrade glycogen appreciably. The anaerobic utilization of G-1-P and G-6-P by tumor homogenates was compared manometrically. G-1-P was utilized as effectively as G-6-P, indicating phosphoglucomutase activity (20-30-yl CO,/mg dry wt. tumor homogenate/h) Addition of 50 zg epinephrine- HCl/ml medium to intact Ehrlich cells and 50 “eg epinephrine- HCl + 25 ug glucagon/m! medium to hepatoma cells did not activate a phosphorylase. Addition of both hormones to mouse-liver slices resulted in a 20 to 60% reactivation of this enzyme. Fig. 1 demonstrates that HeLa-carcinoma homogenates rapidly reactivated added dog dephosphophosphorylase. The activation process was proportional to the amount of homogenate added, was ATP dependent, and no activation occurred when a boiled homogenate was used. Addition of dog dephosphophosphorylase kinase did not greatly increase the rate of activation. Similar results were obtained with Ehrlich carcinoma, hepatoma and normal mouse-liver homo- genates. These data describe a unique type of glycogen storage disease, since the tumors possess the phosphorylase-activating enzymes, vet lack a functional dephosphophosphorvlase. It is not known whether this defect occurs in non-neoplastic cells. All types of tumors tested resemble biochemical mutants insofar as all have a similar block in the normal hormonally-linked sequence of phospho- rylase activation.   800 4 uw +DPP <a 700 KINASE ~ =x & 600 4 2 : =x ~ a 800 Y = = 400 s | 2 300 | ° Z 200 o | x 100 ] 0    0.05 O15 O15 ML HELA HOMOGENATE TRIS, pH 7.4, 0.34 umole ATP, 0.5 umole MgSO,, 0.039 yzmole epinephrine: HCl, whole homo- genate, and where indicated, dog-liver dephosphophosphorylase and dog-liver dephosphophospho- rylase kinase. Total volume was 3-2 ml. Reaction mixtures were incubated at 30° for 5 min. 1 ml of the phosphorylase reagent! containing 2.0 “moles 5’-AMP was then added and the tubes were incubated at 37° for 20 min. Samples were deproteinized by TCA precipitation at o and 20 min. The help of Dr. SAMUEL SPICER for performing the histochemical essays and Dr. EARL SUTHERLAND for generously supplying purified preparations of dog-liver enzymes is very gratefully acknow!- edged. Laboratory of Biochemistry and Metabolism, National Institute of MarsHALt W. NIrENBERG\" Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.) 1M. W. NIRENBERG, Federation Proc., 17 (1958) 283. 2 A. A. Hapjrotov anp K, I, Dancueva, Nature, 181 (1958) 547. ST. W. Ratt, E. W. SutHERLAND AND J. BerTHET, J. Biol. Chem., 224 (1957) 463. 4 E. W. SUTHERLAND AND W. D. Wosirart, J. Biol. Chem., 218 (1956) 459. 5 R. C. Haynegs, JR. anp L. Bertuet, J. Biol. Chem., 225 (1957) I15. 8 D. Lipkin, W. N. Cook anp R. Marxuam, Absir. Am. Chem. Soc., San Francisco, Calif. (1958) 41). 7 W. C. Stapiz, N. HAUGAARD AND J. B. Marsu, J. Biol. Chem., 188 (1951) 167.. § B. B. WesTFa xt, V. J. Evans, J. E. Suannon, Jr. anp W. R. Earte, J. Natl. Cancer Inst., 14 (1953) 655. ®T. TaKeucui anv H. Kouriaki, J. Histochem. and Cytochem., 3 (1955) 153. Received May 17th, 1958", "Nirenberg, Marshall W.", null, "Biochimica et Biophysica Acta", "Amsterdam Elsevier", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-t979_nnck_r327", "00000000-0000-0000-E7D0-432A3564C8E1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Induction of Two Similar Enzymes by One Inducer.  A Test Case for Shared Genetic Information", "101584910X163", null, "1960", "1960", "Marshall Nirenberg argues that genetic information does not appear to be shared, but rather serves as an inducer for two similar enzymes in different metabolic pathways.  This was an important step in delineating the relationship between genetic components and their expression.", "Abstracts (summaries)", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Federation Proceedings.", "Copyright may apply", null, null, "42 g Biochemistry THE INDUCTION OF TWO SIMILAR ENZYMES BY ONE INDUCER. A TEST CASE FOR SHARED GENETIC INFORMATION. Marshall W. Nirenberg (intr. W. B. Jakoby.) Natl. Insts. of Health, Bethesda, Md. This investigation asks whether a portion of one gene con- tains information for the synthesis of a protein subunit which might be an integral part of two or wore enzymes. A strain of Pseudomonas fluorescens formed an inducible 7-hydroxybutyric acid dehydrogenase (Reaction 1) when grown upon y-hydroxy- butyric acid (7-HBA), and an inducible B-hydroxypropionic acid dehydrogenase (Reaction 2) when grown upon A-hydroxypropionic acid (B-HPA) Nirenberg, M. W. and Jakoby, W. B., J. Biol. Chen. (in press.) + 1) 7-HBA + DPN === Succinic Semialdehyde + DPNH + H 2) B-HPA + DEN === Malonic Semialdehyde + DPNH + Ht An attempt was made to induce the reversible 7-HBA dehydro- genase by both the reactant and the product of the reaction; only 7-HBA was effective. A series of mutant strains blocked in reaction 1 were obtained. Strikingly high B-EPA dehydro- genase levels were found in these strains when 7-HBA was added, Analysis demonstrated that 7-HBA at low concentrations induced the formation of 7-HBA dehydrogenase, and, at higher concen- trations, induced the formation of both 7-HBA and B-HPA dehydrogenases, Genetic information did not appear to be shared; instead 7-HBA was found to serve as an inducer for two similar enzymes in different metabolic pathways. Ves", "Nirenberg, Marshall W.", null, "Federation Proceedings", "Federation of American Societies for Experimental Biology", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-paj7_4c9v~zcvi", "00000000-0000-0000-272E-D8DDC8C3003A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "On the Coding of Genetic Information", "101584910X164", null, "1963", "1963", "This collaborative publication is a product of the Cold Spring Harbor Symposium of 1963.  The authors discuss the state of the field in areas of known factors in messenger efficiency of synthetic polynucleotides, coding ratios, the current \"code word dictionary,\" and synthesis of nucleotides.  Tables and figures are included.", "Articles", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "9", "pages", "Text", "English", "Reproduced with permission of Cold Spring Harbor Laboratory Press.", "Copyright may apply", null, null, "On the Coding of Genetic Information M. W. Nirenpera, O. W. Jonzs, P. Leper, B. F. C. Cuark, W. 8S. SLy, anp 8S. PestKa National Institutes of Health, Bethesda, Maryland The process of expressing genetic information by aligning amino acids in proper sequence during protein synthesis usually requires the RNA polym- erase-catalyzed synthesis of a strand of RNA complementary to DNA. Recent experiments re- ported at this Symposium, and elsewhere, suggest that M-RNA (messenger RNA) synthesized in vivo is complementary to only one of the two strands of DNA (Robison and Guild, 1963; Marmur, et al., 1963; Spiegelman, 1963; Wood and Berg, 1963). The M-RNA becomes bound to ribosomes, perhaps forming a polysomal aggregate and the amino acids may be carried to these sites and ordered in correct sequence by specific transfer RNA species. It is possible that M-RNA code words are read from a fixed point by nucleotide sequences in transfer RNA complementary to those in M-RNA code words. Thus, coding errors during protein synthesis may be minimized by the requirement for correct recog- nition at three successive steps; that is at the DNA- M-RNA, M-RNA-transfer RNA (or other inter- mediate), and amino acid-transfer RNA-activating enzyme levels. Little is known about the mechanisms which impart specificity at the last two steps. SOME FACTORS INFLUENCING THE MESSENGER EFFICIENCY OF SYNTHETIC POLYNUCLEOTIDES The effects of base composition, catalytic ability, molecular weight, and secondary structure upon the messenger activity of synthetic polynucleotides will be considered at this time. The messenger activity of a synthetic polynucleo- tide may be related to its molecular weight. In H. colt extracts, poly U containing more than 100 uridylic acid residues per chain has greater template activity than smaller chains (Matthaei et al., 1962), but oligo A fractions containing as few as 9-10 adenylic acid residues per chain have been found recently by Jones et al. (1963) to direct polylysine synthesis. Also, in yeast extracts, oligo U of average chain length 11 actively directs phenylalanine incorporation (Marcus et al., 1963). Although cer- tain oligonucleotides may be degraded by nucleases more rapidly than polynucleotides and thus appear to be less efficient as templates for protein synthesis, RNA chain-length must be considered when com- 549 paring template activities of different RNA fractions. Also, secondary structure of RNA greatly in- fluences its messenger activity. When poly U is mixed with poly A, double- and triple-stranded helices are formed which are completely inactive in directing polyphenylalanine synthesis (Nirenberg and Matthaei, 1961). Oligo A also forms helices with poly U, and the extent of inhibition of polyphenyl- alanine synthesis can be correlated with oligo A chain-length and oligo A-poly U helix stability (Nirenberg et al., 1963). In addition, Singer et al. (1963), have investigated a series of copolymers containing varying amounts of U and G and have found that guanine-rich polymers containing a high degree of ordered secondary structure (perhaps due to G-G interactions) also are inactive as templates for protein synthesis. These results suggest that RNA with a high proportion of helical structure may have little template activity for protein synthesis. Recent experiments have shown that poly U-poly A helices do not bind to ribosomes, and for this reason may be unable to direct protein synthesis (Cukier and Nirenberg, unpublished results). It is possible also that small, localized areas of ordered structure may serve as periods in protein synthesis. It is difficult to compare directly the messenger efficiencies of different polynucleotide preparations because the efficiency is modified by molecular size and secondary structure. However, if the average chain length and secondary structure of different RNA preparations are assumed to be approximately equal, the data of Table 1 suggest that nucleotide content may not influence greatly the overall tem- plate efficiency of M-RNA. Poly U, poly UC, poly ACG, and poly UACG contain 1, 8, 27, and 64 triplets respectively, and the preparations of these polynucleotides shown in Table 1 have been found to direct 1, 4, 9, and 18 amino acids, respectively, into protein. The essential point is that approxi- mately the same total quantity of amino acids were directed into protein by each polynucleotide. Al- though these data must be interpreted with care because the same factor may not limit the incor- poration rate of each amino acid, they suggest that the polynucleotide preparations may have approxi- mately equal template efficiencies and that most nucleotide sequences may be able to code for amino 550 NIRENBERG, JONES, LEDER, CLARK, SLY, AND PESTKA TaBLE 1. TEMPLaTE Activities or 1, 2, 3, anp 4 Bass PoLyNucLEoTIpES     Polynucleotide U uC ACG UACG U 100 47 — 56 Base ratio Cc — 53 . 32 13 Moles-per cent A _ _— 46 5 G —_— —_— 22 25 Possible triplets 1 8 27 64 C4-Amino acids directed PHE PHE - LYS ILEU PRO into protein LEU ALA MET LYS SER ARG CYSH ALA PRO SER VAL ARG THR GLY THR GLU-NH, TRY GLU-NH, ASP-NH, TYR ASP-NH, HIS PHE HIS PRO LEU SER Total C!4-Amino acid incorporation (mymoles) 3.21 2.91 2.22 5.09   See text for details. acids. Although nonsense sequences may exist, thus far none have been demonstrated definitively. Polynucleotides containing all base combinations now have been used to direct protein synthesis in E. colt extracts. A qualitative summary of these data is presented in Table 2. Only those polynucleo- tides containing the minimum bases necessary to direct an amino acid into protein are shown. For example, phenylalanine is directed into protein by poly U and other U containing polymers; however, since other bases are not required, phenylalanine is listed only under poly U. Poly U, poly A, and poly C direct phenylalanine, lysine, and proline, respectively, into protein. Polylysine synthesized in #. coli extracts under the direction of poly A has been found to contain 3-15 lysine residues per chain (Jones, Yaron, Sober, Heppel and Nirenberg, unpublished results). No messenger activity has been demonstrated for poly G (Matthaei et al., 1962), but the highly ordered structure of poly G might mask template activity. However, a poly- nucleotide composed only of hypoxanthine (poly 1) with less secondary structure than poly G, still has not been found to direct amino acids into protein. Code word nucleotide sequences are arbitrary. Since hypoxanthine can replace G in RNA code words, the 2-amino group of G does not appear to be essential for coding amino acids (Basilio et al., 1962; Nirenberg and Jones, unpublished data). Each polynucleotide composed of 2 different bases has 8 triplets, but no polymer has been found to direct more than 6 different amino acids into protein. Poly UC is unique in that it codes for only 4 amino acids, even though all UC triplets appear to function as code words (see Coding Ratio Section). It is important to note also that polynucleotides containing only two different bases direct with great specificity almost all amino acids into protein. These findings undoubtedly reflect basic molecular characteristics of both the recognition process and the general nature of the code. THE CODING RATIO A series of poly AC and poly UC preparations with different proportions of bases were synthesized and their activities in stimulating cell-free amino acid incorporation into protein were determined. As shown in Fig. 1, poly AC directs the incor- poration into protein of proline, histidine, threonine, TaBLe 2. SuMMARY OF Coping Data         Poly U A c G PHE LYS PRO — Poly UA uc UG AC AG cG UAG TYR LEU LEU HIS ARG ARG MET LEU SER VAL ASP-NH, GLU ALA ASP ILEU CYSH GLU-NH, GLU-NH, SER ASP-NH, TRY THR ASP* THRt   Only those polynucleotides containing the minimal number of bases necessary to stimulate an amino acid into protein are shown. Amino acids coded by homopolynucleotides are not listed again under randomly-ordered polynucleotides. * Predicted t Reported by Wahba et al. (1963). CODING OF GENETIC INFORMATION 800 wa +a Threonine @-2--- =o = “8 Profine p 5 ° Asporagine 8 Lysine Histidine   ny ° Oo Glutamine HuMOLES Cl*AMINO ACID INCORPORATED INTO PROTEIN     I 1 t 4 1 1 i J. © 10 20 30, «4050 6 70 60 690 L TIME (MINUTES) Ficurs 1. The rate of C!4-amino acid incorporation into protein directed by poly AC (base ratio = A, 47% and C, 53%). Reaction mixture components are described in the legend of Table 3. Incubations were stopped at the times indicated by the addition of 3.0 ml of 10% TCA at 3°. The samples were heated at 90° for 20 min, chilled and then filtered through Millipore filters and washed with 5% TCA at 3°. Radio- activity measurements were performed in a thin-window gas flow Nuclear Chicago Corp. counter with a counting efficiency of 23%. Each point represents the upmoles of Ci4-amino acid incorporated into protein due to the addition of poly AC. asparagine, glutamine, and lysine at linear rates for 15-20 min. Reactions were terminated after 10 min of incubation, while the rates of incorpora- tion were still linear. , In Table 3 is presented an example of the data obtained for each of the five poly AC preparations tested. The theoretical proportions of the four doublet and eight triplet permutations expected in 551 randomly-ordered poly AC (containing by analysis, 47% A and 53%,C) are shown in the first and second columns respectively. In the third and fourth columns are shown the wumoles of each C4-amino acid directed into protein by this polymer. A total of 1,685 Humoles of amino acids were directed into protein, and the relative propor- tions of each amino acid incorporated, in per cent, are shown in the last column. The 4 doublet permutations do not contain enough specific information to code for the 6 amino acids incorporated, whereas the information con- tent of the 8 triplet words is adequate. The per cent incorporation of lysine, asparagine, glutamine, and histidine agrees well with triplet code word fre- quencies, but not with doublet frequencies. If all triplets were read, some amino acids would respond to 2 or more code words, for 6 amino acids would then be coded by 8 words. In such cases, the sum of the triplet frequencies would have to be com- pared with the corresponding amino acid incorpora- tion data. For example, if CAA and CCA both coded for one amino acid, the sum of their frequencies is 24.9%, which cannot be distinguished from the frequency of the doublet CA (also 24.9%). There- fore, this experimental approach may allow deter- mination of the coding ratio for some, but not all, amino acids. Analysis of a series of polynucleotides with varying base-ratios permits comparisons to be made with greater accuracy. The expected statistical relationship between code word frequency and poly- nucleotide base-ratio are presented graphically in TasBLe 3. CompaRIsoN Brrween Amino Acips INCORPORATED AND RNA CopE Worp FREQUENCIES   Theoretical code word frequency in poly AC containing 47% A and 53% C C'4-Amino acids incorporated into protein Frequency of   “uMoles C4. Amino acids Doublets Triplets Cl. Amino acid incorporated incorporated per cent per cent AA 22.1 AAA 10.4 Lysine 183 10.8 AC 24.9 AAC 11.7 Asparagine 192 11.6 CA 24.9 ACA 11.7 Glutamine 157 9.3 CC 28.1 CAA 11.7 Threonine 444 26.3 Total 100.0 — CCA 13.2 Histidine 159 9.4 ACC 13.2 Proline 550 32.6 CAC: 13.2 Total 1685 Total 100.0 CCC 14.9 Total 100.0   Each reaction mixture contained the following components in a final volume of 0.25 ml: 0.1 M Tris, pH 7.8; 0.01 M magnesium acetate; 0.05 M KCl; 6 x 10-°M mercaptoethanol; 1 x 10-?M ATP; 5 x 10-3 M potassium phosphoenol- pyruvate; 5 ug of crystalline phosphoenolpyruvate kinase (Calif. Corp. Biochem. Research); 0.8 x 10-4M C!4-amino acid; {approximately 30,000—150,000 cpm/reaction mixture); 3.2 x 10-* M each of 19 C}2-L amino acids minus the C4-amino acid; 15 ug of polynucleotide when specified; and 1.1 mg EZ. coli preincubated 8-30 protein (Nirenberg and Matthaei, 1961). Reaction mixtures were incubated at 37° for 10 min. Protein precipitation, washing, and counting were performed as described by Nirenberg and Matthaei (1961). The theoretical frequencies in per cent of doublets and triplets in polynucleotides were calculated as follows: The fre- quency of the triplet AAA in this poly AC preparation would be 0.47 x 0.47 x 0.47 x 100 = 10.4%. The doublet frequency for CA would be 0.47 x 0.538 x 100 = 24.9%. The «moles of each amino acid incorporated in the absence of polynucleotide were: lysine, 30; asparagine, 54; glutamine, 49; threonine, 40; histidine, 26; proline, 36. 552 NIRENBERG, JONES, LEDER, CLARK, SLY, AND PESTKA   T T T T tT T 1 q q v ------ Theoretical Frequency of RNA Codewords —— Observed Frequency of Amino Acid Incorporation 30f AC or       PERCENT CODEWORDS IN POLY AC         %9 10 20 30 40 50 60 70 80 90 100 PERCENT C IN POLY AC Freure 2. Comparison of C!+-histidine, C*-threonine, C**-asparagine and C!4.glutamine incorporation data with the theoretical frequencies of doublet and triplet code words in poly AC preparations. The solid lines represent the experimentally determined incorporation data calcu- lated as described in Table 1. The dotted lines represent the theoretical frequencies of RNA code words. All assays were performed as described in Table 3. Figs. 2 and 3. Theoretical frequencies in per cent of doublet and triplet code words are shown on the ordinate and the base-ratio is shown on the abscissa. Nucleotide sequence is arbitrary, and each curve represents only one of the three possible sequence permutations. As noted before, the sum of the frequencies of the triplets AAC and ACC equals the frequency of the doublet AC. Thus the AC curve represents either the doublet AC, or the sum of the two triplets AAC plus ACC. Also shown are the observed C14-amino acid incorporation data. Each point represents a different poly AC preparation with the indicated base-ratio. As shown in Fig. 2, the observed incorporation of C14-histidine agrees well with the theoretical frequency of the triplet ACC and differs markedly from both the AAC triplet and AC doublet curves. The data also demonstrate that the observed incorporations of both Cl#.asparagine and C!*-glutamine agree well with the frequencies of AAC triplets. In contrast, the incorporation of C14-threonine is similar to the expected frequencies of either the doublet AC, or the two triplets, AAC plus ACC. Therefore, threo- nine appears to be coded either by a doublet or by two triplets, and one cannot differentiate between these alternatives on the basis of these data. In Fig. 3 are presented the template activities of poly AC preparations for C!4-proline and C!-lysine. The experimentally obtained incorporation data indicate that proline is coded either by the doublet CC or by the two triplets CCC and CCA. C\"*-lysine appears to be coded by the triplet AAA. C4_Amino Acip IncorPoRATION DIRECTED BY Poty UC The data of Fig. 4 show that proline is directed into protein either by the doublet CC or by the sum   100 90r 80° TOF 60 SO} 40r 30 20F T 10     ° 100k ' '   90PF\\\\\\\\ q Theoreticol Frequency BOL \\\\ of RNA Codewords _ V\\\\ Observed Frequency of TOR VN Amino Acid Incorporation - ON AA 60F or \\\\ ON AAAFAAC 50-  44A,), Se 4   PERCENT CODEWORDS IN POLY AC 40} 30+ 20r T 10       ~~ ~, { J {>> S=8. ! 50 60 70 80 390 100 30. 40 PERCENT C IN POLY AC   0 ! | oO 10 20 Figure 3. Comparison of C'-proline and C#-lysine incorporation data with the theoretical frequencies of doublet and triplet code words in poly AC preparations. The solid lines represent the experimentally determined incorporation data calculated as described in Table 1. All assays were performed as described in Table 3. CODING OF GENETIC INFORMATION 553 100 T T T 1 1 1   90Fr 7Or SOF 20- LOr     0 100     wo °o > oF It VN tet ee Theoretical Frequency i of PNA Codewords q Observed Frequency of LN y Amino Acid Incorporation o °o T s   PERCENT CODEWORDS IN POLY UC ~ ° T n °o - [\" \\\\ \\\\ or 4 YUUN, \\\\e~Suuu+ vue N X b a oO oO T T uw o T OF       J L ml Qo O 10 20 30 40 50 60 70 80 90 100 PERCENT C IN POLY UC FievurE 4. Comparison of C'4-proline and C14-phenyl- alanine incorporation data with the theoretical frequencies of doublet and triplet code words in poly UC preparations. The solid lines represent the experimentally determined incorporation data calculated as described in Table 1. All assays were performed as described in Table 3.   of the two triplets CCC and CCU. Cl4_ phenylalanine appears to be coded either by the doublet UU or by the two triplets UUU and UUC. It is important to note that if the code words corresponding to these amino acids are triplets, both CCC and CCU would code for proline and both UUU and UUC would code for phenylalanine. In Fig. 5 are shown the poly UC-directed serine and leucine incorporation data. Both serine and leucine appear to be coded either by the doublet UC, or by the two triplets UUC and UCC. Coding of serine or of leucine by one, rather than 2 triplets is not indicated. It is important to note that if serine is coded by triplets, one triplet would have to contain 2 U residues and the other 2 C residues. Triplet words for leucine also would contain either 2U or 2 C residues,           v T T T T T T T T T oOo foocttt Theoretical Frequency of 5 RNA Codewords 7 30k Observed Frequency of J 2 Amino Acid Incorporation z g uc xr or ra e ‘ ‘. “ ‘ . 4 S @ruuceuce ,- Leucine z ire s c 8 7 \\\\ oO ‘ “TT > ~ \"kT ~~ E jo UUe >< vce ~\\\\ z L ’ \\\\ iw 10 “ee ua “S ~N ‘ AN 7 & 4 js “ ~“ AN w \" “ Ny NY - ~ x a / u n \\\\ ; “7 ~S . \\\\ Q fet 1 L L 1 L 1 1 Pa   o 10 20 30 40 50 60 70 80 90 100 PERCENT C IN POLY UC FieureE 5. Comparison of C!*-serine and C14-leucine incorporation data with the theoretical frequencies of doublet and triplet code words in poly UC preparations. The solid lines represent the experimentally determined incorporation data calculated as described in Table 1. All assays were performed as described in Table 3. These experiments strongly suggest that histidine, asparagine, glutamine, and lysine are coded by triplet words and that the RNA code cannot be composed only of doublets. Threonine, proline, phenylalanine, serine, and leucine were found to be coded either by multiple triplets or by doublets. These data are summarized in Table 4. A mixed doublet-triplet code cannot be excluded on the basis of the available data; however, a uniform code containing only triplets would appear more probable. Tue CuRRENT CopE Worp DicrTionaRY Assuming for the present that all amino acids are coded by triplets, current approximations of RNA code words may be summarized as shown in Table 5. Nucleotide sequence is arbitrary. Fifty of the 64 possible triplets have been assigned. Almost all amino acids can be coded by polynucleotides containing 2 different bases. Since polynucleotides containing 3 bases direct’ protein synthesis as efficiently as polymers containing only 2 bases, it TaBLEe 4. Summary oF Copinec Ratio Data     Code Word* Cl*#.Amino acid Triplet Doublet Histidine ACC —_ Asparagine CAA — Glutamine AAC — Lysine AAA — Threonine CCA + ACA or AC Proline cce + CAC + CUC or CC Phenylalanine UUU + UCU or UU Serine cuUU + CCU or CU Leucine vvC + UCC or UC   * Nucleotide sequences are arbitrary. 554 NIRENBERG, JONES, LEDER, CLARK, SLY, AND PESTKA TaBLe 5. SumMary oF RNA CopE Worps   Amino acid RNA code words*   Alanine CCG UCGt ACGt Arginine CGC AGA UGCt CGAT Asparagine ACA AUA  ACUF Aspartic acid GUA GCAf GAAt Cysteine UUG Glutamic acid GAA GAUT GACT Glutamine AAC AGA AGUt Glycine UGG AGG CGG Histidine ACC ACUT Isoleucine UAU UAA Leucine UUG UUC ucc DUA Lysine AAA AAU Methionine UGA Phenylalanine UUU CUU : Proline CCC CCU CCA CCGt Serine UCU UCC UCGTt ACG Threonine CAC CAA Tryptophan GGU Tyrosine AUU Valine UGU UGAT   * Arbitrary nucleotide sequence. + Probable. seems probable that most 3 base words are recog- nized. Tentative assignments are given for such words. It seems clear that most amino acids are coded by multiple words. Furthermore, multiple words corresponding to one amino acid often differ in base composition by only 1 nucleotide. These observa- tions also suggest that nucleotide sequences in multiple words often may be identical. A triplet code may be constructed wherein correct hydrogen bonding between 2 out of 3 nucleotide pairs may, in some cases, suffice for coding; or alternatively, a base at one position in the triplet sometimes may pair optionally and correctly with 2 or more bases. It should be noted that a triplet code of this type in some respects would bear a superficial resem- blance to a doublet code and would be in accord with all of the data available. The coding data obtained thus far clearly indicate that most nucleotide sequences can code for amino acids with great specificity. Weisblum et al. (1962) have reported that multiple species of leucine transfer RNA recognize different code words in synthetic polynucleotides; however, additional data presented at this symposium (not submitted for publication) by Benzer and by von Ehrenstein and Gonano suggest that code word specificity in directing leucine incorporation may be greater with synthetic polynucleotides than with natural M- RNA. Ii is important to emphasize the possibility that randomly-ordered synthetic polynucleotides may test the cell’s potential to recognize code words, and that the entire potential may not be utilized in vivo, except perhaps during mutation. Thus M-RNA synthesized by a cell may not contain as many code words as randomly-ordered polynucleotides. Several groups of amino acids are shown in Table 6 which either are synthesized in vivo from the same precursor, or have similar structures. For example, phenylalanine, tyrosine, and tryptophan are derived from shikimie acid, and isoleucine, valine, and leucine are synthesized from «-keto butyrate. RNA code words corresponding to these amino acids are shown also. Such comparisons suggest that a family of amino acids may recognize a family of code words whose members contain similar bases. Although not. all amino acids fit this pattern, enough additional examples may be cited to warrant the. suggestion that such relationships reflect either the evolu- tionary development of the code, or the recognition of nucleotides in code words by amino acids. The latter has been proposed by Woese (1963) and also is discussed by Weinstein in this volume. OLIGODEOXYTHYMIDYLATE DIRECTED POLYLYSINE SYNTHESIS The chemical synthesis of oligodeoxynucleotides by the method of Khorana and his associates (1961, 1962) and the demonstration of an oligodeoxynu- cleotide-dependent synthesis of polyribonucleotides; catalyzed by RNA polymerase (Furth et al., 1961; Stevens, 1961; Chamberlain and Berg, 1962; Fala- schi et al., 1963), provided an opportunity to study their ability to stimulate cell-free amino acid incor- poration. Since poly A serves as a template for polylysine synthesis (Gardner et al., 1962), oligo dT (oligodeoxythymidylate) has been used to direct poly A, and subsequent polylysine synthesis, as follows:   Oligo dT (1) ATP RNA Polymerase - Poly A + PP (2) Lysine ro A > Polylysine E. coli Extracts, ete. In addition, natural DNA and poly U have been shown to direct polylysine synthesis. Poly A was synthesized in RNA polymerase-oligo dT reaction mixtures (stage I) as described in the TABLE 6. RELATIONSHIP BETWEEN AMINO ACIDS OF SrmitaR MeEtTaBoLIc ORIGIN oR STRUCTURE AND THEIR RNA CopE Worps     Aromatic Dicarboxylic amino Tleu, Val, Leu amino acids acids and amides family PHE UUU_ ASP-NH, AAU ILEU UUA UUC AAC UAA TYR UUA ASP AUG VAL UUG TRY UGG GLU AUG LEU UUG AAG. UUA UUC GLU-NH, AUG uUcc AAC   CODING OF GENETIC INFORMATION 555   @ 5 x) o oOo 6 T T T 4 1 t + + L i 1 a ° T 1 t 1 plus akgo dT 3-14 a oO T + 1 my MOLES C’* AMP INCORPORATED ny ° T 1 minus oligo dT 3-44 4, 4 1 +       i L 1 1 ! 4p °% 10 20 30 40 50 0 20 40 60 8 iO 120 me MOLES (pdT) IN OLIGO dT, 344 MINUTES Figure 6. Characteristics of C!4-poly A synthesis in RNA polymerase (stage I) reaction mixtures. In the figure on the left, reaction mixtures were incubated at 37° for 15 min, then were deproteinized and washed with 5% TCA at 3°. The symbols in the figure on the right represent the following: A, —polymer; , @+1.2 mymoles of base residues in oligo dT,, _,,. Each stage I reaction mixture contained the following in a final volume of 0.125 mi: 4 x 10-?M Tris, pH 7.8;4 x 10-?M MgCl,; 10-? M MnCl,; 1.2 x 10-? M mercaptoethanol; 1.6 x 10-3 M 8-C!4-ATP, tetralithium salt (Schwarz BioResearch, Inc.); and 20 ug £. coli RNA polymerase protein (20 units [Chamberlain and Berg, 1962}).   legend accompanying Fig. 6, and then components supporting amino acid incorporation into protein (stage II) as in the legend of Fig. 7, were added. After further incubation, incorporation of C1l4- lysine into polylysine was determined by precipi- tation with a TCA-tungstate solution (Gardner et al., 1962). The data of Fig. 6 show that C'4.AMP incorpora- tion was dependent upon the addition of oligo aTj3-,4 (13-14 nucleotides per chain) to stage I reaction mixtures, and that C!4-AMP incorporation was proportional to the amount of oligo dT added within the range of 2.4 or less mymoles of nucleo- tide residues in oligo dTy5_ 44. The average chain length of the C!-product synthesized in the presence of oligo dT 3.1, was determined by deproteinizing the reaction mixtures, removing the C!4-ATP by paper chromatography, hydrolyzing the C!*-product in 0.3 N KOH and separating the nucleotides by paper chroma- tography. The radioactivity of adenosine, adeno- sine-3’ (2’)-5’-diphosphate and adenosine-3’ (2’)- ‘monophosphate was found to be 339, 308, and 22,900 cpm, respectively. Thus, oligo dT,,_,, stimu- lated the synthesis of poly A of average chain- length 60-70 (pA) (adenylate) residues: These data confirm similar results obtained by Furth et al. (1961) and Falaschi et al. (1963). Falaschi et al. (1963) also demonstrated that oligo dT chains are not elongated by the addition of (pA) residues to the free 3’-hydroxyl ends of oligo dT chains, and have obtained evidence which suggests that oligodeoxynucleotides serve as tem- plates rather than primers. Although our RNA polymerase preparations were purified 100 to 150- fold (Chamberlain and Berg, 1962), we have detected unprimed nucleotide incorporation under other conditions. Further enzyme purification will be necessary to determine unequivocally whether oligodeoxynucleotides function only as templates in this system. CHARACTERISTICS OF THE C¥4_PoLYLYSINE SYNTHESIS After incubating stage I reaction mixtures at 37°, stage II components were added as described in the legend accompanying Fig. 7. No increase in C14. lysine incorporation was found in the absence of oligo dT, whereas the addition of 1.2 mumoles of (pdT) residues in oligo dT,5_,, stimulated C-lysine incorporation at a linear and almost optimal rate for 30 min. In separate reaction mixtures, C!-AMP incorporation into poly A was determined at the end of the stage I incubation. No incorporation was observed in the absence of oligo dT, whereas the addition of 1.2 mymoles of (pdT) in oligo dT directed the incorporation of 3.8 mymoles of AMP           1000 7 T ns | TT TT T oligo dTi3-14 900+ << 4B mp moles 4 ogo AT 3-14 a 800k 2.4 mp moles - ul - < \\\\ § 7oo}- oligo OT i3-14 a 4 a 12 my moles «x fa} # 600} 4 WwW z g 500h 4 a : @ © 400F 4 w wd 3 300b + = x a 200F, . minus ogo dT 7 = wT ba 100 - 4 0° | i | 1 | i ! 4 ! l o t 20 30 40 50 6 70 B80 30 100 MINUTES Ficure 7. Characteristics of oligo dT,, 1, directed syn- thesis of C14-polylysine. The symbols represent the following: m, minus oligo dT; a, plus 1.2 mymoles (pdT) residues in oligo dT,3_143 © plus 2.4 myumoles (pdT) residues in oligo dT,3_,4; @, plus 4.8 mymoles (pdT) resi- dues in oligo dT,,_,4. Components of stage I reaction mixtures are as noted in the legend of Fig. 6. Stage II reaction mixtures contained, in 0.25 ml: 6 x 10-2 M Tris, pH 7.8, 2 x 10-3 M MgCl; 1.2 x 10-? M magnesium acetate; 5 x 10-4 M MnCl,; 1.2 x 10-? M mercapto- ethanol; 2.8 x 10-?M ATP; 5 x 10-2?M KCl;5 x 10-3M potassium phosphoenolpyruvate; 5 ug crystalline phospho- enolpyruvate kinase (Calif. Corp. Biochem. Research); 2 x 10-4 M each of 19 L-amino acids; 2 « 10-4 M C!4-1- lysine (Nuclear Chicago Corp.) with specific radioactivity of 4-8 meuries/mmole; 20 ug RNA polymerase protein (20 units [Chamberlain and Berg, 1962]) and 1.1 mg E. colt extratt protein (Nirenberg and Matthaei, 1961). Stage I reaction mixtures were incubated at 37° for 15 min before the addition of stage II components. 556 NIRENBERG, JONES, LEDER, CLARK, SLY, AND PESTKA into poly A. Poly A, in turn, directed the incorpora- tion of 0.64 mmoles of C14-lysine into polypeptide. The effect of oligo dT upon the individual incor- poration of 18 other C!4-amino acids (minus cysteine) also was determined. Only C*+-lysine was directed into protein, which demonstrated marked amino acid specificity. Oligo dT was found to be resistant to digestion with pancreatic DNase. DNase partially inhibited C*4-lysine incorporation into protein, whereas puro- mycin and pancreatic RNase were strongly in- hibitory. As expected, no inhibition was observed in the presence of actinomycin D. It is important to note that if stage I incubation was not performed, oligo dT directed little or no C¥4-AMP incorporation into poly A in stage II reaction mixtures, and thus no stimulation of CH- lysine incorporation could be observed. RNA polymerase has been found to catalyze the synthesis of complementary RNA in the presence of either RNA or DNA templates (Weiss and Naka- moto, 1961; Weiss, 1963; Krakow and Ochoa, 1963). Poly A synthesized from poly U templates was found to stimulate C14-lysine incorporation into protein. In other experiments no stimulation of C¥4.lysine incorporation was observed when poly C rather than poly U was added to stage I reaction mixtures. RNA polymerase also has been shown (Chamberlain and Berg, 1962) to catalyze a DNA dependent synthesis of poly A from ATP (in the absence of UTP, GTP, and CTP). Under these con- ditions, poly A synthesized under the direction of calf thymus DNA stimulated C14-lysine incor- poration. Paper chromatography of polylysine, as described in the legend accompanying Fig. 8\\", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-74kc_de96~fnzx", "00000000-0000-0000-7DA1-BFE6D6B32AC5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "A Gradient of Molecules in Avian Retina with Dorsoventral Polarity", "101584910X165", null, "1981", "1981", "Marshall Nirenberg pursued many lines of research in neurobiology in the late 1970s and early 1980s, including the expression of retinal genes.  This collaborative article from the Biochemical Genetics lab at NIH examines the embryological development of different cell lines in chick retina.", "Articles", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "15", "pages", "Text", "English", "Reproduced with permission of Cold Spring Harbor Laboratory Press.", "Copyright may apply", null, null, "A Gradient of Molecules in Avian Retina with Dorsoventral Polarity G. DAVID TRISLER, MICHAEL D. SCHNEIDER, JOSEPH R. MOSKAL, and MARSHALL NIRENBERG Laboratory of Biochemical Genetics National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20205 * Mechanisms that impart positional information for the assembly of the developing nervous system and the establishment of specific synaptic connections have been studied in model systems. The retina is a highly ordered laminar structure due to segregation of different classes of neuronal cell bodies and synapses into separate strata (Karten, this volume). Synaptic connections between retina ganglion neurons and tectum neurons preserve the topographic relations of ganglion neurons in the retina, resulting in a point-to-point retino- tectal map. However, the mechanisms underlying these phenomena have not been defined. Sperry (1963) postulated that two orthogonal gradients of molecules on retina ganglion neurons and corresponding gradients of complementary molecules in the optic tectum might determine the specificity of connections between retina and tectum neurons. Other mechanisms that have been proposed include adhe- sive interactions between migrating retina neurites, myelination of bundles of retina axons, and formation of extracellular channels by glia to guide retina axons (Silver and Sidman 1980). Antisera have been widely used to study retinal structure and function (Goldschneider and Moscona 1972; Thiery et al. 1977; Haus- man and Moscona 1979). Clonal neural retina hybrid cell lines de- rived from single retina cells (as a homogeneous immunogen) have been used to produce rabbit antiserum demonstrating an antigen of the rodent neural retina that was expressed in a restricted topograph- ic domain (Trisler et al. 1979). Hybridoma cell lines were derived by fusing mouse myeloma cells with spleen cells from mice immunized with retina cells to produce monoclonal antibodies to retina neurons or Miiller cells (Eisenbarth et al. 1979; Barnstable 1980). 231 232 Monoclonal Antibodies to Neural Antigens To detect cell-surface molecules with topographic specificity in the retina, hybridoma antibodies, as candidates for neuronal recogni- tion molecules, were obtained by fusing P3-X63/Ag-8 mouse myeloma cells (Kéhler and Milstein 1975) with spleen cells from mice immu- nized with small portions of dorsal or ventral 14-day chick embryo neural retina or clonal retina hybrid cells. A hybridoma antibody was obtained that binds to cell-membrane molecules distributed in a dorsoposterior — ventroanterior gradient in retina (Trisler et al. 1981). DETECTION OF ANTIBODIES WITH TOPOGRAPHIC SPECIFICITY * The rationale for the experiments shown schematically in Figure 1 was to immunize mice with cells from dorsoposterior or ventral 14- day chick embryo left retina to obtain hybridoma cell lines synthesiz- ing antibody with specificity for the retina sector used for immuniza- tion. The choroid fissure was used as a landmark. Female BALB/c   MOUSE IMMUNIZATION WITH CHICK RETINA SEGMENTS D ae v mS / ’ \\\\ f A : P At \\\\P , Ny / Ay Vv Vv   HYBRIDOMA ANTIBODY SPECIFICITY D _ D an Pa ‘ é XV Ai yP ! ‘A P GA A’ \\\\ / X ’ x a” “ ” Vv           v NUMBER NUMBER OF OF HYBRIDOMA HYBRIDOMA COLONIES COLONIES t++tit + 1 + + 5 + + 13 + + 3 _ _ 5A _ _ 101 Figure 1 Strategy for detecting region-specific retina surface-membrane antigens. Lymphocyte hybridomas were derived from mice immunized with cells from dorsoposterior (left panels) or ventral (right panels) retina; hy- bridoma antibody binding to paired cultured-cell monolayers from the portions of 14-day chick embryo retina shown in black in the lower panels was determined. A Gradient of Molecules in Avian Retina 233 mice were immunized on days 0, 7, and 14 intraperitoneally with 8 x 10° mechanically dissociated retina cells and intravenously with 2 x 10° cells from dorsoposterior or ventral retina. On the 17th day, 1 x 10% spleen cells from an immunized mouse were fused with 2 x 10’ P3-X63/Ag-8 mouse myeloma cells as described by Galfre et al. (1977). After fusion, the cells were suspended with 5 x 10° spleen cells from a nonimmunized mouse in 50 ml of medium (Trisler et al. 1981), and 672 wells of 96-well microculture plates were inoculated with cells (74 l/well). Additional medium was added (0.1 ml/well) on the 5th and 10th days after fusion. To detect an antibody to a surface molecule enriched in one region of the retina, tissue culture supernatants from wells with hybridoma colonies were tested by indirect radioimmunoassay against cells cultured for 2 days (Schneider and Eisenbarth 1979);   6-1 oT -7 TOPOGRAPHIC GRADIENT OF ANTIGEN              DORSAL OORSAL 10+   VENTRAL VENTRAL RIGHT RETINA LEFT RETINA pMOL 125]-F(ab'}9 BOUND/mg PROTEIN TOP P3X63 Ag8 BUFFER       | 3 4 RETINAS _ NE Figure 2 Topographic gradient of TOP antigen in (e) right or (A) left 14-day chick embryo neural retina. Equivalent sectors from different retinas were pooled and assayed for ['I|F(ab’), binding after incubation with (e————-e; a——-A) TOP antibody, (--------- ©; A---9----- A) P3- X63/Ag-8 antibody, or (6>——-o, AA) buffer in the absence of antibody. The right half of the figure depicts, within each sector of the retina, the pmoles of specifically bound ['*5I)F(ab’), per mg of retina protein (binding due to antibody to TOP minus binding due to antibody synthesized by P3-X63/Ag-8 parental cells). Assay conditions are de- scribed in the legend to Fig. 3. 234 Moncclonal Antibodies to Neural Antigens cells were taken from the one eighth of the retina originally used as immunogen or from the remaining seven eighths of the retina. Hybri- doma antibody binding to retina cells was detected with an !25]- labeled, affinity-purified F(ab’), fragment of rabbit anti-mouse IgG ((°°I]F(ab’),). A TOPOGRAPHIC GRADIENT OF SURFACE-MEMBRANE MOLECULES * One of 155 hybridoma cell lines examined synthesized antibody to a cell-surface antigen distributed preferentially in dorsoposterior ret- ina (Fig. 2), designated TOP, for toponymic antigen (i.e., a marker of position). Neural retinas from tight or left eyes of 14-day chick                                                                            A. PROTEIN’ | = |B MONOCLONAL ANTI- = (C1250 Fab) ANT. a CONCENTRATION FE gt BODY CONCEN-@ Ji {| MOUSE IgG CONCEN- 6; TOTAL, 7 2 | TRATION 230; TRATIO ; g al iF rae ral SPECIFIC] E6T pops: ié 1 2 Ab 4 7 | RETINA 920 A an 434} 3 | Ww a ao = ob Jaf M410 NONSPECIFICA, - a D9 VENTRAL | oe aa is RETINA | gMENTRAL a NONSPECIFIC | = = 7 a-0h--0--0 J 8 8 0 200 400 600\", 00.001 0.01 0.1 1275 1 2 3°°4 ug RETINA PROTEIN g ul ASCITES FLUID g uM 125)-Flab’)o Qa a Figure 3 (A) Effect of dorsal retina protein concentration (pooled sections 4 and 5 from 14-day chick embryos as illustrated in Fig. 2) on (*1]F(ab’}, binding due to: (A) antibody to TOP; (0) antibody synthesized by P3-X63/Ag-8; (e) specific binding. (B) Effect of monoclonal antibody concentration on (e, A) (**I]F(ab’), specific binding due to antibody to TOP or (0, A) nonspecific binding due to P3-X63/Ag-8 antibody, to cells from {e) dorsal retina sections 4 and 5 or (4) ventral retina sections 1 and 8. (C) Effect of concentration of [¥51]F(ab’), rabbit anti-mouse IgG. For all experiments each reaction mixture contained the following components, except where specified, in a final volume of 50 yl: 150-200 ug of retina protein, 50 ug of gelatin, and 1 yl of hybridoma ascites fluid; the reaction mixtures were incubated for 30 min at 4°C and washed three times. The pellets were resuspended in 50 “I contain- ing 440 nM [”5]]F(ab’), (50,000 cpm), 50 ug of gelatin, and 500 ug of bovine serum albumin in Dulbecco's Phosphate-buffered saline, incubated for 30 min at 4°C, washed three times, and counted. , A Gradient of Molecules in Avian Retina 235 embryos were cut into eight 45° sectors and assayed for TOP antigen. The highest concentration of antigen detected was in dorsoposterior retina and the lowest in ventroanterior retina. Bilaterally symmetric gradients were found in right and left eyes. Other hybridoma anti- bodies, including A2B5 (Eisenbarth et al. 1979), bound equally to cells from each region of retina. The effects of varying concentrations of retina protein, hybri- doma antibody, and [\"5I]F({ab’), rabbit anti-mouse IgG are shown in Figure 3, A, B, and C, respectively. Assay conditions employed are summarized in the legend to Figure 3. To determine whether the TOP antigen gradient is polar (i.e., varies with angle of rotation around the center of the retina), with         0 1 1 1 L 1 r 12345678 RETINA SECTION     pMOL 125-F(ab')g7 BOUND/mg PROTEIN 5 10 50 100 PERCENT OF MAXIMAL DISTANCE pMOL 125)-F(ab’}2 BOUND/mg PROTEIN Figure 4 Orientation of the TOP gradient. Specific binding of [51] F(ab’), (pmoles per mg of protein) is shown within the appropriate segment of retina in A and on the ordinate in A and B. (A) Each left retina was cut into eight 45° sectors (7.25 mm in length), which were divided into central (4.9 mm) and outer (2.35 mm) segments. (B) Demonstration that TOP concentration detected is a function of the square of distance from the ventroanterior margin of the retina. (A) Strips of retina 2.5 mm wide running from the ventroanterior margin (0% distance) to the dorsopos- terior margin of the retina (14.5 mm = 100% of maximal distance), parallel to the choroid fissure, were removed from eight retinas (left eyes) and each was cut into nine 1.5-mm segments as shown. (0) Strips of retina from anterior (0%) to posterior (100%) margins of retina were prepared and assayed as above. (0) The data from A. The length of the arc from the ventral pole of the gradient to the center of each segment was calculated assuming the retina to be a hemisphere and using equations relating angles and sides of spherical triangles. 236 Monoclonal Antibodies to Neural Antigens uniform antigen concentration along any line of radius, or varies with distance from the dorsoposterior to the ventroanterior margins of the retina, left retinas of 14-day chick embryos were divided into 16 sections as shown in Figure 4A and assayed for TOP. Specific {°I]F(ab’), binding varied from 0.35 pmoles per mg of protein at the ventroanterior margin to 15.5 pmoles per mg of protein at the dorso- posterior margin of the retina. Thus, a 35-fold gradient of TOP antigen was detected, aligned with the dorsoposterior-ventroanterior axis of the eye (i.e., oriented parallel to the long axis of the choroid fissure). Antigen concentration detected was a logarithmic function of distance from the dorsoposterior pole of the gradient to the ventroan- terior pole along the circumference of the retina (Fig. 4B). In contrast, little or no variation in TOP antigen concentration was detected along the axis perpendicular to the choroid fissure. The concentration of ['5I]F(ab’).santi-TOP antibody-TOP antigen complex detected (F,) is a function of the square of the circumferential distance (D,) from the ventroanterior pole of the gradient toward the dorsoposterior pole. Thus, TOP molecules can be used as a marker of cell position along a ventroanterior-dorsoposterior axis of retina, i.e., Dy = Dmax (Fy/Fmax)°*, where Dax and Fax are maximal values for retina at the dorsoposter- ior margin of the retina. Under the conditions used for the experi- ments shown in Figure 4, A and B, F,,,, was 20 pmoles of ['?5I]F(ab’), bound specifically per mg of protein, and Dy, was 14.5 mm. Thus, the calculated mean position in retina of cells that bind 5 pmoles of ('5I]F(ab’), specifically per mg of protein is 7.25 mm from the ven- troanterior pole of the gradient, which agrees well with the experi- mentally determined values. EXPRESSION OF TOP ANTIGEN DURING DEVELOPMENT * The antigen was detected in the optic cups of 48-hour chick em- bryos, and evidence for a gradient of TOP in retina was found with 4- day chick embryos, the earliest stage tested, through the adult (Fig. 5). Whereas the amount of TOP detected per mg of ventral retina protein remained constant throughout development, the concentra- tion of TOP antigen in dorsal retina increased threefold between the 4th and 12th days of embryonic development and then decreased somewhat in the adult. In summary, these results suggest that a gradient of TOP molecules is formed early in retina development, during active neuroblast proliferation and neuron genesis, and is maintained after neuron genesis ceases. A Gradient of Molecules in Avian Retina 237                                           2 us TTT < TTT 5 A. TOPOGRAPHIC GRADIENT | 2 B. ANTIGEN AND 4 VS. DEVELOPMENTAL AGE G PROTEIN/RETINA @ 20+ 16 4 = 100 100 3 2 4+ 4 F 1251-Ftab') } > a L 8 < 3 15+ FO > 10F 102 > 4 z LE ta z 0 “44 2 € a S 4 81216A0) 5 : z = 19 os | DB yol PROTEIN | 45 i Ww ” FE c a nN - © wn = T o a 3 E 2 4 2 4 2 § g oN 0 a: 3 a Ooh pi ris 4 0.01 0.01 2912345678 4 8 12. 16 20 ADULT RETINA SECTION DAYS AFTER FERTILIZATION Figure 5 Expression of TOP antigen in chick retina during embryonic develop- ment and in the adult. (A) Symbols represent antigen concentration detected in each of eight radial sectors of retina (shown in Fig. 2) from chick embryos at: (o) day 8, (A) 10, (a) 12, {@ ) 14, (W) 16, (®) 18, and (us) adult. The insert depicts antigen detected in dorsal or ventral halves of retina from 4- through 18-day embryos and adults. (B) (0) TOP antigen detected per retina; (A) protein per retina; (s) [“°I]F(ab’), spe- cifically bound per mg of protein. A chick embryo was found with a third eye in the middle of the forehead (Fig. 6); retinas from the right, middle, and left eyes each had a gradient of TOP molecules with normal symmetry, polarity, and orientation. Thus, a gradient of TOP with normal orientation was generated in the third eye despite the abnormal orientation of the eye in the embryo. ANTIGEN DISTRIBUTION * The antigen was detected by autoradiography (Fig. 7) or indirect immunofluorescence on most, if not all, cell types in 14-day chick embryo dorsoposterior retina. More antigen was detected in the inner and outer synaptic regions than elsewhere in the retina. Little antigen was detected in ventroanterior retina. All mechanically dissociated cells examined from 8-day chick embryo dorsoposterior retina had punctate rim fluorescence; all cells examined from middle retina also N 38 Monoclonal Antibodies to Neural Antigens B 4   q 1 T ui Lerteve | 8-             za 9 - oO a < 4 z ti 5 18+ 4 z 3 ° a 10+ = rs 5 3 a a 5r - 8 3 ° BEAKS l i | l i 1 1 1 = 12345867 8 RETINA SECTION Figure 6 (A) TOP antigen gradients in retinas from the (0) right, (¢) middle, and (A) left eyes of a 14-day chick embryo with three eyes. Total [°F(ab’), bound per retina section is shown. Reaction mixtures contained 2.38 nM [”5I]F(ab’},. {B) Frontal view of head of embryo. The third eye is situated on the forehead and oriented in a dorsoanterior direction. The embryo had two pairs of beaks, two brains in one head, and one body. were fluorescent, but less intensely than dorsoposterior retina cells. No fluorescent cells were detected in ventroanterior retina. The highest concentrations of TOP antigen were found in tissue derived from the forebrain (Table 1): retina > cerebrum > thalamus. Lower levels of antigen were found in optic nerve, cerebellum, dorsal and ventral retina pigment epithelium, and optic tectum. Little or no antigen was detected in heart, liver, kidney, or blood cells. Gradients of TOP molecules with similar orientation and symme- try were detected in turkey, quail, and duck retina (Fig. 8), but the antigen was not detected in goldfish, Xenopus laevis, Rana pipiens, or postnatal Fisher rat retina. PROPERTIES OF TOP ANTIGEN * Incubation of 14-day chick embryo retina cells at 100°C or incuba- tion at 37°C with 11 mM trypsin resulted in loss of antigenic activity from cells (Table 2). To study the possible involvement of carbohy- drate residues, hapten-competition experiments were performed (Table 2). Incubation of retina cells with wheat germ agglutinin (which binds to 8-N-acetylglucosaminyl residues) at 4°C reduced   Figure 7 Autoradiographs of 14-day chick embryo retina. Frozen sections (16 ym) from outer halves of dorsal or ventral retina were treated with antibody to TOP, followed by [!I]F(ab’)., exposed 22 days, and stained with toluidine blue. (A) Dark-field and (B) phase-contrast views of dorsal retina. In A, some silver grains over cel] soma in the inner nuclear layer appear dim due to staining of cells by toluidine blue. (C) Dark-field and (D) phase-contrast views of ventral retina. (R) photoreceptor layer; (OS) outer synaptic layer; (IN) inner nuclear layer; (IS) inner synaptic layer; (G) ganglion cell layer; (A) ganglion cell axon layer. Magnification, 517. 239 Table 1 Distribution of TOP Antigen in Chicken Tissues         OO a 14-Day embryo Adult Tissue (pmoles [!**I]F(ab'), specifically bound/mg protein) Dorsal neural retina 12.0 7.70 Ventral neural retina 1.08 2.70 Cerebrum 3.30 3.12 Thalamus 2.13 _ Optic nerve _ 0.58 Optic tectum 0.15 ~ Cerebellum 0.23 0.46 Dorsal retina pigment epithelium 0.26 - Ventral retina pigment epithelium 0.25 - Heart, liver, kidney, or blood cells 0.002 —0.055               12 T q v T T T TOT TURKEY > 3 =z a Of 8 4 #S oa ae ae uL=> ra £8 ° Oo 4b 4 oa 2 oO 0 1 1 L i i 1 123 45 67 8 RETINA SECTION Figure 8 TOP antigen gradients in retina from: (co) Japanese quail (15-day embryo); (A) White Pekin duck (16-day em- bryo); (0) turkey (17-day embryo); the eggs hatch 17, 28, and 28 days after fertilization, respectively. ['25I]F(ab’), concentrations and «Ci/pmole were 0.26 nM (0.90 wCi/pmole), 0.074 nM (2.92 ~Ci/pmole), and 0.078 nM (2.38 xCi/pmole), respectively. 240 A Gradient of Molecules in Avian Retina 241 Table 2 Effect of Trypsin, Heat, or Lectins on TOP Antigenicity eee eee eee eee e ee cena ee ence eee cpm [{5]]F(ab’),   Treatment of retina cells     Exp. bound specifically no. 0-30 min 30—40 min to retina cells % 1 control + trypsin inhibitor 1700 100 trypsin + trypsin inhibitor 93 6 trypsin + trypsin inhibitor _ 1803 106 2 4°C, 30 min 1607 100 100°C, 30 min 132 8 3 _ control 467 100 50 wg wheat germ agglutinin 271 58 50 wg concanavalin A 951 204 50 wg Ulex europaeus agglutinin I 607 130   Properties of TOP antigen molecules in 14-day chick embryo dorsal retina. In experiment 1, retina cells were incubated for 30 min at 37°C, in either phosphate- buffered saline (PBS), PBS with 11 4M trypsin (crystallized three times; Worthington), or PBS with 11 uM trypsin inactivated with 12 4m soybean trypsin inhibitor (Worthing- ton), then for 10 min with soybean trypsin inhibitor. TOP ascites fluid was diluted 1000-fold; 1.86 nm [\"5I}F(ab’), (9.05 x 10-? «Ci/pmole) was used. In experiment 2, TOP and P3-X63/Ag-8 antibodies were diluted 100-fold; 1.82 nm [I]F(ab’), (9.69 x 107? »Ci/pmole) was used. In experiment 3, retina cells were incubated with lectins (50 ug per 6 x 10° cells) for 15 min at 4°C, washed to remove unbound lectin, and assayed for TOP. TOP and P3-X63/Ag-8 antibodies were diluted 500-fold. antibody binding to 58% of the control value, whereas Ulex euro- paeus agglutinin I (specific for L-fucosyl residues) had no effect, and concanavalin A (specific for a-D-mannosy! and a-D-glucosy] residues) increased antibody binding twofold. Increased anti-TOP antibody binding was also observed after treatment with succinylated concana- valin A. ANTIGEN CONTENT OF CULTURED RETINA CELLS * Cells dissociated from 8-day chick embryo retina with 0.05% tryp- sin (crystallized three times), 0.003% collagenase, 2% chick serum, and 0.001% DNase contain little or no TOP antigen; however, cells cultured for 24 hours in vitro bound 50% as much antibody to TOP as did cells from intact retina in ovo. With cells dissociated with trypsin from dorsal or ventral 8-day embryo retina and cultured for 6 days, approximately the same amount of antigen per mg of protein was 242 Monoclonal Antibodies to Neural Antigens   qv v v ANTIGEN EXPRESSION IN COCULTURES OF DORSAL AND VENTRAL RETINA CELLS          14 DAYS IN OVO          8 DAYS IN OVO + 6 DAYS IN VITRO     pMOL \"75)-F(ab’}, SPECIFICALLY BOUND/mg PROTEIN a qT       PERCENT DORSAL CELLS   PERCENT VENTRAL CELLS Figure 9 TOP antigen in monolayer cultures contain- ing varying proportions of cells dissociated with 0.05% trypsin and 0.005% DNase from dorsal or ventral 8-day chick embryo retina and cultured for 6 days. Each 100-mm culture plate contained 8 x 10’ retina cells and 15 ml of medium (90% Eagle’s minimal essential medium, 10% fetal bovine serum). detected as with dorsal and ventral 14-day chick embryo retina in ovo (Fig. 9). With trypsinized cells from dorsal and ventral retina that were mixed in different proportions and cocultured for 6 days, anti- gen levels detected were nearly additive (Fig. 9; linear correlation coefficient, r’, 0.98). No evidence of induction or suppression of antigen was obtained under the conditions tested. Dorsal, middle, and ventral 8-day chick embryo retina cells were dissociated with trypsin collagenase, chick serum, and DNase, then cultured for 4 days, dissociated a second time by the same method, and cultured for 6 additional days. The antigen contents detected were similar to those found with dorsal, middle, and ventral retina cells that were dissociated only once and then were cultured for 10 days (Fig. 10). Thus, a gradient of antigen can be reexpressed and maintained for at least 10 days in culture. A Gradient of Molecules in Avian Retina 243   ANTIGEN GRADIENT IN 10 DAY CULTURES OF RETINA CELLS TRYPSIN TRYPSIN DAY 0 DAYS 0,4         pMOL \"°1-Fiab’), SPECIFICALLY BOUND/mg PROTEIN i T     | 0 M Dd v OM V RETINA SECTION Figure 10 TOP antigen in monolayer cultures con- taining cells dissociated with 0.05% trypsin, 0.003% collagenase, 2% chick serum, and 0.001% DNase from dorsal, middle, or ventral 8-day chick embryo retina, and cultured for 10 days as in Fig. 9; where indicated, cells were dis- sociated again as above on the 4th day of culture, recovered, and cultured in fresh 100-mm plates for 6 additional days. MONOCLONAL ANTIBODIES TO RETINA HYBRID CELL LINES ¢ As an alternative strategy to identify surface membrane molecules localized to restricted topographic regions of the neural retina, mono- clonal antibodies were generated against clonal hybrid cell lines originated by fusing retina cells from rat, mouse, or Chinese hamster embryos with clonal mouse neuroblastoma N18TG2 or human fibro- blast VA-2-M7 cells. The retina hybrid cell lines used for immuniza- tion of mice were: NCE-9AK (Chinese hamster embryo retina x N18TG-2), N18RE-103 (Fisher rat embryo retina x N18TG-2), N18ME- 1 (C57/B1 mouse embryo retina x N18TG-2), and CHEM7A2al (Chi- nese hamster embryo retina x VA-2-M7). NCE-9AK, N18RE-103, and 244 Monoclonal Antibodies to Neural Antigens N18ME-1 hybrid cells generate action potentials when stimulated electrically, possess tetrodotoxin-sensitive Na* channels, extend long neurites, and resemble neurons in morphology. N18RE-103 cells syn- thesize dopamine, and N18ME-1 cells synthesize norepinephrine (E. Heldman et al., unpubl.). In contrast, neither catecholamine synthesis nor tyrosine hydroxylase activity was detected in parental N18TG-2 neuroblastoma cells. NCE-9AK cells possess large, dense-core vesicles and are more adhesive to 10-day chick embryo retina cells than to cerebral cortex cells or N18TG-2 cells (M. Delong and M. Nirenberg, unpubl). Spleen cells from immunized mice were fused with P3-X63/Ag-8 mouse myeloma cells, and 1136 hybridoma cell lines were obtained; 133 hybridoma cell lines were obtained that synthesize antibodies that bind to the retina hybrid cells used for immunization (11.7%). Twenty-eight antibodies bound to retina hybrid cells but not to N18TG-2 or VA-2-M7 parental cells or other nonneural cell lines tested from the same species as the retina parental cells. CONCLUSIONS ¢ A monoclonal antibody was obtained that defines a cell-surface antigen distributed in a 35-fold concentration gradient in avian ret- ina. The concentration of antigen is highest at the dorsoposterior margin of the retina and lowest at the ventroanterior margin. The antigen was found on most or all cell types in chick retina, and the amount of antigen detected is related to cell position in the retina. The concentration of antigen found is a function of the square of circumferential distance from the ventroanterior pole of the gradient toward the dorsoposterior pole. The dorsoposterior portion of the retina differs from other portions of the retina in neuronal lineage and embryologic development, migration of cells and axons across the midline of the embryo, synapse specificity, adhesive specificity, and function. The function of TOP molecules has not been determined; however, our working hypothesis is that TOP molecules play a role in the encoding or decoding of positional information in the retina. The mechanism of generating and maintaining the highly ordered topo- graphic gradient also remains to be determined. REFERENCES Barnstable, C.J. 1980. Monoclonal antibodies which recognize different cell types in the rat retina. Nature 286:231. Eisenbarth, G.S., F.S. Walsh, and M. Nirenberg. 1979. Monoclonal antibody to a plasma membrane antigen of neurons. Proc. Natl. Acad. Sci. 76:4913. A Gradient of Molecules in Avian Retina 245 Galfre, G., S.C. Howe, C. Milstein, G.W. Butcher, and J.C. Howard. 1977. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266:550. Goldschneider, I. and A.A. Moscona. 1972. Tissue-specific cell-surface anti- gens in embryonic cells. J. Cell Biol. 53:435. Hausman, R.E. and A.A. Moscona. 1979. Immunologic detection of retina cognin on the surface of embryonic cells. Exp. Cell Res. 119:191. Kohler, G. and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495. Schneider, M.D. and G.S. Eisenbarth. 1979. Transfer plate radioassay using cell monolayers to detect anti-cell surface antibodies produced by lym- phocyte hybridomas. J. Immunol. Methods 29:331. Silver, J. and R.L. Sidman. 1980. A mechanism for the guidance and topo- graphic patterning of retinal ganglion cell axons. J. Comp. Neurol. 189:101. Sperry, R.W. 1963. Chemoaffinity in the orderly growth of nerve fiber pat- terns and connections. Proc. Natl. Acad. Sci. 50:703. Thiery, J.-P., R. Brackenbury, U. Rutishauser, and G.M. Edelman. 1977. Adhe- sion among neural cells of the chick embryo. IJ. Purification and char- acterization of a cell adhesion molecules from neural retina. J. Biol. Chem. 252:6841. Trisler, G.D., M.D. Schneider, and M. Nirenberg. 1981. A topographic gradi- ent of molecules in retina can be used to identify neuron position. Proc. Natl. Acad. Sci. 78:2145. Trisler, G.D., M.A. Donlon, W.G. Shain, and H.G. Coon. 1979. Recognition of antigenic differences among neurons using antiserums to clonal neural retina hybrid cells. Fed. Proc. 38:2368.", "Moskal, Joseph R. ; Nirenberg, Marshall W. ; Schneider, Michael D. ; Trisler, G. David", null, "Monoclonal Antibodies Against Neural Antigens", "Cold Spring Harbor Reports in the Neurosciences", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ptqm-7ekh.8e4y", "00000000-0000-0000-1299-17F37DB6A450", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Synapse Formation by Neuroblastoma Hybrid Cells", "101584910X166", null, "1983", "1983", "This article maintains that both biological and environmental factors are important to consider in neural synaptic formation.  Using the clonal line of neuroblastoma and somatic hybrid cells developed by Nirenberg and the biochemical genetics team at NIH, the experiment demonstrates that synaptogenesis can be influenced by the activation of the enzyme adenylate cyclase.", "Articles", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "9", "pages", "Text", "English", "Reproduced with permission of Cold Spring Harbor Laboratory Press.", "Copyright may apply", null, null, "Synapse Formation by Neuroblastoma Hybrid Cells M. NIRENBERG, S.P. WILSON, H. HIGASHIDA, A. ROTTER, K. KREUGER, N. BUSIS, R. Ray, J. KENIMER, M. ADLER, AND H. FUKUI Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205 The most widely held hypothesis concerning the mechanism of synapse formation during the last 20 years has been the chemoaffinity hypothesis of Sperry (1963), i.e., that neurons distinguish appropriate from inappropriate synaptic partner cells by interactions be- tween molecules that code for cell recognition. Al- though Sperry avoided detailed models, many in- vestigators have assumed that neurons or other cells that possess different cell-recognition molecules are generated and that the cells then sort out in appropriate sequences by differential adhesiveness before synaptic connections form. Another quite different hypothesis is that neurons may form relatively stable intercellular attachments during differentiation and that environmen- tal factors and/or transynaptic signals may regulate the expression of genes and thereby determine the final pathway for differentiation and the type of neuron and synapse to be expressed. These hypotheses are not mutually exclusive; both may be involved in the assembly of synaptic circuits. We have established clonal lines of neuroblastoma and somatic hybrid cells that express neural properties and form synapses with cultured striated muscle cells, and we have used the cells as model systems for studies on the effects of extracellular molecules on the expres- sion of neural properties and on synaptogenesis. We find that the expression of stimulus-secretion coupling and synaptogenesis can be regulated in neuroblastoma hybrid cells by receptor-mediated events that are cou- pled to the activation of adenylate cyclase. We also find that many *S-labeled glycoproteins are affected when cells are shifted to a synapse competent state. RESULTS Synapse Competent and Defective Cell Lines Twenty-six neuroblastoma or hybrid cell lines were tested for their ability to synthesize and release acetyl- choline (ACh) and form synapses with cultured rat striated muscle cells (Higashida et al. 1978; Wilson et al. 1978). Synapses were detected by intracellular recording of miniature end-plate potentials and evoked muscle responses. Photomicrographs of cells from four of the five cell lines found that form abundant synapses are shown in Figure 1. NBrlO-A and NBr20-A cells originated by fusion of mouse neuroblastoma N18TG-2 (Minna et al. 1972) with clonal BRL-30E rat liver cells; NCB-20, by fusion of NI8TG- 2 cells with fetal Chinese hamster brain cells; and NG108-15 (Nelson et al. 1976), by fusion of NI8TG-2 with C6BU-1 rat gli- 707 oma cells (Amano et al. 1974). Muscle responses to ACh secreted by the neural cells were inhibited rever- sibly by 5 um d-tubocurarine and irreversibly by 10 nM a-bungarotoxin. Omission of Ca** ions in the me- dium reduced the frequency of miniature end-plate po- tentials (Nelson et al. 1978). The expression of some neural properties by the cells is dependent on prolonged elevation of cellular cAMP. For example, untreated cells have few or no neurites and relatively small soma. However, treatment of cells for 7 days with 1 mM dibutyryl cAMP promotes neurite extension, and decreasing the concentration of fetal bovine serum reduces neurite retraction (Seeds et al. 1970). Neurites are produced in profusion under these conditions as shown in Figure 1. Other cell lines were found that synthesize ACh and adhere well to myotubes, but do not form synapses. The phenotypes of the cell lines are summarized in Table 1. Cells from five lines synthesize ACh, take up Ca** ions from the medium and secrete ACh when depolarized by 80 mM K* ions, possess small clear vesicles 60 nm in diameter and large dense-core vesicles 180 nm in diameter, synthesize and secrete a protein that stimulates the aggregation of nicotinic ACh receptors (AChR) on myotube plasma membranes, and form many synapses with cultured myotubes. Cells from three lines take up Ca** ions slowly and to only a small extent, secrete relatively little ACh when depolarized, and form few synapses with muscle cells. Two cell lines lack functional voltage-sensitive Ca* + channels and therefore do not take up “Ca*+ or secrete more ACh when depolarized and do not form synapses. Cells from five lines take up Cat* when depolarized, but do not respond to the increase in cytoplasmic Ca*+* concentration by secreting ACh, and form few or no synapses. These cells release ACh into the medium in the basal unstimulated state but lack a Ca**-dependent ACh secretion reaction(s). Three cell lines possess small clear vesicles, but lack both large dense-core vesicles and functional AChR ag- gregation protein, and form few or no synapses. Nine additional cell lines have little or no choline acetyltransferase activity and thus synthesize little or no ACh and do not form synapses with striated muscle cells. Regulation of Synaptogenesis Neuroblastoma and hybrid cells have PGE, receptors that mediate the activation of adenylate cyclase (Sharma 708 NIRENBERG ET AL.   NBr20A     Figure 1. Neuroblastoma hybrid cells from lines that form many synapses with cultured myotubes were treated for 7 days with | mm dibutyryl cAMP, and the concentration of fetal bovine serum was reduced from 5% to 1% between the fifth and seventh days (H. Higashida et al., in prep.). Bar, 50 um. et al. 1975b, 1977). Celis were cultured in the presence of PGE, to increase the endogenous rate of synthesis of cAMP and elevate intraceliular levels of cAMP (Matsuzawa and Nirenberg 1975; Sharma et al. 1975a). Treatment of cells with PGE; has no immediate effect on cell membrane potential or rate of ACh secretion (McGee et al. 1978). In Table 2 are shown the effects of treating NG108-15 cells and rat myotubes with PGE), or a cyclic nucleotide phosphodiesterase inhibitor such as dibutyryl cAMP or theophylline, on the percentage of myotubes tested that were innervated and the frequency of miniature end-plate potentials of myotubes. The cells were cocultured and treated with the compounds shown in Table 2 for 5-7 days before myotubes were assayed for synapses by intracellular microelectrode recording. Treatment of cells with | mM dibutyryl cAMP, | mM theophylline, or 10 4m PGE, increased the percentage of muscle cells tested that were inner- vated from 15% to approximately 60% and also Table 1. Cell Line Phenotypes       K * -dependent Vesicles AChR ACh large aggrega- Cell forma “Cat+  HJACh small dense tion lines tion uptake release clear core protein Synapse 5 + +++ +++ + + + +++ 3 + + + + + + + 2 + - - + + + - 5 + ++ - + + + -or+ 3 + ++ + + - - -or+ 9 - - Data from Higashida et al. (1978), Rotter et al. (1978), Wilson et al. (1978), and N.A. Busis, M.P. Daniels, H.C. Bauer, P.A. Pudimat, P. Sonderegger, A.E. Schaffner, and M. Nirenberg. SYNAPTOGENESIS BY NEUROBLASTOMA CELLS Table 2. Effect of Culture Conditions on Synaptogenesis and ACh Secretion by NG108-15 Cells     % Synaptic myotubes —_responses/ Culture with min/ conditions synapses myotube Control 1S 0.7 Dibutyryl] cAMP (1 mm) 55 14 Theophylline (1 mm) 64 10 PGE, (10 pm) 63 1] PGE, (10 pm) + theophylline (1 mm) 98 32   Each value is the mean of values obtained from more than 75 myotubes (Higashida et al. 1978). resulted in 14- to 20-fold increases in the mean miniature end-plate-potential frequencies (Higashida et al. 1978). Presumably, each miniature end-plate poten- tial reflects the response of a myotube to the spon- taneous release of ACh from a single NG108-15 vesi- cle. The effects of PGE, and theophylline were ad- ditive or synergistic since 98% of the myotubes tested were innervated and a 45-fold increase in the miniature end-plate-potential frequency was observed. As shown in Figure 2, the effects of PGE, theophylline, or dibutyryl cAMP on synaptogenesis are expressed slowly. Half-maximal increases in myotube innervation required 1-2 days of treatment. In other ex- periments not shown here, cells were incubated with PGE, theophylline, dibutyryl cAMP, or PGE, and theophylline for 5-7 days; the cells were then incubated for an additional 4-14 days in the absence of the com-              LG T T T if T T T T T  |@) REGULATION OF SYNAPSE FORMATION <—t Z joo) PGE! + THEOPHYLLINE | wn x = 80r DIBUTYRYL cAMP 4 ep) 5 ay 6OF / 4 oO / wa 3 40, / CONTROL 1 3 if _ CONTROL = 20f /// + OQ “4 38 1 j 1 1 1 8 | 2 3 34 =5 oO} DAYS Figure 2. Effects of PGE), theophylline, or dibutyryl cAMP on the percentage of myotubes innervated by NG108-15 cells are shown as a function of time of coculture. At zero time, 2x 10° mechanically dissociated NG108-15 cells were added to each 35-mm petri dish, which contained well-differentiated myotubes that had formed from myoblasts dissociated from newborn rat hind-limbs during 9 days of culture. The cells were cocultured in Dulbecco’s modified Eagle's medium, 5% horse serum, 100 pm hypoxanthine, 16 »m thymidine, and the following when indicated: Control, no addition; 1 mM theo- phylline; 10 am PGE; 10 xm PGE, and 1 mm theophylline; or | mM dibutyryl cAMP. Synapses were detected by in- tracellular microelectrode recording of miniature end-plate potentials. Each point is the mean of values obtained from 10-20 myotubes. (Data from Higashida et al. 1978.) 709 pounds to determine whether the effects on synapses were reversible. On cessation of treatment, synapses and ACh secretion rates slowly returned to control values over a period of 7-10 days. Thus, the effects of the compounds on synaptogenesis were expressed slow- ly, and on withdrawal of the compounds, the effects were reversed slowly. Treatment of NG108-15 cells with PGE, and Ro20-1724, a cAMP phosphodiesterase inhibitor, also increased the number of synapses. How- ever, no reversal was detected due to withdrawal of these compounds. Effects of Culture Conditions on ACh Storage and Secretion As shown in Figure 3A, intracellular ACh levels of NG108-15 cells increased eightfold and threefold when cells were treated for 3 or more days with 10 uM PGE, and 1 mM theophylline or with 1 mM dibutyryl cAMP, respectively. The cells were incubated with 30 um (?H]choline for 17 hours to label intracellular ACh. Cells were then washed, and intracellular [H]ACh was extracted and quantitated. More than 90% of the in- tracellular ]H}ACh was found to be particulate. Treat- ment of cells for 5 or more days with dibutyryl cAMP (Daniels and Hamprecht 1974) or with PGE, and theo- phylline (Wilson et al. 1978) resulted in marked in- creases in the abundance of small clear vesicles and large dense-core vesicles. These results suggest that the increase in intracellular ACh is due, at least in part, to             z  I@®Palachin — [B)ceLcucar camPlC)PRoTEIN/FLASK a NGIO8-IS CELLS Zc 5 “g & 300° pce + + ® PGEL+ t CONTROL @ © 9 a THEOPHYLLINE THEOPHYLLINE u £ a y x « s \\\\ a @ 2001 +h 1 fo dag a \\\\ Ww x | 2 & [ | ee ; i 3 «0 / Bt, cAMP | , S 100 F y ye % ir, a CONTROL ‘ > 5 CONTROL / e ao 4 PGEI + : = THEOPHYLLINE. 4 1 4 4 1 1 1 4 1 1 1 £ tt LL. be k 1 1 1 QO O13 5 70! 38 5 7O!1 3 5 7 DAYS Figure 3. Effects of dibutyryl cAMP or PGE, and theophyl- line on intracellular PHJACh levels of NG108-15 cells (A) and intracellular cAMP (8); (C) cell proteins per flask are shown as a function of time (Wilson et al. 1978). (A) NG108-15 cells were incubated in culture medium containing approximately 30 um [methyl--H]choline chloride for 17 hr to enable cells to synthesize [SHJACh. Cells were washed 3 times with isotonic buffer, and then intracellular FHJACh was extracted (Toru and Aprison 1966) and separated from other radioactive compounds by high-voltage paper electro- phoresis (Potter and Murphy 1967). Where indicated, the medium was supplemented with the following: Control, no addition; 1 mm dibutyryl cAMP; or 10 wm PGE; and 1 mm theophylline. (B) cAMP was extracted from cells and purified as described by Matsuzawa and Nirenberg (1975); the amount of cAMP in each sample was determined by inhibition of the binding of H]cAMP to cAMP-dependent protein kinase. (C) Protein was determined by a modification of the method of Lowry et al. (1951). 710 an increase in the number of ACh storage vesicles in cells. In the presence of 10 yM PGE, and | mM theophylline, cAMP levels of NG108-15 cells increased markedly and then decreased somewhat during the re- mainder of the incubation period due to partial desen- sitization of PGE, receptors (Fig. 3B). However, cAMP levels of treated cells were higher than those of control cells throughout the 7-day period examined. The rate of cell division also decreased after the first day of treatment with PGE, and theophylline; thus, the rate of accumulation of protein per flask decreased in the presence of PGE, and theophylline (Fig. 3C). The effect of treating NG108-15 cells for 0, 1, 3, or 5 days with 1 mM dibutyryl cAMP on the ability of cells to secrete ACh in response to a depolarizing stimulus is shown in Figure 4A. The cells were in- cubated with 27 uM (H]choline for 1 hour, washed by perfusion, and then depolarized with 80 mm K+ (replacing 80 mM Na‘) during the period shown. [HJACh secreted into the medium was separated from other *H-labeled compounds and quantitated as described by McGee et al. (1980). Logarithmically ‘dividing, untreated control cells did not respond to depolarization by secreting -ACh. However, cells treated 1, 3, or 5 days with 1 mM dibutyryl cAMP became increasingly responsive to depolarization with respect to the amount of ACh secreted (McGee et al. 1978). Cells treated with PGE, and theophylline for 7 days secreted twice as much ACh when depolarized as did cells that had been treated with dibutyryl cAMP (Fig. 4B). REGULATION OF K*-DEPENDENT Bulach RELEASE FROM CELLS           T r T r T T Zz - z a LLS TREATED WITH OIBUTYRYL| CELLS. TREATED WITH - | cAMP FOR O- 504s BND THEOPHYLLINE ee! e fe 250P 4500 = sais rows | OE ~ 200+ 4400 5 e lw lu R 3DA) a © 150b 4300 a # 3 ij uy J 100F 4200 ¢ x x 3 {DAY g 4 50 5100 a 0 ma a ° AT 3 $ eet e 1. lo = oO 2 4° 6 8 0 2 4 6 8 10 MINUTES Figure 4. HJACh secretion by untreated control NG108-15 cells or by cells treated for 1, 3, or 5 days with 1 mm dibutyryl! cAMP (A) or by cells cultured for 7 days with or without 10 uM PGE, and | mm theophylline (B) is shown in response to depolarization of cells with 80 mm K* (replacing 80 mm Na* in the medium). The cells were incubated with [merhylH]choline chloride to enable them to synthesize PHJACh, and then washed and depolarized. (Data from McGee et al. [1978]; the figure was redrawn.) PHIACh was separated from other radioactive compounds and quantitated as described by McGee et al. (1980). The basal rate of [HJACh secretion by unstimulated cells (5.4 mm K*) was subtracted from each value shown. NIRENBERG ET AL. Conditional Expression of Stimulus-Secretion Coupling in NBr10-A Cells Depolarization of neuronal terminals is known to ac- tivate voltage-sensitive Ca** channels; Cat* ions then flow into the neuronal terminals and increase the rate of secretion of neurotransmitter. Thus, the effects of treating cells for 7 days with PGE, and theophylline or dibutyryl cAMP on Ca** uptake via voltage- sensitive Ca* + channels were determined. “Ca** ions rapidly bound to NBr10-A cells initially and/or entered cells incubated in medium containing 5.4 mM K* ions, but uptake soon plateaued. Depolarization of cells with 80 mM K* resulted in a marked increase in “Ca** up- take. Half-maximal inhibition of depolarization- dependent uptake of “Ca** was obtained with 9x 10-7 M D600; La3+, Cot+*, or Ni+* ions also in- hibited K*-dependent “Ca++ by cells. Electrophysio- logical studies revealed Ca** spikes; action potentials also were obtained when strontium or barium ions were substituted for Ca*+* ions (not shown). As shown in Figure 5, logarithmically dividing, un- treated control NBrl0-A cells tacked functional voltage-sensitive Cat* channels and did not take up Ca++ ions when cells were depolarized. However, cells treated for 7 days with 10 uM PGE, and 1 mM theophylline or with 1 mM dibutyryl cAMP took up 4Ca*+ via voltage-sensitive Cat * channels when cells were depolarized with 80 mm K* (Rotter et al. 1979).                   = |K'-DEPENDENT SPECIFIC ““ca** 8 |UPTAKE BY CELLS EXPOSED s 6 TO DIFFERENT CONDITIONS Loe 1 > PGEI + THEOPHYLLINE G ot oe 1 Kb Oo Zot / 7 . Je / te OIBUTYRYL cAMP ~ 5 , x <—t a 20.5 + a 9 we | vy CONTROL CELLS 5 20 4 6 8 q MINUTES Figure 5. Effect of culture conditions on the expression of func- tional voltage-sensitive Ca*+* channels of NBrt0-A cells. “Ca++ uptake was due to activation of voltage-sensitive Ca* + channels of untreated, logarithmically dividing control NBr10-A cells and cells cultured for 6 days with 1 mm dibutyryl! cAMP or 10 wm PGE; and | mM theophylline. The cells were depolarized with 80 mm K* (in place of 80 mm Na*). Values for “5Ca*+* binding to cells and/or uptake at 5.4 mm K*, which were not inhibited by 100 um D600 and were not mediated by voltage-sensitive Ca* + channels were subtracted. Ca** uptake dependent on depolarization was completely inhibited by 100 um D600. (Data from Rotter et al. 1979) SYNAPTOGENESIS BY NEUROBLASTOMA CELLS 711                PERCENT OF NBriOA CELLS|@) RATE OF RISE OF Ca2* Orne ACTION ‘POTENTIAL ® ACTION POTENTIALS Ca2* CHANNEL ACTIVITY oor DIBUTYRYL camp | 7° a 80 448 DIQUTYRYL cAMP a 6 wn 360 13> oO Wn © NONE 5 td Oo a 40 425 Zz <x ty 20 none 7! = § Oa a st 2 3 4 § 6 ©   DAYS Figure 6. Acquisition of functional voltage-sensitive Ca* * channels by NBr10-A cells cultured with or without 1 mm dibutyryl cAMP is shown as a function of time of treatment. Ca** action potentials were detected by intracellular micro- electrode recording; each point is the mean of values obtained from 20 NBr10-A cells. Logarithmically dividing NBr10-A cells teached confluency on approximately the fourth day of culture. The medium contained 5 ym tetrodotoxin to inhibit voltage- sensitive Na* channels. (Data from Rotter et al. 1979.) Four methods were used to measure Ca*+ uptake de- pendent on cell depolarization: “Ca++ flux was measured; net uptake of Ca** ions from the medium by cells was determined using a Ca‘ *-specific elec- trode; Ca** ion concentrations were determined by a spectrophotometric assay with murexide (P. Darvenezia and M. Nirenberg, in prep.); and Ca** action potentials were demonstrated by electrophysiological methods with intracellular microelectrode recording. Results obtained by each method showed that the expression of voltage-sensitive Ca* * channels is increased by treat- ment of cells with compounds that elevate cellular cAMP levels. The acquisition of voltage-sensitive Ca*+* channel activity by NBr10-A cells measured with intracellular microelectrodes is shown in Figure 6 as a function of days of culture of cells with or without 1 mm dibutyryl cAMP. Cells were stimulated electrically with depolarizing pulses of current in the presence of 5 pM tetrodotoxin to inhibit voltage-sensitive Nat channels. Voltage-sensitive Ca** channel activity was deter- mined at the resting membrane potential and at a mem- brane potential of —90 mV adjusted with steady cur- rent. Only 10% of logarithmically dividing, untreated cells at low density possessed functional voltage- sensitive Ca*+* channels, and the Ca** spikes that were detected were relatively weak. The proportion of cells with Ca** spikes increased from 10% to 50% as untreated cells multiplied and formed confluent monolayers, whereas 100% of the cells tested that were treated for 3 days with dibutyryl] cAMP generated Ca* + spikes when stimulated electrically. The mean maximum rate of rise of Ca** spikes, a measure of Ca*++ chan- nel activity, increased approximately 20-fold when cells were treated with dibutyryl] cAMP (M. Adler and M. Nirenberg, in prep.) (Fig. 6B). A smaller increase was observed when logarithmically dividing, untreated con- trol cells formed confluent monolayers, revealing a small cell concentration or contact-dependent regula- tion of Ca** channel expression. These and the previous results show that when cAMP levels are elevated, cells acquire at similar rates voltage-sensitive Ca** channels, depolarization-dependent ACh secre- tion, and synapses. Nitrendipine and other dihydropyridines have been shown to reduce voltage-sensitive Cat * channel activ- ity in smooth muscle (for review, see Triggle and Swamy 1983), and specific binding sites for [*H]ni- trendipine have been found in smooth muscle (Bolger et al. 1982), cardiac muscle (Ehlert et al. 1982), and brain (Murphy and Snyder 1982). The receptors for nitren- dipine are thought to be either part of the voltage-sen- sitive Ca++ channel complex or regulators of channel activity. As shown in Table 3, few or no specific bind- ing sites for [*H]nitrendipine were detected in washed membranes prepared from logarithmically dividing control NBrl0-A cells, whereas specific binding sites were found in membranes prepared from NBrlQ-A cells that had been treated for 8 days with 10 4M PGE, and | mm theophylline (M. Nirenberg, L. Anderson, and A. Rotter, in prep.). Scatchard analysis revealed a single class of specific binding sites for [*HJnitrendi- pine with a dissociation constant of 2 x 10-'° M, which agrees well with values reported for other tissues (Bolger et al. 1982; Murphy and Snyder 1982). The maximum number of specific nitrendipine-binding sites in membranes from NBri0-A cells treated with PGE, and theophylline was found to be 61 fmoles/mg of membrane protein, which corresponds to approximate- ly 16,000 sites for nitrendipine per cell. SB37-B neuroblastoma x L cell hybrid cells synthesize ACh but have little or no voltage-sensitive Ca** channel activ- ity and thus do not secrete ACh when cells are depolar- ized and do not form synapses with muscle cells. Spe- cific binding sites for [3H]nitrendipine were not detected in membranes prepared from SB37-B cells grown with or without PGE, and theophylline. These results show that the cAMP-dependent expression of Table 3. PGE, and Theophylline Regulate the Number of Nitrendipine-binding Sites Expressed by NBrl0-A Cells Treatment of cells   fmoles (7H]nitrendipine   (5-8 days) specifically bound/mg protein NBrl0-A control <2 10 um PGE, + 1 mM theophylline 17 SB37-B control <2 10 um PGE + 1 mm theophylline <2   Data from M. Nirenberg, L. Anderson, R. Rotter. and R. Ray (in prep.). The reaction-mixture components and con- ditions were similar to those reported by Murphy and Snyder (1982). Each reaction mixture contained 0.2 nm (PH)nitrendipine and approximately 250 yg of protein (a washed membrane fraction pelleted at 48,000g). 712 NIRENBERG ET AL. voltage-sensitive Ca** channels in NBrl0-A cells is accompanied by an increase in the number of specific binding sites for [*H]nitrendipine and suggest that elevation of cellular cAMP results in an increase in the number of Ca** channels in cells. cAMP-dependent Changes in Glycoproteins of NG108-15 Cells Elevation of cAMP levels of neuroblastoma or hybrid cells gradually results in the acquisition by cells of functional action potential channels for Na*, K+ (Nelson et al. 1978), and Ca++ (Rotter et al. 1979), which suggests that cAMP regulates the synthesis or catabolism of channel molecules. We therefore deter- mined the effect of PGE, on glycoproteins expressed CONTROL PGE1 by NG108-15 cells. Logarithmically dividing control NG108-15 cells and cells treated with 10 uM PGE, for 7 days were incubated for 18 hours with [**S]methi- onine. S-labeled glycoproteins soluble in 1% Triton X-100 were fractionated by chromatography on wheat germ agglutinin-, Lens culinaris agglutinin-, and Ric- inus communis agglutininy-agarose columns. *S-la- beled glycoproteins were eluted with 0.5 M N-acetyl- glucosamine, 0.2 M a-methylmannoside, and 0.2 M lactose, respectively, desalted, and subjected to two- dimensional polyacrylamide gel electrophoresis and autoradiography (Fig. 7). Autoradiographs of duplicate two-dimensional gels (triplicates in other experiments), exposed for 7 hours and I, 2, and 4 days, were com- pared with the aid of a computer and programs de- signed for analysis of two-dimensional gels (Vo et al. 1981). Computer-generated plots based on the 48 auto- radiographs analyzed in this experiment were obtained.   Figure 7. Two-dimensional electrophoresis of *§-labeled glycoprotein fractions. NG108-15 cells treated for 6 days with 10 um PGE, and   M,x10°3 logarithmically dividing, untreated control cells were incubated for [8 hr in Dulbecco's mod- ified Eagle's medium that contained 2% fetal bovine serum and 5 uM L-(*5S)methionine (100 pCi/ml of medium). The cells were harvested and washed in isotonic phosphate-buffered saline. The peileted cells were then lysed, and the proteins were solubilized by the addition of a solution containing 10 mm HEPES (pH 7.4): 150 mm NaCl; | mm CaChk, | mm MgCl, | mM MnCh; | mm _ phenylmethylsulfony! fluoride, and 1% Triton X-100. Cell lysates were centrifuged at 100,000g for 1 hr at 3°C, and glycoproteins in the supernatant portions were fractionated by wheat germ agglutinin-,       L. culinaris agglutinin-, and R. communis ag- glutininy-agarose column chromatography. *5S- labeled glycoproteins were eluted with 0.5 M N- acetylglucosamine, 0.2 M a-methylmannoside, or 0.2 M lactose, respectively, dialyzed against 0.01 mM NHsHCOs, and lyophilized. Two por- tions from each sample were subjected to two- dimensional polyacrylamide gel electrophoresis on duplicate 7.5% acrylamide gels according to the method of O'Farrell (1975) with 1.0 x 10° cpm (10-20 ug of protein) applied to each gel. Autoradiographs were exposed for 7 hr and 1, 2, and 4 days; those shown were exposed for 2 days. Arrows indicate some of the cAMP-de- pendent differences in *°S-labeled glycopro-       teins, i.e., control cells vs. cells treated with PGE). (Data from K.E. Krueger, M.I. Miller, and M. Nirenberg, in prep.) SYNAPTOGENESIS BY NEUROBLASTOMA CELLS In most cases, different species of S-labeled glyco- proteins were eluted from the three lectin columns. Most species of **S-labeled glycoproteins were syn- thesized by both control cells and PGE,-treated cells. However, numerous cAMP-dependent changes in the 35§-labeled glycoproteins were found as summarized in Table 4. Twelve *°S-labeled glycoproteins were ex- pressed by PGE,-treated cells that were not detected in extracts of logarithmically dividing control cells. In addition, the radioactivities of 29 S-labeled glycopro- teins expressed by cells treated with PGE; were 2.5- to 10-fold higher than those of control cells. cAMP- dependent decreases in the *5S-labeled glycoproteins were also found. Forty-eight S-labeled glycoproteins were expressed by control cells but not by cells treated with PGE). In addition, the radioactivities of 25 75S- labeled glycoproteins expressed by control cells were 2.5~-6.5-fold higher than those of cells treated with PGE). No changes in the *S-labeled glycoproteins were detected when cells were incubated with 10 um PGE; for 20 minutes (not shown here). DISCUSSION Neuroblastoma hybrid cells were obtained that syn- thesize ACh and form synapses with cultured striated muscle cells. Other cell lines were found that syn- thesize ACh, adhere to myotubes, but form few or no synapses. Different kinds of presynaptic defects were identified, such as cell lines without voltage-sensitive Ca** channel activity, large dense-core vesicles, depolarization-dependent ACh secretion, or active pro- tein that increases the aggregation of nicotinic AChR on myotube plasma membranes. No immediate effect of cAMP was detected on voltage-sensitive Ca** channel activity, ACh secre- tion, or the percentage of myotubes that were inner- vated, In contrast, these properties and synaptogenesis were regulated by prolonged elevation of cAMP levels of hybrid cells. Activation of adenylate cyclase with 10 uM PGE, for 5 days, or treatment of cells with | mM dibutyryl cAMP, resulted in 10-100-fold increases in voltage-sensitive Ca*+ * channel activity, 15-45-fold in- creases in spontaneous secretion of ACh at synapses, and 5-10-fold increases in the number of muscle cells innervated. Treated cells also contained considerably 713 more large dense-core vesicles and small clear vesicles and had higher levels of intracellular ACh than did un- treated cells. Treatment of NBrl10-A cells with 10 pM PGE, and 1 mM theophylline resulted in the appearance of speci- fic binding sites for [*H]nitrendipine, a putative antag- onist of voltage-sensitive Ca** channels. Specific ni- trendipine-binding sites were not detected in SB37-B cells, which lack voltage-sensitive Ca* * channel activ- ity. These results suggest that cells acquire more voltage-sensitive Ca** channels when cellular cAMP is elevated for a number of days. By regulating the expression of Ca*++* channels in hybrid cells, cAMP would be expected to function as a conditional switch that modulates the activities of many Ca**-dependent reactions in the neural cells and couples these reactions to the cell membrane potential and to transynaptic or hormonal stimuli that affect adenylate cyclase activity. Glycoproteins of NG108-15 hybrid cells grown in the presence of [*°S}methionine with or without 10 »M PGE, were solubilized and fractionated by wheat germ agglutinin, lentil lectin, or ricin column chromatog- raphy and by two-dimensional gel electrophoresis. Ele- vation of cellular cAMP levels resulted in the disap- pearance of some glycoproteins, the appearance of new glycoproteins of different relative molecular weights, large increases or decreases in the radioactivities of some **S-labeled glycoproteins, and changes in the iso- electric points of still other *S-labeled glycoproteins. These results agree with and extend previous reports of differentiation-specific changes in neuroblastoma pro- teins or glycoproteins (Truding et al. 1974, 1975; Charalampous 1977; Garvican and Brown 1977; Prashad et al. 1977; Akeson and Hsu 1978; Prashad and Rosenberg 1978; Rosenberg et al. 1978; Zisapel and Littauer 1979; Littauer et al. 1980; Atkinson and Bramwell 1981; Sugiyama et al. 1980; Imada and Im- ada 1982). Treatment of neuroblastoma or hybrid cells with di- butyryl cAMP has been shown to alter the levels of some species of polysomal mRNA (Morrison et al. 1980; U.Z. Littauer, pers. comm.). When ‘‘undif- ferentiated’’ neuroblastoma cells were shifted to a more differentiated state by altering various growth condi- tions, some species of polysomal poly(A)* RNA were no longer expressed (Felsani et al. 1978; Grouse et al. Table 4. cAMP-dependent Changes in S-la", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mvky~7vwq-c4ef", "00000000-0000-0000-54A4-DC78F1518AD0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Synapse Selection Based on Differences in Synapse Turnover", "101584910X167", null, "1983", "1983", "As a continuation of Marshall Nirenberg's work in neurobiology in the early 1980s, the study in this article shows different degrees of synapse formation in neurons and muscle tissue cells.  The experiment shows that \"rat retinal neurons form transient synapses with rat muscle cells only during a discrete period in development, whereas chick embryo spinal cord neurons form synapses with rat muscle cells at all developmental stages tested.\"  Findings suggest that different rates of turnover with synapses can result in the selective retention of one population of synapses and loss of the other.", "Articles", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "6", "pages", "Text", "English", "Reproduced with permission of Elsevier Scientific Publishing Company.", "Copyright may apply", null, null, "Int. J. Devi. Neuroscience, Vol. 1, No. 1, pp. 25-30, 1983 0736-5748/83 $3.00+-0.00 Printed in Great Britain. Pergamon Press Ltd. ISDN SYNAPSE SELECTION BASED ON DIFFERENCES IN SYNAPSE TURNOVER JEFFREY M. THomMpsoN,* GEORGE S. EISENBARTH,+ Rosert R. RuFFOLO, JR.¢ and MARSHALL NIRENBERG§ Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A. Abstract — Rat retinal neurons formed transient synapses with rat muscle cells in culture only during a discrete period in development, from the 20th day of embryonic development to the 7th neonatal day. In contrast, chick embryo spinal cord neurons formed synapses at all developmental stages tested, from the 2nd to the 18th day of embryonic development. The percentage of cells from the spinal cord that formed synapses with muscle cells was maximum at 4 days of embryonic development and decreased thereafter. However, the number of synapses with muscle formed by cells from 8-day embryonic spinal cord did not decrease during 14 days of culture. Under identical conditions, all synapses formed between rat retinal neurons and muscle cells were terminated during 7 days of culture. These results show that differences in the rates of turnover of two populations of cholinergic synapses can result in the selective retention of one population of synapses and the loss of the other, and thereby alter the specificity of synaptic connections. Key words: Synaptogenesis, Retinal neurons, Spinal cord neurons, Tissue culture, Synapse specificity, Development. Neurons dissociated from chick embryo retina rapidly form synapses with striated muscle cells in culture only between the 6th and 15th day of embryonic development. '®'® On the other hand, evi- dence from other systems has been reported which suggests that during development some neurons retain the ability to form synapses. '-? In adult vertebrates, denervation of striated muscle often leads to the formation of many new motorneuron—muscle synapses, but the specificity of the original synaptic circuits in most cases is not restored.” Chick retinal neuron-muscle synapses are transient and terminate with a half-time of 21 h, as opposed to synapses with neurons from chick embryo spinal cord which are retained for 14 days or more.'® Evidence for transient synapses in other systems has also been reported.'” For example, during development of the retinotectal projection in Xenopus, synaptic contacts between retinal ganglion neurons and tectal cells progressively shift.!°-!9 Neurons in the adult snail Helisoma form transient inappropriate electrical synapses which are broken upon establishment of stable, appro- priate synapses.” In the lateral vestibular nucleus of the rat,2° evidence has been reported for de- generation of neuronal connections. Some rat and chick striated muscle cells have been shown to be innervated by multiple neurons initially, but by only single neurons later in development.” In this report, we show that rat retinal neurons form transient synapses with rat muscle cells only during a discrete period in development, whereas chick embryo spinal cord neurons form synapses with rat muscle cells at all developmental stages tested. Spinal cord neurons form synapses with muscle which are selectively retained while retinal synapses are lost. METHODS Cultures of striated muscle cells from hindlimbs of newborn rats or 20-day embryos and neurons from rat retina or chick spinal cord at different stages of development were prepared as described previously.'? Neuron-myotube synapses were detected by recording spontaneous depolarizing synaptic muscle responses, usually 0.5 mV in amplitude and 10 ms in duration, with intracellular   Present addresses: * Department of Anatomical Sciences, Schools of Basic Medical Sciences and Life Sciences, University of Illinois — Urbana/Champaign, Urbana, IL 61801; + Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215: + Department of Cell Biology, Lilly Research Laboratories, Eli Lilly and Co.. Indianapolis, IN 46206, U.S.A. § Correspondence to: Marshall Nirenberg, Bld. 36, Rm. 1C06, Nationale Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20205, U.S.A. 25 26 Jeffrey M. Thompson et al. microelectrodes filled with 3 M KCI solution as described by Nelson et al.!3 Only myotubes with stable membrane potentials of -40 mV or more were used. A myotube was considered to be inner- vated if at least three spontaneous synaptic responses of the muscle cell, judged free of artifacts, were identified during a recording period of 2 min. The length of the recording period was extended when the frequency of spontaneous muscle responses was low. RESULTS AND DISCUSSION Neurons dissociated with trypsin from rat retina at different stages of development were added to monolayers of differentiated rat striated muscle cells that had been cultured for approximately 9 days. After 24 h of incubation, myotubes were assayed for synapses. Synapses were first detected with neurons from retina of 20-day rat embryos (Fig. 1); the proportion of retinal cells which formed synapses with muscle was maximal 1 day after birth, and then declined. No synapses were found between neurons from 7- to 18-day postnatal rat retina and muscle cells. The frequency of spontaneous synaptic muscle responses followed a similar developmental sequence. Activation of glutamate receptors of rat retinal neurons by local application of 5 mM sodium glutamate in culture medium by diffusion from a micropipette resulted in an increase of acetylcholine secretion from the neurons and as much as a 30-fold increase in the frequency of synaptic muscle responses (not shown). Application of glutamate to muscle cells in the absence of neurons did not evoke depolariz- ing muscle responses.   RAT RETINA NEURON-MUSCLE SYNAPSES - 1 Dd ao 1 = °o MUSCLE RESPONSES/MINUTE PERCENT MUSCLE CELLS WITH SYNAPSES     1 i 1 1 +.   200 2 4 6 AGE OF RETINA NEURONS (DAYS) Fig. 1. Synapse formation between neurons dissociated from rat retina at various stages of de- velopment and rat striated muscle cells. Twenty million cells dissociated from rat retina with trypsin were cocultured for 24 h with a monolayer of rat striated muscle cells in a 35 mm Petri dish. For all experiments described in this report, the growth medium consisted of Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum. The medium was changed 1 h prior to assay. Symbols represent the following: (*), % of muscle cells tested with synapses; (©), mean number of spontaneous synaptic responses/min of innervated muscle cells. Each point represents 20-30 muscle cells (2-3 dishes) assayed for synapses; a total of 190 cells were tested. We estimate’®’® that each culture dish contained 20,000 myotubes which occupied 20% of the surface area of the dish (9.6 cm’). Since 60% of the muscle cells were innervated (12,000 synapses per dish), we calculate that 0.06% of the cells plated from 1-day neonatal rat retina formed synapses with muscle celis. Probably 0.3-0.6% of retinal neurons may be able to form synapses with muscle cells, since only a fraction of the retinal cells was in contact with muscle cells and some muscle cells probably were innervated by 2 or more neurons. Synapse selection 27 The ability of neurons dissociated from chick embryo spinal cord to form synapses with rat striated muscle is shown in Fig. 2 as a function of developmental age. Spinal cord neurons dis- sociated on the 2nd day of chick embryo development formed synapses with muscle cells. The maximum number of synapses with muscle cells was found with neurons from 4-day embryos which agrees well and extends the findings reported by Berg & Fischbach.’ We estimate that 0.29% of the spinal cord cells from 4-day embryos formed synapses with muscle cells. If we allow for multiple synapses and for a fraction of neurons which are not in contact with muscle, then 1.5-3% of the cells from spinal cord may be able to form synapses with muscle cells. The number of synapses with muscle formed by cells from 4- to 14-day chick embryo spinal cord decreased via an apparent first- order process with a half-time of 3.8 days (Fig. 2, inset). Synapses between neurons dissociated from 18-day embryo spinal cord and muscle cells were detected after 51 h of coculture, but not after 24 h of coculture. The maximum frequency of spontaneous synaptic responses of muscle cells also was obtained with neurons from 4-day chick embryo spinal cord; the frequency of responses de- creased with cells from 4- to 10-day chick embryo spinal cord and remained constant thereafter. Results of Obata! suggest that the decrease in frequency of muscle responses may be due to the formation of inhibitory synapses. In other experiments (not shown), local application of 5 mM glutamate to neurons from 6-day chick embryo spinal cord cultured with muscle cells resulted in 2- fold increases in the rate and amplitude of muscle responses.                80 + 7 , + + 40 SPINAL CORD NEURON-MUSCLE SYNAPSES wn 1 2 004 FA 60 30 = E = a 3 3 2 wy 40 20 3 2 20 oe 3 RESPONSES 2        4 6 8 10 iz 14 16 AGE OF SPINAL CORD NEURONS (DAYS /V ovo) Fig. 2. Synapse formation between neurons dissociated from chick embryo spinal cord at various stages of development and rat striated muscle cells. Five million cells dissociated from chick embryo spinal cord with trypsin crystallized 3 times were cocultured for 24 h, (@), or 51h, (A), with a monolayer of rat striated muscle cells in collagen-coated Petri dishes 35 mm in diameter (9.6-cm? surface area). Symbols represent the follow- ing: (¢, &), % of muscle cells tested with synapses; (©, A), mean number of spontaneous synaptic responses/min of inner- vated muscle cells. The data shown in the inset are the % of maximum innervation of muscle cells plotted with a logarithmic ordinate. t,,2 represents the half-time for decrease in the % of muscle cells innervated. Each point represents approximately 30 (range 10-60) muscle cells assayed for synapses; a total of 311 cells were tested. Choline acetyltransferase (EC 2.3.1.6) specific activity from 6- and 18-day chick embryo spinal cord before and after dissociation with trypsin and after coculture of dissociated spinal cord cells with striated muscle for 1 day is shown in Table 1. Relatively high specific activities of choline acetyltransferase were present in 6- and 18-day embryo spinal cord and were reduced by dissocia- tion with trypsin. However, cells dissociated from 6- or 18-day chick embryo spinal cord which were cocultured for 1 day with muscle cells still possessed relatively high choline acetyltransferase activities. During 1 day of coculture with muscle cells, the enzyme activity of 6-day embryo spinal cord neurons increased, whereas the activity of neurons from the 18-day embryo decreased. These results suggest that cholinergic neurons were abundant in cocultures of both 6- and 18-day embryo spinal cord and muscle cells, and that neurons from 18-day embryos may have been injured more easily by the process of dissociation than neurons from 6-day embryos. 28 Jeffrey M. Thompson et al. Table 1. Choline acetyltransferase activity of spinal cord neurons     Age of chick embryo spinal Total activity Specific activity cord (days from Treatment (pmol '*C-acetylcholine (pmol '*C-acetylcholine Protein fertilization) formed/min/dish) formed/min/mg protein) — (mg/dish) 6 Before trypsinization _ 437 _ 6 After trypsinization 234* 124 1.95* 6+ 1 Day in vitro with muscle 401 176 (393)+ 2.28 18 Before trypsinization _ 695 _— 18 After trypsinization 1010* 310 3.30\" 18+ 1 Day in vitro with muscle 501 171 (300)+ 2.93 Muscle in vitro (no neurons) _— — 1.26   Spinal cords were dissected from 6- or 18-day chick embryos, trypsinized to obtain dissociated cells or minced and sonicated to prepare homogenates. Fifteen million spinal cord cells were added to rat myotubes in a 60 mm plastic tissue culture dish. After 1 day, cells were removed from the dish by scraping and then were sonicated. Freshly trypsinized spinal cord cells were also sonicated to determine choline acetyltransferase activity prior to culture. There were 3.6 X 10° cells recovered from a 6-day embryo spinal cord (0.96 mg protein/cord) and 7 x 10° cells recovered from an 18-day embryo spinal cord (5.05 mg protein/cord). Choline acetyltransferase activity of homogenates was determined as described by Wilson et al. 1 * Amount of choline acetyltransferase activity or protein, per 15 x 10° spinal cord cells, added to each dish. + Specific activity based on neural protein only (calculated by subtracting the protein determined from muscle cells cultured without neurons from protein in dishes with cocultured neural and muscle cells). In other experiments (not shown), cells were dissociated from 6-day chick embryo spinal cord and cultured with muscle cells for 1, 3 and 6 days (first stage). Then the cultured cells were dis- sociated again with trypsin and the number of cells plated initially were plated on fresh muscle cells. After coculture for one additional day (second stage), muscle cells were assayed for synapses to de- termine whether neurons previously cultured and dissociated could form new synapses. The percentage of muscle cells with synapses found in the second stage was 83, 25 or 10% with neurons cocultured in the first stage for 1, 3 or 6 days, respectively. Thus, the proportion of myotubes inner- vated by spinal cord neurons decreased with age both in ovo (Fig. 2) and in vitro. Whether the de- crease in the number of myotubes with synapses detected is due to a decrease in the ability of neurons to form synapses, to an increase in fragility of older neurons to dissociation by trypsin, or to programmed cell death”®!!'> is unknown. Neurons from chick embryo retina form synapses with muscle cells between the 6th and 15th day of embryonic development,'® whereas neurons from rat retina form synapses between the 20th em- bryonic and 7th neonatal day. At later developmental stages, both chick and rat retinal neurons lose the ability to form synapses with muscle cells. In contrast, chick embryo spinal cord neurons acquire the ability to form synapses with muscle cells by the second day of embryonic development, and continue to form some synapses throughout the developmental period tested (2-18-day embryos). The sequence of neuron genesis, the availability of target cells, their relative positions and the duration of the synapse-competent state may determine, in part, the kinds of synaptic connections that form. Retinal neurons from 1-day neonatal rats formed transient synapses with striated muscle cells (Fig. 3). Synapses were detected 2 h after retinal neurons were added to muscle cells (not shown), reached maximum abundance by 24 h, and then declined progressively as a functional of time of coculture. However, after 7 days of coculture, no synapses between rat retinal neurons and muscle cells were detected. The transient nature of the rat retinal neuron synapses with muscle cells is similar to that reported for synapses between 8-day chick embryo retinal neurons and muscle cells which disappear after 8-10 days of coculture with a half-time for loss of synapses of 21 h.'©!8 The decrease in the number of retinal neuron- muscle synapses during 7 days of coculture is due to the loss during development of the ability of neurons to form synapses with muscle cells and to the ter- mination of synapses with myotubes which have formed. Neurons from spinal cord, which presumably include motorneurons that normally synapse with striated muscle cells, also formed synapses within 2 h with cultured muscle cells; the maximum per- centage of muscle cells innervated (60% ) was achieved by 1 day of coculture. However, the number Synapse selection 29 of synapses between spinal cord neurons and muscle cells did not decrease appreciably during 14 days of coculture (Fig. 3). These results show that cholinergic spinal cord neurons and retina neurons form synaptic connections with striated muscle cells, and that synapses formed by spinal cord neurons are retained whereas those formed by retina neurons disappear by a process of selec- tion based on differences in turnover. Whether synapses between spinal cord neurons and muscle   SELECTIVE TERMINATION OF SYNAPSES PSES 8 SPINAL CORD NEURONS RAT RETINA NEURONS PERCENT MUSCLE CELLS WITH SYNA     EY a CE DAYS OF COCULTURE   10 12 14 Fig. 3. Synapse turnover by cells dissociated from 8-day chick embryo spinal cord or 1-day neonatal rat retina. (#), 5X 10° rat retinal neurons per dish cocultured with muscle cells; (©), 5 x 10° chick spinal cord neurons per dish cocultured with muscle cells. Ten mM _ 5- fluorodeoxyuridine and 100 »M uridine was added to prevent rapidly dividing cells from overgrowing cultures. Each point represents 20 muscle cells assay for synapses, a total of 170 cells. Data for spinal cord— muscle synapses are from Ruffolo et al. 18 cells continue to turn over, and have attained a steady-state wherein the rate of synapse formation is equal to the rate of synapse termination, or whether spinal cord neurons form stable, long-lived synapses with muscle cells as suggested by Changeux & Danchin® remains to be determined. Both possibilities seem probable since spinal cord neurons were shown to form synapses with muscle cells throughout the developmental period studied (2-18-day embryos). However, the results also suggest that the synapses between chick embryo spinal cord neurons and muscle cells may be either longer-lived or more stable than those between rat retinal neurons and muscle cells, and thus turn over at a slower rate. Although the mechanism has not been determined, the results demonstrate that differences in the rates of turnover of two populations of cholinergic synapses can result in the selective retention of one population of synapses and the loss of the other, and thereby alter the specificity of synaptic connections. REFERENCES 1. Barondes S. H. (ed.) (1976) Neuronal Recognition. Plenum Press, New York. 4. Bennett M. R. & Pettigrew A. G. (1974) The formation of synapses in striated muscle during development. J. Physiol. 241, 515-545. 3. Bennett M. R. & Pettigrew A. G. (1975) The formation of neuromuscular synapses. Cold Spring Harbor Symp. Quant. Biol. 40, 409-424. 4. Berg D. K. & Fischbach G. D. (1978) Enrichment of spinal cord cell cultures with motoneurons. J. Cell Biol. 77, 83-98. 5. Bulloch A. G. M. & Kater S. B. (1981) Selection of a novel connection by adult molluscan neurons. Science 212, 79-81. 6. Changeux J. & Danchin A. (1976) Selective stabilisation of developing synapses as a mechanism for the specification of neuronal networks. Nature, Lond. 264, 705-712. 7. Chu-Wang I. & Oppenheim R. W. (1978) Cell death of motoneurons in the chick embryo spinal cord. 1. A light and electron microscopic study of naturally occurring and induced cell loss during development. J. comp. Neurol. 177, 33- 58. 8. Chu-Wang I. & Oppenheim R. W. (1978) Cell death of motoneurons in the chick embryo spinal cord. Il. A quantitative and qualitative analysis of degeneration in the ventral root, including evidence for axon outgrowth and limb innervation prior to cell death. J. comp. Neurol. 177, 59- 86. 9. Cotman C. W. (ed.) (1978) Neuronal Plasticity. Raven Press, New York. 10. Gaze R. M., Keating M. J. & Chung S. H. (1974) The evolution of the retinotectal map during development in Xenopus. Proc. R. Soc. Lond. B. 185, 301-330. 30 11. 12. 13. 14. 15. 16. 17. 18. 19, 20. 21. Jeffrey M. Thompson et al. Hamburger V. (1975) Cell death in the development of the lateral motor column of the chick embryo. J. comp. Neurol. 160, 535-546. Mark R. F. (1969) Matching muscles and motoneurones. A review of some experiments on motor nerve regeneration. Brain Res. 14, 245-254. Nelson P. G., Peacock J. H., Amano T. & Minna J. (1971) Electrogenesis in mouse neuroblastoma cells in vitro. J. Cell Physiol. 77, 337-352. Obata K., Oide M. & Tanaka H. (1978) Excitatory and inhibitory actions of GABA and glycine on embryonic chick spinal neurons in culture. Brain Res. 144, 179— 184. Oppenheim R. W. & Chu-Wang I. (1977) Spontaneous cell death of spinal motoneurons following peripheral innerva- tion in the chick embryo. Brain Res. 125, 154-160. Puro D. G., DeMello F. G. & Nirenberg M. (1977) Synapse turnover: the formation and termination of transient synapses. Proc. natn. Acad. Sci. U.S.A. 74, 4977- 4981. Purves D. & Lichtman J. W. (1980) Elimination of synapses in the developing nervous system. Science 210, 153-157. Ruffolo R. R., Jr., Eisenbarth G. S., Thompson J. M. & Nirenberg M. (1978) Synapse turnover: A mechanism for acquiring synaptic specificity. Proc. natn. Acad. Sci. U.S.A. 75, 2281-2285. Scott T. M. & Lazar G. (1976) An investigation into the hypothesis of shifting neuron relationships during develop- ment. J. Anat. 121, 485-496. . Sotelo C. & Palay S. L. (1971) Altered axons and axonal terminals in the lateral vestibular nucleus of the rat: possible example of axon remodeling. Lab. Invest. 25, 653-671. Wilson S. H., Schrier B. K., Farber J. L., Thompson E. J., Rosenberg R. N., Blume A. J. & Nirenberg M. W. (1972) Markers for gene expression in cultured cells from the nervous system. J. biol. Chem. 247, 3159-3169.", "Thompson, Jeffrey M. ; Eisenbarth, George S. ; Ruffolo, Robert R. ; Nirenberg, Marshall W.", null, "International Journal of Developmental Neuroscience", "Pergamon Press, Inc.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-juwd~yfjz.pqay", "00000000-0000-0000-39D1-82DD290FB6C8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Pages from Nirenberg's laboratory journal titled \"Major Problems [of Neurobiology]\"", "101584910X169", null, "1966", "6 September 1966", "Nirenberg lists some of the many different biological systems he considered using to study neurobiology.  Squid axons, crayfish, nematodes, tissue cultures, and many other systems are all listed along with what Nirenberg believed to be the major problems facing neurobiologists in 1966.", "Laboratory notes", "Neurobiology,Research Design", "Transition to Neurobiology, 1965-1969", "10", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "AW Dalayoum ° 1oPLY LITHOGRAPHED IN USA             DN ane - > |     2S es oe       ) Mp e aN NEI       — a ob Ey teh DA mm                                                           ——.- 4. ce ae _ ~ a a }. i —— = ep _ T 7 i ee abe _— i 6 / —— a Te _ _ ee 4. _ _ _ uf pe —_. _ nea _ _ _               4                                                                                                                                                                           ss AW Datafarm 1OPLV           A ADDISON WESLEY PUBLISHING COMPANY. INC... READING. MA ii '| @ hid Py rbd tf fo} | PVT M@EP ETE tp rt iqyiqay « pa hop PEE EE pap pp ee tb Pe pp pF ee ee ee ee | Pp Ep | PoE Ee pp PE j OS Aare (aa sot) fpr ate                          rid d MASS AW Daldyoim, ~ 1OPLV 1 | 1                                 | PrET Td tt | | ss AW Dalayaum * 'OPLV [|       FOPLV AW Dalyan ° ISON WESLEY PUBLISHING COMPANY INC... READING. MASS LOTHOGRASHED IN US a ALD : Oo : : s aa mores ne - uy oN, nen m4 Dy a Dg, 6 ee eee en, 3 é an \" - we", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qxrw_5qku_458b", "00000000-0000-0000-AD41-6C93CEA2803E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Pages from Nirenberg's laboratory journal titled \"Ideas\"", "101584910X170", null, "1967", "10 October 1967", "These pages from Nirenberg's journal list some of his early ideas about neurobiology.  He discusses the kinds of codes, synapses, and logical relationships utilized by the nervous system to function.", "Laboratory notes", "Neurobiology,Synapses,Neurons", "Transition to Neurobiology, 1965-1969", "4", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "LITHOGRAPHED IN U.S.A ADDISON WESLEY PUBLIGHING COMPANY. INT READING. MAS 4140 Dalaprim OPLYV ; ¥ Bee ene oa \\\\ | Poy of ' mo | Sp Pott foo i i . i | i : ! : : . { i : i i i | i t : : bo Pp GG i : Poo| se ! DG ! ii bob 4 BG            ||   & — ®& ~~     | _ Pa be Pe ! bo ‘ ae eb oe i | | i pb EP pp ee EP pp da Se ba Ee EP Dah Ped | Pha db da Pet   AIdO: unaluysg Ae SSYW ONIDVAY  ONI ANVdAOD SNIHSIIG Ne ABTS UMW NGS lady v¥STR Nt OBHdVHOOHIG", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-eq4e-egbq.a4k3", "00000000-0000-0000-E9FD-0209EDC02035", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "NRP [Neurosciences Research Program] Talk", "101584910X171", null, "1966", "July 1966", "Marshall Nirenberg often attended the NRP meetings sponsored by the Massachusetts Institute of Technology.  This draft of a talk he was to give at the meeting is on \"the basic concepts of the genetic code.\"  Nirenberg concludes the draft by linking the genetic code discussion with neurobiology, speculating that neurobiology and molecular biology have a great deal to offer each other.", "Speeches, Drafts (documents)", "Genetic Code,Codon,Neurobiology", "Transition to Neurobiology, 1965-1969", "11", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, ": BiG — se + a (4) per S pee o> {4yu Powe RP PIS                       = A A, no                                                       we =     ~~ ate 2 apecr-r. $1                         y Dees LA     a | | (3000: I Bt : — Pd = - het PLL ~ 1X Lech     it oh, “¢ RE momar", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ytx7~z6f7_w44i", "00000000-0000-0000-FDD1-FF61C83D47F1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Pages from Nirenberg's laboratory journal on New Years Day", "101584910X172", "101584910X173", "1966", "1 January 1966", "After deciding to turn his research over to neurobiology on New Year's Eve, 1965, Nirenberg scheduled his next six months to outline how he would slowly turn his attention and his laboratory over to the new scientific endeavor.", "Laboratory notes", "Neurobiology", "Transition to Neurobiology, 1965-1969", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "—     Di. ~49- 9-10.'30 Pm >} 1030 ~— 12/08            >     tafe va AR Tp ( Y Pe the mas Sp a phe 6 ph mok |", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hccr.8a7w_f3in", "00000000-0000-0000-2562-4C5CF39293CC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Pages from Nirenberg's laboratory journal titled \"Brain\"", "101584910X173", "101584910X172", "1965", "31 December 1965", "These pages from Marshall Nirenberg's laboratory journal are the earliest signs of his interest in turning his research over to neurobiology, but this early consideration is only labeled \"Brain\".  Nirenberg wrote down this interest in changing fields on December 31, 1965.", "Laboratory notes", "Neurobiology", "Transition to Neurobiology, 1965-1969", "4", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Deus ns Bos S| May ot wh fit + ct ma mm . Aa on On @é 2) Neon mnt de flitobat eal tf? SREP, ba a fhe oop soy Note AD ys Lk Ty ae he - of x hth $US De eR — Pat hese. * ters onan,   ras B eer Tey at nes — Math, MY ed toh pe on LI ieee. oaaas opener a 3 a Pie ALL, £1 olen A. NAL gp DUAR ed Ne pe.   LibtiOGRAOCHRED IN USA ADDISON - WESLEY PUBLInaIM SG Co tRPANY INC READING. MASS 1 | iC 5 ee ee : Lf poh eG SoG bop oR Eon | A poof} ft : Po H ' I : os ‘ L i; t i : t i : . : . : { ‘ Hl ' i i : ' u", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wic7_w3aq~qzw2", "00000000-0000-0000-1140-4FC5E028B76B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Pages from Nirenberg's laboratory journal titled \"Basic Questions [of] Neurobiology\"", "101584910X174", null, "1967", "12 July 1967", "Marshall Nirenberg often outlined his thoughts on research projects in his laboratory journal.  These pages, from July 1967, list many of the topics Nirenberg believed to be the central questions of neurobiology.", "Laboratory notes", "Neurobiology", "Transition to Neurobiology, 1965-1969", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "|| af Oy Ty | P| cide | L                 | AVG P| wo Hl i i ! i i } fereg w-¥ SSVW ONIOVEY ON   ANVdWiOD DONIHSINE Md Adds    bj | BMW NOSIUOY   VE f NE   ! ] : i po i j ] | GSBHdVHOOHLIG t       feces see ae eel on", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-djcn.3z3d~ugp6", "00000000-0000-0000-E4E6-DF4DE7DAD4FE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Pages from Nirenberg's laboratory journal titled \"Codons vs. Neurons\"", "101584910X175", null, "1967", "10 October 1967", "In 1967 and 1968, Marshall Nirenberg compared the genetic code with the neural code.  This page from his laboratory journal details one aspect of that comparison by contrasting codons from the genetic code with neurons from the neural code.", "Laboratory notes", "Neurons,Codon", "Transition to Neurobiology, 1965-1969", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-spcu-p2n7.8hrc", "00000000-0000-0000-EA67-559C1DB9871A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes from Neurosciences Research Program meeting", "101584910X176", null, "1968", "4 February 1968", "The Neurosciences Research Program met several times throughout the year in order to allow scientists from various fields to interact and discuss issues related to neurobiology.  In these notes from one of those meetings, Nirenberg lists various types of biological systems that could be used to study neurobiology.  He also writes of the various questions and problems to consider when studying neurobiology.", "Notes", "Neurobiology,Genetics,Research Design", "Transition to Neurobiology, 1965-1969", "14", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "PA HRP ae Tice “yt 4) Ma duf Vay CS EES ma 7 |) dds — 680 c) pp — (Fen A 9) enon — forked orp Yh coll, - ( 4 _ ~ Somgen \\\\ A EE fo x ‘ iC                  2 Pa c = = a iy F Ht \\\\ hs pea ey         lo Bice Z ] “2, i - ‘ ‘ | Oo Loe <b >200 beh | Dow C5000 o) Chik. <8, 009 y Neen. LYIMDH    ft, “ at BR pong. . FOL                        nahh cast 4 ye > ss clef fees Ce crt Ue Ean pn ”                 1 t * \\\\ .                                UP Cosa ek ty Fed) 245 ra ( foly bop hs:       5, Woe 1 DSP 00 O ‘/ ; Man a O04 Ge ¢g )Dangheakn. c) _~ 500, Uae ce           A if a x ihe po Saehch 71 ta-4¥ oe. OH HER menial beta — SNe ene ee _L) ——>", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hfwm~nnmd_rwgm", "00000000-0000-0000-BDF6-D16BD0ED4C7F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Pages from Nirenberg's laboratory journal titled \"Genetic vs. Neural Codes\"", "101584910X177", null, "1968", "9 March 1968", "In 1968, Marshall Nirenberg compared the genetic code to the neural code.  These pages from his laboratory journal give detailed examples of his thought process when considering the two codes.  Nirenberg writes about the logic, generalizations, and universal aspects of the codes.  He also considers the biological basis of memory, the prospect of other codes, and the calculations necessary to predict the number of synapses in the brain.", "Laboratory notes", "Neurons,Synapses,Genetic Code,Research Design", "Transition to Neurobiology, 1965-1969", "18", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "RZ x00 oc, Jom | payin a meaty Rede _ ayh cot fr TA ol - etd 0% ee joo. 3 = ihns mo ati Mex, ot 4 oP yy ats fall ab pi Aedty ® é cf IF | 2 | Uy 3} oo C 4} | xo 5] Dba   7 eel emake’ | AA GM Yoh fi tia] Poy f = ( 2. ) r ett | Ua kk Ce binge: ben b> dhs / Ce | rae ; Y bie Aoife on Math. of joe         Do He N~</ ye UA 12a } H// by Je Gk teg Sig : Pe- oi Ro                                             oe iF tok TeaLy ~ : ( O", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-sbjq-suxf-vdn3", "00000000-0000-0000-4025-44505CB614AC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Theodore Melnechuk to Marshall W. Nirenberg", "101584910X178", null, "1968", "17 December 1968", "This letter from Theodore Melnechuk of the Massachusetts Institute of Technology includes a transcription of an interview with Sydney Brenner discussing the work of Marshall Nirenberg, Sydney Brenner, and Seymour Benzer in neurobiology.   An abstract of Brenner's main points from his Warren Triennial Prize lecture, including his thoughts on the future of molecular biology, is followed by notes on his genetic work in neurobiology with nematodes and transmitter biochemistry, ascarids and neurophysiology, and his philosophy of science and genetics.", "Letters (correspondence), Transcripts", "Neurobiology,Research Design", "Transition to Neurobiology, 1965-1969", "11", "pages", "Text", "English", "Reproduced with permission of Theodore Melnechuk.", "Copyright may apply", null, null, "December 17, 1968 Dear Marshall: Here is a transcription of my notes of a conversation with Dr. Brenner.  I'm sorry that the press of work delayed my completion of it.  And even I had trouble reading my scribbled notes. The transcription also includes Dr. Benzer's recent comments on his work.  Together, I hope they are useful to you in connection with the remarks or report that will kick off the last day of the next Stated Meeting.  I'm sorry neither Brenner nor Benzer could attend.  But then you have that much les introducing to do as chairman. Also enclosed are our Polaroid copies of the slides shown in conjunction with Dr. Gajdusek's talk at an earlier Stated Meeting. Under separate cover I am sending a copy of the tape recordings that include Dr. Gajdusek's presentation at an earlier Stated Meeting.  Wardy Holman included in his package of the two tapes the track and foot count of the start and finish of the presentation. With best Holiday wishes, I am Sincerely, Theodore Melnechuk Director of Communications Enclosures: 2 T. Melnechuk December 16, 1968 Notes of a Conversation on the Morning of October 30, 1968 at the NRP Center Between Professor F.O. Schmitt and T. Melnechuk and guest, Dr. Sydney Brenner of the Medical Research Council Laboratory of Molecular Biology at Cambridge, England I.   Background At the Stated Meeting of NRP Associates in February 1968, Dr. Marshall Nirenberg gave a brief account of the research strategy he thought he might follow in studying the behavior genetics of simple neural systems.  Believing it would be interesting to hear similar accounts from two other eminent molecular geneticists, Drs. Seymour Benzer and Dr. Sydney Brenner, Professor Schmitt invited the latter two investigators to speak at the Stated Meeting in February 1969, with Dr. Nirenberg chairing.  Neither invitee could be present at that time, but each responded with an account of their strategy, and Dr. Brenner included a status report as well. Dr. Benzer's account was in the form of a paragraph in a letter to Professor Schmitt dated December 3, 1968.   I cite the paragraph in full: The work my group and I are engaged in is the isolation of behavioral mutants of Drosophila, particularly in respect to the phototactic response, with the idea of obtaining genetic blocks at the various steps involved.  We look at the mutants for anatomical or developmental defects affecting the nervous system and also at electrical events in the visual system, as well as biochemical aberrations.  Much of the work is still very preliminary, but we have begun to find interesting electroretinogram abnormalities in certain visual mutants and are writing up that work for publication.   I will send you a copy of the paper when it is ready, for your information. It is also possible that Dr.  Benzer will describe his neuro-biological work at a symposium on  \"Control Processes in Multicellular Organisms\" scheduled to be held in New Delhi next March 16-20 by The Ciba Foundation.  In a preliminary program dated March 14, 1968, Dr. Wolstenholme has Dr. Benzer slated to speak on \"Genetics of Brain Function\" at 4:30-5:00 on Thursday, March 20, 1969. Dr. Brenner's comments were made in the course of a two-hour conversation with F. O. Schmitt and T. Melnechuk on October 30, 1968.  Before reporting that conversation, however, it seems helpful to abstract the main points made by Dr. Brenner two days earlier at the Massachusetts General Hospital in giving the Warren Triennial Prize lecture. II.   \"The Future Of Molecular Biology\" Under the title just given, Dr. Brenner made several major points: 1.  Scientists tackle problems that they consider important.  Of these, they tackle those problems they consider solvable.  And problems become solvable through the onset of new techniques, or new conceptions, or both. 2.  Current major problems include the physical mechanisms of: a.  Polypeptide folding b.  Enzyme action c.  Protein repression and induction 3.  The nature of a scientific explanation is different in physics and biology.  In physics, an explanation is ultimately a differential equation.  But in biology, an explanation is ultimately a list -- for example, a program. 4.  Only Max Delbruck expected that molecular biology might discover new principles of physics, according to Gunther Stent's \"That Was the Molecular Biology That Was\" (Science, Vol. 160, pages 390-395, 26 April 1968; a copy is attached as an appendix). 5.  The goal of molecular biology is to be able to compute an organism from a knowledge of its genes. 6.  The Monod-Jacob notion of repression-and-induction is a sufficient key to understanding development. 7.  Just as \"Project K\" -- to know all about the genetics of the biochemistry of E. Coli -- is feasible, so is \"Project Man,\" about whose physiology much is known. 8.  In order to yield as complicated and hierarchically arranged an organism as man, a genetic program must surely have parts, so there is some hope of getting at its logic before getting a thorough knowledge of its details.  By \"part of a program\" is meant such a sub-program, focused on an organ or a function or constituents, as would permit the eye to evolve without concomitant evolution of the kidney. 9.  Molecular Biology is essentially the Chemistry of Natural Selection, so it must eventually deal with the interplay of the genetic program with the accidents of history. III.   Correlative Neurobiology Unfortunately, Dr. Brenner's remarks at the NRP were not tape-recorded.  What follows is an edited transcription of T. Melnechuk's notes, which were not made with this eventual use in mind: Staff.- Drs.  Brenner and Crick now have a staff of 14 scientists.  After a building delay, they will have 28, who will work in three groups, one on molecular genetics, one on cell biology, and the largest on neurobiology.  The technical staff includes technicians trained to photograph anything at scales from the visible down to the EM level. Subject.- Brenner's own work is in neurobiology: not so much molecular -- that is, not on membranes, and not on biochemistry, except where relevant to the genetic problem, -- as on the genetic control and specification of structures in the nervous system: what they are and how they got there.  He wants to know what single genes can do, both structurally and behaviorally. Organism.- He prefers to work at this time on a system with no learning, one that is rigorously specified, controlled from within.  Therefore, he does not work with goldfish, but with nematodes. The small nematode he works with most is 500-700 mu long in \"adolescence\" and 1 mm long when adult.  The specimens are \"identical\", being naturally inbred, for in their 3-day life cycle, each nematode begets 300, so that in a week, one becomes 10^5; all on one Petri dish. Neuroanatomy.--The small nematode probably has less than 500 neurons.  Brenner is cutting serial sections 10 mu thick, using EM with 30 A resolution.  The job is being done broadly first, to get technical facility.  The entire sensory system has been defined, the neurons counted, and the dendrites pictured, but the hook-up of the sensory system to the central nervous system is not yet known.  All the sensory neurons have dendrites like cilia, with microtubules. Work continues on the motor side.  Work on the innervations of the esophagus is half done.  He has identified 92 muscle cells, though not what these cells do.  Each set of 16 muscle cells constitutes a ring.   Such muscle cells modulate like neurons.  They have oblique striations, rather than sarcomeres. The nematode's head, which narrows down, receives projections from 121 cells.  To get a three-dimensional grasp of the neural processes, Brenner prefers getting a mental picture by handling the actual cross-sectional EM pictures.  Neither a cinemicrotome approach a la Livingston nor an analog of the X-ray crystallographer's overlay method work for him; he wants to reconstruct the nervous system in his own mind. Behavior.- Nematodes have a limited behavioral repertoire.  It is difficult to describe their behavior scientifically.   Brenner takes cine pictures and does a frame-by-frame analysis. Of the 200 mutants he has observed, most are behavioral -- usually forms of paralysis.  He picks the mutants by a brute-force method:  he examines a colony, and picks up the cripples with a toothpick. Genetics.- Brenner is doing an extensive genetic analysis.  The behavioral alterations are produced by mutations of 22 easily recognizable genes; this number will go up to about 30.  The genetic map is saturated; 1 gene has 8 mutants.   The X chromosome is mapped, and the autosomes identified as linkage groups.  This work will take 2-3 months more to finish. The next step is to identify the structural defect in each mutant, so that if there is a change, it can be seen.  This will give some idea of what single genes can do, both structurally and behaviorally. Transmitter Biochemistry.- To do the biochemistry of nematode transmitters,  large numbers of animals are needed, so that mass culture techniques are necessary.  Work is almost ready to begin.  The idea is that if one is defective, you have \"the edge of the wedge\".  For example, you could study what the behavior would be if there were no inhibitory transmitter -- which, by the way, GABA is suspected to be in this organism. Brenner named three people who will be working on nematode neurochemistry: Tony Stratton or Stretton, who was with Vern Ingram; Slater or Slayter, and Dennis Bray, both of whom have been with Steve Kuffler. Neurophysiology.- Unfortunately, the particular animal chosen for being appropriate to genetics and electron microscopy is not appropriate for neurophysiological studies.  They are too small for the insertion of electrodes. However, large nematodes exist: the ascarids.   Brenner believes that their basic structure is similar enough to that of the small nematodes so that the only difference is one of scaling up the 5 or 6 levels from the free-living nematodes.  He says that Goldschmidt's old anatomical work is mostly wrong, by the way. Because of their complicated parasitical life cycle, ascarids can't be grown in the laboratory.  But neurophysiological studies can be performed, and the findings presumed transferable to the smaller species. Philosophy.- Brenner said he was not for, indeed was against, the idea of a changeable genome.  Instead, he was for answering the question:  Given the wiring diagram, what else must be known in order to be able to compute the organism's behavior? Next, a deeper problem:  How is it constructed?  The accurate computation of structure in development argues an utterly deterministic, digital program.  The specificity and precision of detail could not result from analog methods, such as gradients of concentration.  But only the genes are digital.  There must, therefore, be accurate gene switching. But the interplay of the long phylogenetic and ontogenetic process with history and its accidents during the prolonged development of elaborate organisms argues that such complicated systems must be subject to additional, new constraints, such as economy and continuity in evolution (as in the case of the persistence of cilia in sensory receptors).  Complicated systems may, therefore, employ analog principles as well as the digital principles of simpler systems. Brenner believes that storage is not in molecules but in cell assemblies, by means of synaptic switching.  Given the switches, their logic, and the plasticity of the complicated nervous system, he wants to know how it works.   He sees the need of an analysis of function space. IV.   Other Lines Permeability.- Brenner considers this question too wide for him. Molecular Biology of Transmitters.- Here, Brenner would like to see the protein receptor molecules isolated, and has two chemists coming to work at this problem in a professional manner.  He wants to work on Miledi's preparation, denervating a muscle and watching the spread of sensitivity.  He thinks too many people are already working on Torpedo. In his terms, he is after \"the machine language of learning.\"  Of the receptor molecule species he would like to know: Is it localized?  If so, how does it get localized?  Where and by what is it made?  How fast?  Does it turn over?  Etc. Neuronal Cell Transplants and Cultures.- Brenner believes that while good work has been done, it has not yet been quantitative.  For example, Jacobson's work on the retina:  how many cells?  1,000?  10,000?  100,000? Brenner would like to learn how many neurons in culture it takes to get functioning synapses.  What does it require to get at least one criterion of change?  What nutrition?  Perhaps the geometrical requirements may be odd: monolayers versus impregnation of collagen sponges? He sees the need for some analogy to the one-step growth curve of Delbruck's work on phage.  He is aiming for a set of cells (an explant, a clone) that can be assembled in vitro.  Then he thinks the proper tool is the EM. Quantitative Neuroanatomy.-  Brenner would like to see more quantitative human morphology.   He would also like to see produced a Handbook of Wiring Diagrams, containing those circuits that have been established critically in various organisms, as by Trujillo-Cenoz in the retina and others in the cerebellum.", "Melnechuk, Theodore", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3zkb.9jvq_mswy", "00000000-0000-0000-8354-06A2D87D80EF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Dictation of plans for a potential neurobiology conference", "101584910X179", null, "1969", "16 October 1969", "Marshall Nirenberg relayed his daily requests to his laboratory with the use of audio recordings called \"belts\".  These recordings would then be transcribed into notes for the various members of the laboratory.  In this belt, Nirenberg plans a conference at NIH on neurobiology to include many of the scientists that migrated from molecular biology, including Seymour Benzer, Sydney Brenner, Gunther Stent, Max Delbruck, and Julius Adler.", "Transcripts, Notes", null, "Transition to Neurobiology, 1965-1969", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "[Handwritten: Neurobiology Conference - Fogerty In[ . . . ]] 10/16/69 Benzer's suggestions - Limit conference to following or less: 1.  Benzer 2.  Brenner 3.  Barondes 4.  Sidman 5.  Stent 6.  Changeux 7.  Delbruck 8.  Adler 9.  Kuffler 10.  Kandel 11.  Nirenberg Belt #1 1. Ask Wanda to Xerox and also give him original of a paper by Weidman and Kuwabara in Investigative Ophthalmology - a recent issue.  Very dark Xerox copies otherwise the photos do not come out at all. 2. How does Wanda get instructions?  Make three copies of transcription. 3. Norma to take out of library a book by Burn, K. H., entitled The Autonomic Nervous System, published by F. A. Davis Co., Philadelphia, 1963.  Would like to order a copy for the lab and one for the library. 4. Order two copies of a book by Deutsch, F. entitled Models of the Nervous System by Wylie, 1967. $9.95. 5. Order two copies of a book by Morgan, C., entitled Physiological Psychology, 3rd Edition, McGraw-Hill & Co., 1965. 6. Order two copies of a book, also check out of library, by Friede, R. entitled Topographic Brain Chemistry, Academic Press, 1966. 7. Check out of library a book by Causey, G. entitled The Cell of Schwann, E. & F. Livingston, London, 1960. Xerox chapter on tissue culture and also give the book to MN. 8. Wanda to Xerox a paper by Kornguth et al. in Experimental Cell Research, Vol. 45, page 656, 1967; also one by Mahaley, Cancer Research, Vol. 26, page 195, 1966. 9. Norma to get a paper in Cancer Research, Norman Anderson is one author, not senior author, but Mahaley will also be on the paper. Don't know title but it will be about an electron microscope study of particles that have been isolated from brain tissue by zonal centrifugation.  Also a Xerox of a paper by Aldrich and Johnson in Biochem. J., Vol. 73, page 270, 1969, and one by Holmstedt and Toschi, in Acta Physiologica Scandinavia, Vol. 47, page 208, 1969. 10. Xeroxes of following papers:  Weber, in Bulletin Schweiz. Acad. Med. Wiff.  Vol. 8, 263, 1952. Content of Cerebral Tumors Bulbring et al. in Lancet, i, 865, 1953. Content of Cerebral Tumors Belt #2 12. Check to see if I ordered the following book: McIlwain, Henry, entitled Biochemistry and the Central Nervous System, 3rd Ed., if 4th Ed. available want that, Little, Brown and Co., Boston, 1966.  One for lab and one for MN. 13. Also order a book by A. V. Palladin, translated by Freisinger, Gilman and Woodman entitled Problems of the Biochemistry of the Nervous System, published by Pergamon Press, New York. 14. Xerox a dark copy of a paper by Koelle in the J. of Comparative Neurology, Vol. 100, page 211, 1954. 15. Also one by Szilard in Proc. Nat. Acad. Sci. Vol. 51, page 1092, 1964.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-vue3.siex-c7hs", "00000000-0000-0000-F741-575324B43013", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Dictation of a Howard University talk acceptance letter", "101584910X180", "101584910X156,101584910X181", "1969", "21 January 1969", "Marshall Nirenberg relayed his daily requests to his laboratory with the use of audio recordings called \"belts\".  These recordings would then be transcribed into notes for the various members of the laboratory. In this belt, Nirenberg writes a draft for a letter to Howard University.  This letter announces his lecture at Howard University titled \"Genetic Versus Neural Information Processing Systems\".", "Transcripts, Notes, Drafts (documents)", null, "Transition to Neurobiology, 1965-1969", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Belt 1 1/21/69 1. Loretta have we ordered a book called \"Teratology Of The Central Nervous System\" that is from the University of Chicago Press, this brochure here describes it.  I may have already ordered it but if not, then we should order 1 for myself and 1 for the Lab.  I don't know the cost of it though. Now this memorandum from Evelyn Attix about Foreign Travel for 1969-1970 I don't expect to do any foreign travel then and you should ask Alan Peterkofsky and Tom Caskey; also pass this information on to Evelyn Attix. And this request from Mundschenk - just send a reprint of the Cold Spring Harbor paper for 1966 and also if we have a manuscript a copy of it from the one that went off to the Journal of the American Medical Association that review that I wrote recently send a copy of that also to him. Post this announcement on the bulletin board from the Laboratory of Neurophysiology. Also, for outgoing letters and things which I am supposed to sign I think it probably would be best if I sign them because that is the only way I have of ever knowing that the letter has gone out. Then I would like to send a letter to: Dr. Angella Ferguson College of Medicine Department of Pediatrics Howard University Washington, D.C. Dear Dr. Ferguson: I shall be glad to participate in the Seminar on Genetic and Developmental Aspects of Mental Retardation as we have discussed previously, but I do not wish to have my talk published.  As I mentioned earlier, I would be glad to discuss the work that we are trying to do but the work is not far enough along to have it published in any form.  It really would not be appropriate to publish this type of presentation.  So I would be able to participate in the Seminar only if the understanding that my remarks would not be published.  If this is agreeable, the title of my talk is \"Some Thought on Genetic and Neural Information Processing\".  \"Genetic versus Neural Information Processing Systems\" Sincerely yours,", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9rps_y5ya-8eh6", "00000000-0000-0000-913F-17549D0D051E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Neuroblastoma research data", "101584910X188", "101584910X189", "1970", "22 April 1970", "Takahiko Amano, a post-doctoral fellow in Marshall Nirenberg's laboratory, prepared this sample of neuroblastoma cells.  These pages outline the preparation that the neuroblastoma cells undergo before they are made into tissue cultures.", "Laboratory notes", "Neuroblastoma", "Neuroblastoma Research, 1967-1976", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "ea Ta | Deserinhion of Sample Name. | | Hr/ 70 ' Tf. Lowry Protein Assay Exp. ‘Sol. oO: N >I Bale Dilotian of Sample} H,0 Sample, Fold Components of Reactions Diluted | 30 _dlilstion PARP |. Bh A SAN Aik. Folin MOON eae coli                                                                       . — iS | Na Water +: TH lyse 8 ! iy Mo len a 4. Water So | | | | | ie Lt. a | Water | kee (SD° uo ! od i | 4 _ 5a, Albonin Sted. 43) (ea c / $® Tok dena 5 lonq Albumin Std mo ASS) | eet °° fe 6 15 ua. Albormin se 83h | Sp, S| aD nf cnn arp pe aa a 7 20. yg. Albumin Std. #53! Roe aa | ve. PBS Chaba] 96097, 3g [ay weH Hob] |) abe) | Pn | ro SF : : fo | | $s | o 7 wees : : ; . lot ee . [ _ sl SP 1 Z a oo |S) _ ta , fey _ ao 413 . ‘ | fo | So : 14 | a od eee po NP Is i Z ! 2p, SO | i | , yO y. i a ‘ i - | po a } _ ee oe ak _ pe oe ! : | | : miofee lt -— ne ce ce . . 2 oo i po ! i | - Tp - - —- wee : a + f lo oT a) fe _ _ | i | ! +— _ | an _ jo _ | ' I | pot | pepe dione ra — de )-~— . oe ; —— [                                                                       2, | | : ; - - Se Se - Mecud nee : a io none ~ none po | . : . | yd Bf ao eho doe fd = / ; | | i i ; | ° whee ee to + — a . . ae joo ee 5 : - po Le ch ne eee ! ' , ‘ i i \" Hl ' E Do Poy | - - ‘- : ! 1 ! : ; \\\\ ! 0 | poop ! Pp fo | : | ky oo. Pod | a | ' \\\\ : a To pment fated ete a i ! : i : i : : Sep mre perma nenree i | ! : ' by pO! P| A | | ! ee : ; 7 : . _ i : i ! E oy f 4 - ce fe ee Pe t t i : } | | P| | i | | ! i ; | ‘ H | | : { i { y i : { , a if ‘ i | i | i : ; et} | Poy! ! Ff \\\\ 3 , a — = “F _— be. i = - _ ! 22 i _ i ! : : ; i i oo -c ry } : ; i = t 5— ! | ' fo. | iy ppt pt Se lh ! : : | i i i ' PJs. i | | : | ' i - - : i i i ag | | bo Bt | | 4 c = y | ' i ! ! : i < -@9 ‘ : : 1 | Raa nr ee Lat . an Le $ ve | | ! ins 34 po pod a : Pade. ; ae FY hoe Po Spry bob a — ood P| fp  Y, eR pe Le 8 i : : i : ‘ : ! i ; | i i i ee bt pt _ e ! | | | | : | po PO - | --—} beef fee pe fp. | | gg. roe | t t bo mf anf ee ee bee fc gee a oO i ; : ~ pz 7 +, oO TM Ye eg, 29 = %: r 4 ; ee - 3 i fo             9 w oO Ce —_ ° . s = 4 . ° / 9 Q re) 9 S 9 9 ~ c O oO ~~ SNM! aaWN C Zz HONI Nad OZ x OZ ¢ \"00 N3OZL310 3N30N3 ia wadvd HdvWeo N3ISZLBIG OZ-ire ‘ON ws d", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4pia-6x2h~cbyj", "00000000-0000-0000-2B3F-C22513684030", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Dependence of Cell-Free Protein Synthesis in E. coli upon RNA Prepared from Ribosomes", "101584910X168", null, "1961", "1961", "In this article, the work for which was supported by a NATO post-doctoral fellowship, Marshall Nirenberg and Heinrich Matthaei reveal their stable cell-free system in which their protein synthesis studies would culminate with the landmark poly-U experiments.  Here, Nirenberg and Matthaei present their finding that incorporation of amino acids into protein is dependent on the addition of ribosomal RNA preparations.", "Articles", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "5", "pages", "Text", "English", "Reproduced with permission of the Academic Press.", "Copyright may apply", null, null, "Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS OTHE DEPENDENCE OF CELL-FREE PROTEIN SYNTHESIS IN E. COLI UPON RNA PREPARED FROM RIBOSOMES, st Heinrich Matthaei* and Marshall W. Nirenberg Laboratory of Biochemistry and Metabolism, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland Received March 22, 1961 A stable cell-free system has been obtained from E. coli which incorpor- ates c 4 evaline into protein at a rapid rate. This system is of particular interest since certain parameters resemble protein synthesis in intact cells (1). The purpose of this communication is to describe a novel characteristic of the system; that is, a requirement for high molecular weight ribosomal RNA, needed even in the presence of soluble BNA and ribosomes. Washed E. coli W3100 cells harvested in early log phase were disrupted by grinding with alumina and enzymes were extracted with 2.5 volumes of buffer containing 0.01 M Tris, pH 7.8, 0.01 M Mg acetate, 0.06 M KCl, 0.006 M mercapto- ethanol (standard buffer). The extract was centrifuged at 20,000 x g for 20 minutes; supernatant fluid was decanted and 3 yg DNAase per ml was added to reduce its viscosity. The supernatant fluid was centrifuged again at 20,000 x 8 for 20 minutes and the precipitate was discarded. The remaining solution was centrifuged again at 30,000 x g for 30 minutes. This supernatant layer (s-30) was recentrifuged at 105,000 x g for 2 hours to sediment the ribosomes. The supernatant solution (S-100) was aspirated and the ribosomes were washed in standard buffer and centrifugation as before (W-Rib). Fractions 8-30, g-100, and W-Rib were dialyzed against standard buffer overnight at 5°, and were stored at -15°. —— * Supported by a NATO Postdoctoral Research Fellowship 404 Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS In most cases fresh $-30 was incubated for 40 minutes at 35° under the conditions described in Table 1, except that ct valine was present in place of u-c!4y-valine. After incubation the reaction mixture was dialyzed 10 hours at 5° against standard buffer which was changed once. The enzyme was stored at -15°. (Incubated-S-30). RNA fractions were prepared by three phenol extractions at 24° of fresh washed ribosomes; 1, 1/2 and 1/2 volumes of H,O-saturated phenol were used which were concentrated by precipitation with 2 volumes of ethanol and exhaustively dialyzed against standard buffer minus mercaptoethanol. In Fig. 1 amino acid incorporation into protein is presented as a function of time. In the absence of E. coli ribosomal RNA, amino acid incorporation ceases after 30 minutes. Saturating concentrations of E. coli soluble RNA were present in every reaction mixture. Increasing the concentration of soluble RNA 3-fold did not increase the amino acid incorporation into protein. However, when ribosomal RNA was added, a marked increase in amino acid incorporation occurred. At low concentrations of ribosomal RNA, maximum amino acid incorpor- ation into protein was proportional to the amount of ribosomal RNA added, suggesting a stoichiometric rather than catalytic action of ribosomal RNA. In contrast, soluble RNA has recently been shown to act in a catalytic fashion (3). In Table 1 are presented the characteristics of cl 4_1-valine incorporation into protein due to the addition of ribosomal RNA. Amino acid incorporation was dependent upon the addition of ATP and an ATP-generating system and was com- pletely inhibited by the addition of RNAase but not DNAase. Heating ribosomal RNA to 100° for 10 minutes did not destroy its activity. Amino acid incorporation was inhibited by 0.15 ymoles/ml chloramphenicol and 0.20 pmoles/ml puromycin. The addition of 20 different L-amino acids markedly stimulated the incorporation of cl _valine into protein. Therefore, amino acid incorporation due to the addition of ribosomal RNA had many characteristics expected of protein synthesis. In all experiments amino acid incorporation was dependent upon the addition of both ribosomes and 105,000 x g supernatant fluid, indicating that contamination 405 Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   1000 }- 4 900,- 7 800)- 700 600 500 400 300 200 100 0.60 mg. RNA 0.30 mg. RNA — COUNTS / MINUTE 0.12 mg. RNA Minus RNA -       tL Jt | | | jf jf {| 4 0 10 20 30 40 50 60 70 80 90 MINUTES Fig. 1 - Dependence of cl4-t-valine incorporation into protein upon ribosomal RNA. The reaction mixture is presented in Table 1. Each reaction mixture contained 0.98 mg of E. coli soluble RNA (saturating concentration) and 4.4 mg of Incubated-S-30 protein. ———_— of enzyme preparations by intact cells was highly unlikely. Ribosomal RNA had no effect upon amino acid incorporation into protein in the absence of ribosomes and the RNA could not be replaced by the addition of equivalent concentrations of poly- anions such as polyadenylic acid, highly polymerized salmon sperm DNA, or a high molecular weight polymer of glucose carboxylic acid. Pretreatment of ribosomal RNA with trypsin did not affect its biological activity. However, treatment of the ribosomal RNA with RNAase (followed by removal of protein), or alkali, com pletely destroyed its biologic activity. Thus, all of the activity appeared to be associated with RNA. Preliminary attempts at fractionation of the ribosomal RNA have. been per formee : f by means of density-gradient centrifugation in sucrose (4). Biological activity ° 406 Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Characteristics of cl 4_y-valine Incorporation into Protein     Expt. No. Counts/minute/mg protein 1 - Ribosomal RNA 42 + w “\" 204 + \" \" + 0.15 pmole Chloramphenicol 58 + \" \" + 0.20 pmole Puromycin 7 + \" \" Zero time 8 2 - Ribosomal RNA 35 + 1\" \" 101 + \" \"= ATP, PEP, PEP kinase 7 + \" \" + 10 pg RNAase 6 + \" \"+ 10 pg DNAase 110 + Boiled Ribosomal RNA . 127 + Ribosomal RNA, Zero time 8 3 - Ribosomal RNA 34 - 7 \" = 20 L-amino acids 21 + tt “\" 9 9g + \" \"= 20 Leamino acids 52   The reaction mixtures contained the following in pmole/ml: 100, Tris(hydroxy- methyl)aminomethane, pH 7.8; 10 Mg acetate; 50 KC1; 6.0 mercaptoethanol; 1.0 ATP; 5.0 phosphoenolpyruvate, K salt; 20 pg phosphoenolpyruvate kinase, erystalline; 0.05 each of 20 L-amino acids minus valine; 0.03 each of GIP, CTP and UTP; 0.019 cl4.,-valine (# 70,000 counts); 3.1 mg E. coli ribosomal RNA where indicated, and 1.0 mg E. coli soluble RNA (saturating concentrations)3 3.2, 3.2 and 1.4 mg of Incubated-S-30 protein were present in Expts. 1, 2, and 3, respectively. In addition 4.4 mg protein of W-Rib were added in Expt. 3. Total volume was 1.0 ml. Samples were incubated at 35° for 20 minutes, were deproteinized with 10% trichloracetic acid and the precipitates were washed and counted by the method of Siekevitz (2).   the RNA did not follow absorbancy at 260 mi, instead the activity was concentrated around a fraction sedimenting about three times as fast as soluble RNA. Investi- gation of ribosomal RNA preparations with the Spinco Model E ultracentrifuge showed two major peaks with Soow values of about 23, 16 and a minor one at 4.4, correspond~ ing to those described by Aronson and McCarthy (5). Addition of RNAase, but not trypsin, destroyed these peaks. It is possible that part or all of the ribosomal RNA used in our study corresponds to template or messenger RNA. The function of the ribosomal RNA in our system and its further purification are being studied now. In summary, a stable, cell-free system has been obtained from E. coli in which 407 Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS the incorporation of amino acids into protein was dependent on the addition of heat- stable ribosomal RNA preparations. Soluble RNA could not replace ribosomal RNA fractions. In addition, the amino acid incorporation required both ribosomes and 105,000 x g supernatant solution. The kinetics of the incorporation suggested stoichiometric rather than catalytic activity of ribosomal RNA. The ribosomal RNA-dependent amino acid incorporation also required ATP and an ATP-generating system, was stimulated by a complete mixture of L-amino acids, and was markedly inhibited by puromycin, chloramphenicol and RNAase. References 1. Nirenberg, M. W., and Matthaei, H. (In preparation). 2. Siekevitz, P., J. Biol. Chem., 195, 549 (1952). 3. Hoagland, M. B., and Comly, L. T., Proc. Nat. Acad. Sci., 46, 1554 (1960). 4, Britten, R. J., and Roberts, R. B., Science 131, 32 (1960). 5. Aronson, A. I., and McCarthy, B. J., Biophys. J., 1, 215 (1961). 408", "Nirenberg, Marshall W. ; Matthaei, Heinrich", null, "Biochemical and Biophysical Research Communications", "The Academic Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j44r.zmi4-qrvf", "00000000-0000-0000-02E1-C807C40C41E5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Genetic Versus Neural Information Processing Systems", "101584910X181", "101584910X180,101584910X156", "1969", "October 1969", "Marshall Nirenberg gave this lecture at a seminar titled \"Genetic, Metabolic, and Developmental Aspects of Mental Retardation,\" held at Howard University in 1969.  This talk was Nirenberg's most complete account of his considerations of the neural code and its relationship to the genetic code.   As it includes a number of speculations on the relationship between genetics and nervous system function, among others, Nirenberg insisted that it not be published.", "Lectures", "Selection, Genetic ; Transcription Factors", "Transition to Neurobiology, 1965-1969", "10", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "7 | Howarp UNIvErsity -1766   i A WASHINGTON, D. C. 20001 o~ | COLLEGE OF MEDICINE DEPARTMENT OF PEDIATRICS MEDICAL GENETICS UNIT   October 28, 1969 Marshall Nirenberg, Ph.D., Chief Laboratory of Biochemical Genetics National Heart Institute National Institutes of Health Bldg. 10, 6-D-18 Bethesda, Maryland 20014 Dear Dr. Nirenberg: Enclosed is a copy of the text of the talk you gave at our seminar \"Genetic, Metabolic and Developmental Aspects of Mental Retardation\". I understand that it is not, to be published, but we would like an accurate copy Yor our files on this conference. Please review it and make any corrections or modifications you see fit. I will see that you receive a final copy of the talk for your own records. Thank you for your participation and cooperation in this matter. Best regards, Kebuid 4 Mduaay fr: Robert F. Murray, 'Jr., M.D. Chief, Medical Genetics Unit RFM:ctm Enclosure           Dr. Marshall Nirenberg (Ab ere? ' Genetic Versus Neural Information Processing Systems . Ag de wa e a I'd like to talk very, very informally and do something that a biochemist should never do, does always in private and never publically if talking and speculate about something which one doesn't know very much about. Ido this all the time with friends, especially in discussions but not in public so I want to emphasize that,rather than misleading anybody. I spend most of my time thinking about genetic information processing, mechanisms, and so forth and only recently have I really begun thinking seriously about information pro- cessing by neurons and so [ just wish to say that it may sound a little rash to speculate, but, at any rate, if one views a simple cell, suchas E. coli, I guess E. coli, one sees the flow of materials and interconversion of molecules of various sorts, a flow of energy and also a flow of information. Now the infor- mation processing machinery in rapidly growing bacteria will account for 50 or 60% of the total dry weight of the cell. The information is encoded, as you know, ina special class of molecules. It's memory has a specific address and DNA is a very long macro-molecule consisting of only 4 kinds of characters in repeating sequence. A linear string of characters. In E. coli the information may be approximately 3 million letters in length in bacterial chromosomes. A human cell with DNA would consist of approximately 1,000 or 1,500 times as much information. The sequence of the letters in DNA correspond to the sequence of amino-acids in protein and as you know there are 20 amino-acids in protein, 4 bases in DNA and the information in DNA is decoded as a linear form, a one dimensional form and it's decoded by the reading apparatus starting at one point -2- in reading a word sequentially from left to right. The average protein contains some 400 amino acids in length so bacteria may contain about 3,000 kinds of proteins,probably maximally,and for each chromosome there is approximately a million and a half molecules with protein in bacteria so the number of mole- cules in each kind of protein may vary quite considerably from one molecule tp to 100,000 or more molecules. The properties of the template, of the DNA template or an RNA template are really very, very simple. The nucleic-acid sequence, base sequence specifies a kind of object; one molecule of a particular kind. It differentiates, it selects an example of that kind of molecule from many other types of molecules. It specifies the relative position of the molecule, that is relative to the previous molecule it selected and it also specifies the time of the event, relative to the previous event that occurred. So the template really serves both as a template for other molecules and as a biological clock because the words are read in sequential fashion. The principle really isa \\\\ Touring machine principle. That is in the 30's you say British mathematician, Og, Charles Touring devised a very simple kind of machine that would have a reading head that was attached to a tape. The program would travel along the tape and would follow only one instruction at a time and it could read only one space at a time. Basically, this is the same principle employed in all of genetic coding processing and it has some really unique characteristics, for example, one can program one kind of reading machine, Touring machine, to perform any kind of task that any other Touring machine can be programmed for ~ One of the major principles is that there are relatively few kinds of units,and that great diversity is achieved, and also great complexity is obtained simply by varying the sequence of the units. The units are rather standard ones. I should emphasize that all the information available today indicates that the language is largely a universal language, that all species employ essentially almost the same genetic language. I think that this is important when one considers the origin and the evolution of the language and also, considering the problem of neural memory,’ information processing by the nervous system. The origin of the code is an interesting question. One may ask why is the code set up the way it is? Is there any rational reason for it, or is it the result of a unique set of circum- stances that happened only once in the dim and distant past? Then after this unique set of circumstances the code that we're aware of may have evolved from this but the original event may still have control of the format or the form of the code as we see it today. The oldest fossils that are known are micro- organisms. They've been estimated to be 3 billion years of age, found by Barcorn and his colleagues. Fossil micro-organisms 2 billion and ] billion years old have also been reported but the first abundant fossils are found only about 600 million years ago. About 500 million years ago all the invertebrate phyla had evolved and the first vertebrates were formed and mammals rather soon after the code evolved, that the code was probably fixed, the language was fixed because once a sufficient amount of information had been put into the system to form a bacterium, I think that probably it wowld be difficult to make terribly major changes in the code without destroying the information that -4- had already been put into the system unless it was possible, simultaneously to alter the translation apparatus so that the information that's stored can be retrieved. I think this is a fairly reasonable argument and one may well apply this same type of thinking; I wonder if it can be applied at least, to the problem of information processing by the nervous system. There are several differences, I think obvious differences: First, the genetic code must have evolved before any neural code could have evolved. The genetic code probably evolved as the first primitive cells evolved, perhaps between 600 million and 3 billion years ago. Whereas the neural code or neural information processing mechanisms must have evolved at a later date when cells were more adept. They were probably quite sophisticated chemically during the transition, perhaps between single cells and multi-cellular forms of life. And this has another consequence, ‘that is, that almost surely, I think, the basic mechanisms for processing neural information undoubtedly were selected from a large population of precursors or precursor mechanisms whereas one doesn't really know. If this is true in a case of genetic code it could have been one extremely rare set of circumstances. Probably the basic mechanisms for processing neural information had evolved and perhaps were probably fixed by 500 million years ago. All of the information that is available that I can see, at least that I'm aware of, which is not very much, but in regard certainly to the neural information processing mechanisms, but from all the available evidence that I see indicates rather strongly that universal mechanisms are at work, that the neural transmitter substances, neural hormones one finds in very simple forms and pretty much the same substances are found in flatworms as one finds in mam- mals. And similar kinds of mechanisms; synaptic vesicles and nerve endings and -5- so forth. So I would think that the basic mechanisms, there are relatively few basic mechanisms, operative, that they must be systematic mechanisms, and quite simple and logical mechanisms, and that probably they're almost universal similar to the genetic code and that after enough information had been put into this system, into the nervous system, in terms of basic mechanisms, that it's difficult; one can add on new mechanisms, yes, but to change old mechanisms would be difficult because you destroy all stored information if you did do this. I suppose one of the most important aspects of living organisms is the reliability of the mechanisms, of the machinery, and the accuracy also of the machinery. It's really remarkable if one considers that the average protein is 400 amino-acids long, in any series of sequential calculations obviously the accuracy decreases quite markedly as the number of serial steps increases. The accuracy of protein synthesis, from everything that we know is usually quite high and I think that the basic reason for this is, well,there are two basic strategies, I think, that the cell employs to enhance the accuracy of protein synthesis: ditst, fedundancy ks a logical kind of redun- dancy, alternate words, in most cases, have identical first and second letters but only alternate, vary, the third letters so it's systematic redundancy. Secondly, one must consider the number of serial steps and the number of parallel steps here. Protein synthesis is largely a parallel operation. There are serial steps, but interestingly the serial steps enhance the accuracy of protein synthesis. There's one very, very interesting example of this that was reported by Berg and Norris. It was an error-correcting mechanism. As you -6- are probably well aware, the amino-acids are activated and the cell contains that enzymes/are specific to each amino-acid, different enzymes, one for each amino acid. The enzyme is a device that selects a particular, appropriate species of amino acid and the appropriate species of transfer RNA, the adapter molecule and also ATP. It catalyzes first the formation of an intermediate, an activated amino acid. Then the same enzyme will select a species of transfer RNA and it will transfer the amino acid from the activated state that's in to the transfer RNA. Bergan Nomis found that a mistake could be made in the first step, the enzyme would make a mistake and it would recognize the wrong amino acid. In the second step it would correct this error by hydrolyzing, by breaking down this erroneous intermediate. It would not catalyze the transfer of the wrong amino acid to the correct tRNA. This is a beautiful error-correcting mechanism and a perfect example of a mechanism whereby two serial steps enhance the accuracy rather than decrease the accuracy of protein synthesis. Although the other steps are sequential steps, in almost all cases the accuracy of selecting one amino acid or adapter molecule with an amino acid linked to it is independent of the previous selections so an error in one selection usually does not affect the accuracy of further selections. I think that this is the crux of the strategy, right here. There are certainly places where the accuracy of selecting one amino acid does affect the accuracy of selecting subsequent amino acids. This is in initiation, in starting. If the first three bases are incorrectly selected, incorrectly phased, then all subsequent reading will obviously be out of phase and will be » erroneous. Secondly, if an error is made anda word that corresponds to stop, terminate or a protein synthesis is terminated because a word is recognized incorrectly, obviously subsequent selections, there will be no reading, so this will affect it. But, in all other cases that I'm aware of an error of selecting one amino acid will not affect the accuracy of subsequent amino acids . The efficiency of this system is quite remarkable when one considers that the average rate of reading of the reading head, the ribosome along the tape, is relatively slow. The rate of reading is somewhat faster than the average person reads, perhaps in the neighborhood of 1,000 words a minute are read. The efficiency is enhanced very greatly because the cell contains many ribosomes, many reading heads, about 15,000 sites for protein synthesis per bacterial chromosome and one message can be read simultaneously at many different sites, by many dif- ferent ribosomes. Ina relatively short. time a great many words are translated. E. coli have a generation time, under optimum conditions, of 20 to 25 minutes. During this time, 25 minutes, about 500 million amino acids are incorporated to protein. Soit'sa relatively efficient process. We come to the question, really, of learning and of memory. As I said, in the genetic memory, memory has a specific address, corresponding to each memory is a specific molecule, I don't know what the answer is, obviously, when applied to the nervous system. In recent years there has been a great deal of speculation and extrapolation that it may be each neural memory corresponds to a specific molecule of RNA or a kind of molecule of RNA. I don't know, as I say, the answer, but my bias is probably against this possibility although I think it's too early, really to evaluate. I doubt if there's sufficient information available to judge. All cells require nucleic -8- acid, have protein synthesis. It's possible, for example, that a specific permease of some sort can be induced, that one kind or just a limited number of kinds of molecules, proteins must be synthesized to facilitate memory, to lay down the memory traits. It's clear that a chemical reaction of some sort, I would think it highly likely, must take place. The nature of the chemical reaction remains to be defined. In genetics, for many years there was a great deal of confusion about the various theories of learning. One can think of learning on the genetic level, memory, of immunologic memory, and memory in the nervous system. The two basic theories were the instructive theory and the selective theory. By instruc- tive theory one means that something must be present. I think that the easiest thing to do is just to give an example of it. This was clarified by a very famous experiment by Luria and Delbruck during the '40's. This experiment was to take a petri dish, to put some E. coli on this and to grow up a uniform lawn of the E. coli on the petri dish. It was known that if ina flask, for example, in a liquid medium that if one adds an antibiotic streptomycin to a flask, that it will initially kill most of the bacteria, the next morning one sees resistant population of bac- teria. So the question really was, is the streptomycin necessary for the apparent mutation that took place? In a very clever and simple experiment they showed that the mutation would not require, that rather the mutation was independent of the stréptomycin. Streptomycin was not required for this. They grew up a lawn of streptomycin sensitive to E. coli. Then on two plates here which contain strepto- mycin - no streptomycin in this plate, then by pressing a velvet cloth on this and then going directly here and directly here one would transfer colonies from -g- here to here to here, and here. And the orientation of colonies would be similar. One found on a plate like this that one little colony, a clone, the descendents of a single cell would grow, let's say here, here, and here. Then on this plate, with the same orientation, one found exactly the same orientation of the three bacteria that had spontaneously become resistant to streptomycin in the absence of streptomycin. It was very clear that the resistant bacteria were already there and were simply selected for by the streptomycin. This is a very simple experi- ment but it's almost precisely the same kind of situation that can be found in the antibody production mechanism. There seems to be cells that are present. Their descendents are selected for and the antibody production is due to the selection of the population of cells. One wonders, really, whether learning is i to selection. process also as it is in genetic information processing and immunologic information processing. The philosopher, Locke, thought of the mind as a completely blank slate that experience writes on but Socrates thought Se that you can't learn anything that's not already in the mind. This is very similar to a selective type of hypothesis, that one simply selects out from some- thing that's already there. The reason he thought this was because by questions only one can teach. I think that these are problems that one should think about. Thank you very much.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qnmz_ru66_h37m", "00000000-0000-0000-7CB9-E84010E95852", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Neuroblastoma research data", "101584910X190", "101584910X191", "1969", "11 September 1969", "These pages, prepared by post-doctoral fellow Takahiko Amano, outline the preparations of tissue culture neuroblastoma cells.", "Laboratory notes", "Neuroblastoma", "Neuroblastoma Research, 1967-1976", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "date Tit Cloning record Loot ist Cloning » end ik designavion;. n of cells “Sure started / tt : 4g es of DaASSARESs WH! Media used 3 media - | HS : & FCS % 6) passed in agar f= 033%/05%    e/ eeee eeoee 7) . Rémarks; cell moronolesy cf ist 2ne. 3rd SoSH STEHT OH SHEE OEATHOSC SETS EOHSCTHEHA DAHER ETH EME SEH S THEO E OHH ESC SHEATHS AS 8) Preparation of cell suspension Gace Le 9) last media change 5 / eocetile ) last subeulture / ) HO aters pooled, ‘eombined with stickexs ee Te er washed with. % of trypsin in....Dl or D2 LOCH cseeccecvscccces eeeettl. madc on Hes x jv %& — 13) incubated with ...ml. of .....trypsin 2 14) spin [cao LPRecccseeiOle Jonin. at 4- C CENULLLULS ave wvevcccees 15) resuspended in.....ml of media DMEM. Lott 1O3O.. ( HS 200 000% Lotwec Tb bbSieece FCS. coe elo eS LO Gite « Uri « « 16) cell count x 16 “4% Gil factor » fom. Viable/deadercecvccccss/eecscucusvevees 17) diluted COcccccceccecec Sse /al. Wath media . i . 18) plate used 4002 ) ececccsevd eee an so. 3002 ot eG &- im Ath TA eaA 4 werd tet, ~ wee oe 5 see ee Ue a ee wb OL VRP GLbeowes : Caen mT ew de 4 miovce suave 12 e elk => de te oy Plat 7. 3 dae ine, - Sele wee e ee: Cob eee weer eee GSC e cece ee PLE UW site ces ee Ce eee eee ee eees of plating of cylinder | v DL oad eis, eplind 2 Gee Dele Or Guan. . tah Nib: DeLee yD. “the : — t.* . Celery te             cul CG 2 betti: D-wib-g. tS CO7StK ) ' DECI 3S) , tes Clesf )", "Nirenberg, Marshall W. ; Amano, Takehiko", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mya3-dchu~c3gu", "00000000-0000-0000-DC68-8C78DD775547", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Neuroblastoma research data", "101584910X192", "101584910X193,101584910X194", "1969", "30 December 1969", "These pages from a research journal provide the details of how neuroblastoma tumor cells were prepared for tissue cultures.", "Laboratory notes", "Neuroblastoma", "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "N T. Amano r 4 Cell Designation... 1. Je.eeee     Ampole designation... Geeees | Box Bojer eee. . Total # of vials date of freezing..12..29761.-: | Passage Hove Frees weeee 7 | | 1) date of primary culture | | “ : ! 7 | ' I 2) origin of cells ! ! \\\\ { i : : : . 3) date of last subculture | cell # per flask ml of media! type of flsk / | ! : ! | : Media itr (671025 ) Horse serum ; Fes (¢ 1s p dB sg /6_ ml 7 : C3) | ! date of freezing | {co % confluent color of media © | Trypsin used ( sol # — |) ~ % in media....... | sol.# ) i ¥ i FES A i , | oe , Centrifugation | foeerpe. fo | ' Suspended in. — {.O. ml of Media ( iy Freezing media media i % of serum [ [oh DMSO ( lot# ) Fite. AHS of | | oa af | ; : ' argoules: _ type bmxX/o approximate number of cells/ml/vial | ak. : , ; ne te ce ee aoe - Loe Le te - i —_ Freezin g method | | | woe ee r ee ee ee pov tte eee ee *. 4c i [eee hours) a.m. ; i a a.m. o : | P.m p.m. e200 f AG hours | asm. pom. \\\\- _ a.m., pom -170 C pO bees .. hours 2M) Dem 4 a.m, p.m , | : Pd | | ; Peak _ | . Remarks; | 4 , ‘ . | |   ‘ : 1 Numbe =~ of vials taken......; we DYsccccdeccects GMOs cceeccebevecseceee | Cells In Freezer 7) Cell designation... VAG becececeeeees 2)date of primary culture ) origin of COLLIS. sesceeseesoous eeee 2) date of the last subculture. .i/%Vecscres 5) date of last medium CNANZGcccccsccccecece 6) Media Fiz % lot# ( o4te2s ) j2f3e [64 Date.crcscece ES lot# FCS % lot# cell morphology € confluency pH color 7) Trypsin used Oe | ~ in. Rt lot# MAGC.cccccscceccece BOls#ecccccscccecces 8) Centrifugation owe rpm of min. . > (© @ pxso 9) suspended fe in media Fi % lot Lot#esveccoee HS YY  S £ lot# FCS c # lot# % Glycerin lot eeeeeeoeaeee cell count /al. total..../,? Co 7 11) Freezing method eoeoe M1. Of ampoules a d. freezing rate C/fmin. 12) h ampoule # / “eq aw eecveceels PeMe / -20 C a.m eovvceeehle Pelle o / -170 Cc ae. eoecvceeNhs Pem. Remarks: YE 13) Box#ecesccbeccccceeee date of steril autoclaved, in freezer or in freezer in freezer", "Nirenberg, Marshall W. ; Amano, Takehiko", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5dhb-jh8w~antn", "00000000-0000-0000-A9B7-BABB029B1C1E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francis Crick to Marshall W. Nirenberg", "101584910X195", null, "1972", "27 July 1972", "This letter from Frances Crick asks Marshall Nirenberg to nominate Sydney Brenner for the Prix Charles-Leopold Mayer Award. Included with the letter is a list of Brenner's publications.", "Letters (correspondence)", "Awards and Prizes", "Neuroblastoma Research, 1967-1976", "8", "pages", "Text", "English", "Reproduced with permission of Francis Crick.", "Copyright may apply", null, null, "a 2. XC MRC Laboratory of Molecular Biology iy) Jy i | | University Postgraduate Medical School on , U Hills Road, Cambridge. CB2 2QH L/ a England Medical Uw Council telephone Cambridge 48011 27th July, 1972 Dr. M. Nirenberg, Laboratory of Biochemical Sciences, National Heart Institute, National Institutes of Health, Bethesda, Md. 20014, U.S.A, Dear Marshall, I am putting up Sydney this year for the Prix Charles-Leopold Mayer. Could I persuade you to do the same? I enclose a list of his public- ations. You will recall the recommendations have to be in by 15th September. Rog hole } yar eotr, — 4 ts F,H.C. Crick Encs, 1, 2, 3. 4, 5. 6 7. 8. 9. PUBLICA TIONS Gillman, J., Gillman, T. and Brenner, S. Vitamin A and porphyrin- fluorescence in the livers of pellagrins, with special reference to the effects of a high carbohydrate diet and methionine. s. Afr. J. Med. Sci., 10, 67 (1945). Gillman, J., Gillman, T. and Brenner, S. Porphyrin fluorescence in the livers of pellagrins in relation to ultraviolet light. Nature, 156, 689 (1945). Brenner, S. The chromosome complement of Elephantulus. S. Afr. J. Med. Sci., ll, Biol. Suppl., 71 (1946). Brenner, S. Ceroid in the human ovary, with special reference to its mode of formation. Ss. Afr. J. Med. Sci., il, 173 (1946). Brenner, S. Chromosome studies in Elephantulus with special refer~ ence to the allocyclic behaviour of the sex chromosomes and the structure of heterochromatin. M. Sc. Thesis. University of the Witwatersrand, (1947). _ Brenner, §. The non-specificity of the nadi reaction for the cytochrome oxidase-cytochrome c system. S. Afr. J. Sci., 43, 320 (1947), Brenner, S. The identity of the microsomal lipoprotein- ribonucleic acid complexes with cytologically observable chromidial sub- stance (cytoplasmic ribonucleoprotein) in the hepatic cell. S, Afr. J. Med. Sci., 12, 53 (1947). Brenner, S$. The demonstration by supravital dyes of oxidation~reduction systems on the mitochondria of the rat lymphocyte. S. Afr. J. Med. Sci., 14, 13 (1949). Brenner, S. Multipolar meiosis in Elephantulus. Nature, 164, 495 (1949). 10. il. 12, 13. 14, 15, 16, 17, 18, 19, 20. Brenner, S. Supravital staining of mitochondria with amethyst violet. Stain Technol., 25, 163 (1950). Brenner, S. Spectrophotometric studies on the combination of trypan blue and related dyes with the plasma albumin of the rat, guinea~ pig and the baboon. S, Afr. J. Med. Sci,, 17, 61 (1952). Brenner, S. and Gillman, J. The structural specificity of dyes prod- ucing reticulo-endothelioses in rats. S. Afr. J. Med. Sci., 17, 81 (1952). Brenner, S. The chromatic nuclear membrane. Exptl, Cell Research, 9, 257 (1953). Brenner, S. Supravital staining of mitochondria with phenosafranin dyes, Biochim. Biophys. Acta, 11, 480 (1953).   Brenner, S. and Allison, A.C. Catalase inhibition: a possible mech- anism for the production of Heinz bodies in erythrocytes. Experientia, 9, 581 (1953). Brenner, S. The physical chemistry of cell processes. A study of bacteriophage resistance in Escherichia coli., strain B. D. Phil, Thesis, University of Oxford (1954),   Brenner, S. The adsorption of bacteriophages by sensitive and resistant cells of Escherichia coli., strain B. Proc. Roy.'Soc., B, 144, 93 (1955).   Brenner, S. and Stent, G.S. Bacteriophage growth in protoplasts of Bacillus megaterium. Biochim, Biophys. Acta, 17, 473 (1955).   Brenner, S. Tryptophan biosynthesis in Salmonella typhimurium. Proc, Nat, Acad. Sci., Wash., 41, 862 (1955).     Brenner, S. The effects of ribonuclease and deoxyribonuclease on bacteriophage formation in protoplasts of Bacillus megaterium. Biochim. Biophys. Acta, 18, 531 (1955) 21 22, 23 24 25 26, 27 28, 29, 30. ~3- Brenner, S. and Levy, P.R. Non-conversion of B-hydroxypyruvic acid to serve in auxotrophs of Salmonella typhinurium, S. Afr. J. Med. Sci, 20, 92 (1955).   Brenner, S. Genetic control and phenotypic mixing of the adsorp~ tion cofactor requirement in bacteriophages T2 and T4. Virology, 3, 560 (1957). Brenner, S. On the impossibility of all overlapping triplet codes in information transfer from nucleic acid to protein. Proc. Nat. Acad. Sci., Wash., 43, 687 (1957).   Brenner, S., Benzer, S. and Barnett, L. Distribution of proflavin- induced mutation in the genetic fine structure, Nature, 182, 983 (1958). Horne, R.W., Brenner, S., Waterson, A.P. and Wildy, P. The icosahedral form of an adenovirus. J. Mol. Biol,, 1, 84 (1959). Brenner, S., Streisinger, G., Horne, R.W., Champe, S.P., Barnett, L., Benzer, S. and Rees, M.W. Structural com- ponents of bacteriophage. J. Mol, Biol,, 1, 281 (1959). Brenner, S. and Smith, }.D. Induction of mutations in the deoxy~ ribonucleic acid of bacteriophage T2 synthesized in the presence of chloramphenicol. Virology, 8, 124 (1959). Brenner, S. Physiological aspects of bacteriophage genetics. Adv, Virus Research, 6, 137 (1959).   Brenner, S. and Horne, R.W. A negative staining technique for high resolution electron microscopy of plant viruses. Biochim. Biophys. Acta, 34, 103 (1959).   Brenner, S. The mechanism of gene action, CIBA Symp. on Human Biochem, Genetics, 304-317 (1959).   31 32 33 34, 35, 36. 37. 38, 39. 40, -4- Brenner, S., Sanger, F., Barnett, L. and Segall, B. Chemical and genetical studies on the head protein of bacteriophage T2 and T4. Brookhaven Symp. on Biol. (1959).   Horne, R.W. and Brenner, S. A negative staining technique for high resolution of viruses. 4th Int. Cong. on Electron Microscopy, (1958). Springer Verlag, (1960) _Brenner, S., Jacob, F. and Meselson, M. An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature, 190, 576 (1961). Brenner, S. RNA, ribosomes, and protein synthesis. Cold Spr. Harb. Symp. Quant. Biol., 26, 101 (1961).   Brenner, S., Barnett, L., Crick, F.H.C. and Orgel, A, The theory of mutagenesis. J. Mol, Biol,, 3, 121 (1961). Orgel, A. and Brenner, S. Mutagenesis of bacteriophage T4 by acridines. 2 j. Mol, Biol, 3, 762 (1961). Crick, F.H.C., Barnett, L., Brenner, S. and Watts-Tobin, R.J. General nature of the genetic code for proteins. Nature, 192, 1227 (1961). Stent, G.S. and Brenner, S. A genetic locus for the regulation of ribonucleic acid synthesis. Proc. Nat. Acad. Sci., Wash., 47, 2005 (1961).   Shedlovsky, A. and Brenner, Ss. A chemical basis for the host-induced modification of T-even bacteriophages. Proc, Nat. Acad, Sci., Wash., 50, 300 (1963).   Jacob, F, and Brenner, S. Génétique Physiologique- sur la regulation de la synthése du DNA chez ies pactdéries: Mhypothése du replicon. C.R. Acad, Sci., 256, 298 (1963). -5- 41, Sarabhai, A.S., Stretton, A.O.W. and Brenner, S. Co-linearity of the gene with the polypeptide chain. Nature, 201, 13 (1964), 42, Brenner, S, and Stretton, A.O.W. The Amber mutation. J. Cell and Comp. Phys. Suppl. 1, Vol. 64, 43 (1964).   43, Kohiyama, M,, Lanfrom, H., Brenner, S. and Jacob, F. Genetique Physiologique~ Modifications de fonctions indispensables chez des mutants thermosensibles d'Escherichia coli. Sur une muta- tion empechant la replication du chromosome bacterien. C.R. Acad, Sc. Paris, 257, 1979 (1963),   44, Jacob, F., Brenner, S. and Cuzin, F. On the regulation of DNA replication in bacteria, Cold Spr, Harb, Symp, Quant. Biol,, 28, 329 (1963).   45, Stretton, A.O.W. and Brenner, S. Molecular consequences of the amber mutation and its suppression. J. Mol, Biol, , 12, 456 (1965). 46, Brenner, S. and Beckwith, J.R. Ochre mutants, a new class of suppres~ sible nonsense mutants, J. Mol. Biol,, 13, 629 (1965). 47, Signer, E.R., Beckwith, J.R. and Brenner, S. Mapping of suppressor loci in Escherichia coli, J. Mol. Biol., 14, 153 (1965), 48, Brenner, S., Stretton, A,O.W. and Kaplan, S. Genetic code: The “nonsense triplets for chain termination and their suppression. Nature, 206, 994 (1965). 49. Kaplan, S., Stretton, A.O.W. and Brenner, S. Amber suppressors: Efficiency of chain propagation and suppressor specific amino acids, J. Mol, Biol., 14, 528 (1965). 50. ol, 52, 53, o4, 95, 56, 57, 58, 29, ~6- Brenner, S. Collinearity and the genetic code. Proc. Roy. Soc. B., 164, 170 (1966),   Brenner, S., Kaplan, S. and Stretton, A.O.W. Identity of N2 and ochre nonsense mutants. J. Mol, Biol,, 19, 574 (1966). Crick, F.H.C. and Brenner, S. The absolute sign of certain phase- shift mutants in bacteriophage T4. J. Mol. Biol., 26, 361 (1967). Sarabhai, A. and Brenner, S. Further evidence that UGA does not code for tryptophan. J. Mol, Biol., 26, 141 (1967). Sarabhai, A, and Brenner, S, A mutant which reinitiates the polypep~ tide chain after chain termination. J. Mol, Biol., 27, 145 (1967), smith, J.D., Abelson, J.N., Clark, B.F.C., Goodman, H.M. and Brenner, S. Studies on amber suppressor tRNA. Cold Spr, Harb. Symp. Quant. Biol., 31, 479 (1966),   Stretton, A.O.W. and Brenner, S. Spontaneous revertants of amber mutants, J. Mol. Biol,, 26, 137 (1967). Barnett, L., Brenner, S., Crick, F.H. Cc. » Stuiman, R.G. and Watts-Tobin, R.J. Phase-shift and other mutants in the first part of the rlIB cistron of bacteriophage T4, Phil, Trans, Roy. Soc, B., 252, 487 (1967), Brenner, S,, Barnett, L., Katz, E.R. and Crick, F.H.C. UGA: A third nonsense triplet in the genetic code. Nature, 213, 449 (1967). Sambrook, J. F,, Fan, D.P. and Brenner, S. A strong suppressor specific for UGA. Nature, 214, 452 (1967). 60, 61. 62, 63. 64, 65, -7- Abelson, J., Barnett, L,, Brenner, S., Gefter, M., Landy, A., Russell, R, and Smith, J.D. Mutant tyrosine transfer ribo- nucleic acids, FEBS LETTERS, 3, 1 (1969). Russell, R. L., Abelson, J.N., Landy, A., Gefter, M.L., Bremner, S. and Smith, J.D. Duplicate genes for tyrosine transfer RNA in Escherichia coli. J. Mol. Biol. , 47, 1 (1970). Goodman, H.M., Abelson, J., Landy, A., Brenner, S. and Smith, J.D. Amber suppression: a nucleotide change in the anticodon of a tyrosine transfer RNA, Nature, 217, 1019 (1968), Smith, J.D., Barnett, L., Brenner, S. and Russell, R.L. More mutant tyrosine transfer ribonucleic acids. J. Mol, Biol., 54, 1 (1970). Altman, S., Brenner, S, and Smith, J.D, Identification of an ochre-suppressing anticodon. J. Mol, Biol, , 56, 195 (1971), Brenner, S. Nonsense Mutants and the genetic code, J.A.M.A., 218, 1023 (1971).", "Crick, Francis, 1916-2004", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ftb5-adac_cgpc", "00000000-0000-0000-B271-22A132421232", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Lisa Sherman to Marshall W. Nirenberg", "101584910X196", null, "1967", "24 May 1967", "This letter from a nine year old student from New York is the product of a letter writing club listing Nirenberg as a \"noteworthy person\" to write to.  The author requests a letter and a photo and congratulates him on his election to the National Academy of Sciences.", "Letters (correspondence)", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Text", "English", "Reproduced with permission of Lisa Sherman-Cohen.", "Copyright may apply", null, null, "184 Elmwood Street Valley Stream, N.Y May 24, 1967 Dear Mr. Nirenberg, I belong to a Letter Writing Club and I chose you to write to from a list of noteworthy people. I hope I recive [sic] a letter and a picture of you wich [sic] I will put in my album I am nine years old. Congratulation for being elected to the National Academy of Science My father is a doctor. Was it hard getting the Academy? Were you scared? Happy Memorial Day. What Collige [sic] did you go to? Sincerely, Lisa Sherman", "Sherman, Lisa", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rngr_g67x~pn9c", "00000000-0000-0000-9A5B-00180DF8D2A3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Neuroblastoma Problems", "101584910X198", "101584910X199,101584910X200", "1969", "[16-19 April 1969]", "These pages outline Marshall Nirenberg's approach to neuroblastoma research.  He lists the problems to solve, the goals of the research, and the experiment methods to use.", "Laboratory notes, Excerpts", "Research Design,Neuroblastoma", "Neuroblastoma Research, 1967-1976", "38", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "+ ee 6 jhe dQ                               26                     . pent fe ran aay, OL aL   = eater nrerremienrnciaeen oS Cv H                                             Us CR or XG (RL 3070 PK + J,0 afr fe ~ Pro Yt ie m           Pine \\\\c gle fe — freee te at Oy. | _ i e , tae) AL Ve ON \\\\ ote [4 } Q -C 29a Mb                                                                                                                                                                                                                                                                                                                                     Rec. ne eee 0 pa ru “ AI — an Pm Le At LIM fe CE: : Gre LA B “Th — | —                                             31                                                                                                                                                                  wy. , ~~ wd ldugr Xa fy epi e                                                                                               ee         35                                                           36 Vb td be OL; ~ Se Lip Pash (3) Wag Bi f > os ) PS RT g MA 3 7 — Jjod om Tyee                   fe ere (En | a fa Tain H 1 Com a, ley re te.             x) Chaar? . { rte) Cake KP               a ee De { voll Sacef eS 2 a ae! gle’, § ic mite   | 37               2 ——— Dac Nea mee << Nes =f f= te No = ~ ben: Lh he =e e Vo srr ar~ Meo he ff Ay. Ce ake, pte wre 23 we Go BEChe Op Ace i :       a ; _. oN 5 sh Lait. Unter 2 ghee a, 0 o pe] r                                 We 4 i = * ls & os | “ , a ¢ 7 MK t PR, 7 vw * \"r & tre i ‘ ‘ i j | a 4 ‘ A :                         HK @, Corte ( Ke. ho AT eae ) ~ Gif , ae Na a eS re 2h df Woes ee ane me et <) a AT MES aor (3 ie. spe Fate lip i - go Fe Ed to 2 /0¢) ES — (3 = LS tse. Se nk ae Det AZ Preston                          )-y 6 be See                                   oe nen we if a Pet is ue oe . H os ' - \\\\ 4 iol a Pigs a MNY s} 4 \" Ty A st s if § - Sy oe . 3 , wo ~ ee “p> , ~ DW C a Ke io . oo 7 7 WwW nt       E>. 1 meen —— ‘ i * : Yoke .. hg . Sf i; ‘ fi i i . _ ‘ Joy , “gy ‘ 1s : i pte et bh ged tS irl —y- Ay et yd “eK Mas eS co                               ty £ J 4 CPt hy” ¢ i f 4 ~——                                         41                            12 =——=py~ —2D p ff hee B Sys           () roe / sf “$4, . WoW whee Xe str es A fhe A                                  » f ta y f- rch ten, W VV . Kal? Wwdg lead A Ble artbratnee ON  e U Eres 2X ait 4 5 j oy of er Poe q 7 : (pes = Moe phic ko — ki iv Dash to i pte \\\\ 7 7 { on ‘ = hee bay Kk i . ~ OF . { iy t TONS ¢     42, | dj ee car,   pee AI pl —                         2%. 2. he at op oF ae #5 ko TK TR a       2,. oe a aaa =i Te)                                               { 2 KY Oa Pa AY vee al Cheer er—of — é a.             Y. a AED   JS = (eee on: Gon + arn es _                                                                                                 46 oe Foes “hh em A WA a oe aes Ceol ac. — p a PIT he Cae a.         eee     oem terest oem me ee ae hcl ete pes Geng, s) Were. fli l Oe ee d) \" dt af i ) J, Va) ey pe OTOL,           “) (Loepes | = ol? Nude f oll Tae Ducfoes sta eae 2p                                   TS af al ites pennant ananaR =f ER wl i eel a, - —____— ~. hs eel ~~                   rT Y — ae ia wt ~ be Oe . i) Naroging — >} Pil” Mea < Ta,                                         j LU 4 4 : “i i     i = = ee ee | = —— —   49   i UP 1 Base fen ~ x NM a [ +                                                                                                                         | ‘ ~~, | ) rok —_—_— Hes eget hte tk ie Lot eS a =                        hi 4 4 » . | 3 Fos od FCA OMY __f der . =; Cc a! fcr “SUA ~ : Ad at | . \" i re Ae cde Brea Aue                                 FAI 52   —* BAL he aed rdf           FS                            * e. Weg he ee ge eee a an a” x * -   et! — cia aad tA: wi Af = f= AM _ : 2, i os 4 4h, ooh L x _ it’ ae fol RRL es Le n u x? ieee 4 olla all aH ak BM Be pd Ab fe. cake 2 ei ee [. fo ee FE De eh son eek gl — 6 To — YO EOPL audlc hook he ad. i “ ~ ( [ “I igs ck wh? At Y sae a Avi ny ———     ao =e H ee pe tek i uy A, Aedes oe, (on Meo Me des otf ety Fos 4                   nce a , oa aay mentees                                                                                     : 4                                     i : » oe cle en \\\\ NA) wee pd on NV 6 V Hh a x] ee a A” = — bpp es   ' BrP tC ek) Lobel.   2 |   J f                       55           Sy pe LLL rete Re 2 Nimwecg _ '     3, LARA u ay Z a 4s   ow Be Le. Go ae 2 COME = - pat                                                               56 Sa ea ne, fe                                                     57                                           0 THA oeeetiag St 3 oc LA                                                      , a live 9 tell oe Be, Ha ie! 8 PRES oh L So ) Phe                     ) 7 Zz A TE Le AA a 2 LK, 2 if LCL Meck 2. (AD > Wen Hye 4 Sas :                     = Va CORO IE Net ; Ge ante Bote tie phe AF Le \\\\ oo 3, CL a oe           Ao bh                                 a eo? Tape = \"5 wre   - 0 . ge re CX Mag acento enpaet 7 : \\\\eaAy LOOO Ad, Deu, 7                                                             ‘ = aera tr ‘ EO . ‘ _ as               b Pevre ren, br h ee aval ae’ Oa   NO = We ono Al a 120: a t Spt pee   2- f) een Tone SoA 5sxi0° AR at TLo$ Pa                         b. Heke hein tT afl oem as                                                       ales —— Ee PR XA _! in von a Ue ws _A jf Gre 2 Adopt | So _ ei a L a ae JUS i col", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9dkd_ddbs~tffx", "00000000-0000-0000-F063-E758A63C25F0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Ideas for Neuroblastoma Experiments", "101584910X199", "101584910X198,101584910X200", "1969", "16 May 1969", "In these handwritten journal pages, Marshall Nirenberg lists many of his early ideas, questions, diagrams, and tables intended for use in the study of neuroblastoma systems. Nirenberg often outlined his thoughts for experimental research in these laboratory journals.", "Laboratory notes, Excerpts", "Neuroblastoma,Research Design", "Neuroblastoma Research, 1967-1976", "20", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "1) Ep gd 2 =P Sef ) Epes EAR “] )éo x DZ or sb) i a : s) Deoh-3 | | L) 6-Off - be -) ‘ ’ > Ah Cone PE Reh     82   1 Clk, he “tiles 1 Bee Ree                                                                                                                                                                                                                                                 85                               _— OP = bY Die Lend pacbap oo [Rpg rata 9 —_—_ (pee (eR el Ss LA ze S _——— a | oo NY 4 i _— [ oe : _—— RY os SY : i cee _ He O71\" ut cae. rl 1 < fee ep _ aes Ae (o-7 emacs , _— pL Cen akg wa choy 2)             2) @ Qe   ee ic te eee. PE aN NE cps ape. W or Vat Cle ol = 2b, X yee, {         2) 7                86                                                                             87     Q 23 @ zs -K           (- — 1 AEH i e eK ZS De he \\\\ lS LO \\\\ HET Cre SE                                                                                                         89                             NC     f ate 2                                                                                                    \\\\ ‘Aad \\\\9 ysohon Wear i Fo . Bx : eee - EG oll . sag thigh fm Lh aes , (=f) Adkillag. / | — $o4-—B. —— gl C a7 I acl fll gig ol HO — Cnr Pb pd 25) Odo i — Nhe J/g _ — THK Lah AN 4   Meyae—— J g ~ i, a by gr oF > Cun ok ap : o4—.(/ ~ Acs} ve “ 4 “A. 91 5 £) . Pen fen - 5S - - 7 be a AS QO» Qt, | rR CO ORIEN Ee NY                               : D, | ~s 1c Cet = ecco) Honda fel # fc ry Np ic Fi fi De, x! le 4.0 wLbfed ou JiAtnt\"                   92 ; Of A, _ —— Nes — te e e f =                                                                         93                     yf =U A ASS Fee n Poenmacly Sar l 7 C lp, Wu. ticked face oli ES Ma “ Sria~t gig ka qT a fe ] > = 7 Va oe} ach cate _ l Vv     Cok che Woe | wO6 . Ke 75 RT Ee let oS vA Tease RO \\\\ _ \\\\ . / ee Eat tee — [wv _, _ L CAI (Nobuo 7} / Qe                                   E Le thd 759 ; | “7 ~ Mga, > ¥ al PA o4— “SO 2 2. \" OL Limp dy Nea \\\\ Sg! WU? * Xv : Nf ole, 0 0, —> ee, HotRS ag m7 { re 3                                     95   am ae E De Fab ne ~~ GAR ¢                                                                                       C TA 5) aK bee LA J 7S h \"7 x 2\\\\ 4 ; \\\\ oe pt — 7 Be h c — oe a                                           iE cl Soe LG ial a aM aan Pica fC Sane Ea (aaah                               98                       rT a i i he New oo) yA Spe L- 12 Herne oe! - : sie oe | mo Di Blog — 2 YAW\" | ca x \\\\ FereAQ b | 22 Z Boon i a . o             : f fy                       + ic Pal Ds A N) gooemra dpa ; 7 POLE NY 2 : ) \\\\ —+               99 a wT KO yp [PM               Lo Jo. fu BZ iio - 20,022 nC Lends Pieree ~— 20s te 22 on, Ud. on } = 7G, By . O02 en (af ke oo One ated psec 23s). so ~0, 0004 eV p abl . ws                                                                           | U_) 410.4 oe cl pe yy Sf sol \\\\ ad / rf : v / _ r| LA Me 2 2 2h TD mS in Lanes Yeahs se eM, 4 \"A 2p ne DSO tO ER $b blprare Lot OO, OFC vi) / [Mv _\\\\ ¥ Ue Amen \\\\ | TTT V7 t                   0, my : Th iy alba . M, . No. onal A af   Vv   al =aane Fat LAR £                       on pe Ie al CS : me _ \\\\A- ae tel a // J yore =", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-r6m4-fub8_bx7r", "00000000-0000-0000-F71C-3DC51D1EFC30", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Conversation with Phillip Nelson", "101584910X200", "101584910X199,101584910X198", "1969", "10 June 1969", "Marshall Nirenberg collaborated with NIH colleague Phillip Nelson when he was studying the neuroblastoma system.  These pages list the questions and answers from a conversation he had with Nelson about the neuroblastoma research.", "Laboratory notes, Excerpts", "Neuroblastoma,Research Design,Synapses", "Neuroblastoma Research, 1967-1976", "11", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "137 ofl [4 il. Bra v pum aR     CO eee we Ate =                         , o , SP > “ “Dh ns shape Oy WLS pun as ( ie U po Ir ak oe pens es ———                                                                                                     - ; : Me, f a                                                   7 pe pep ees eh ava _——————] py Painnarnens                                             140       ~ eng Vee. Cr —_Newact bs ke dpe Seve = Ay y) ¥ a       n fy, = J \\\\/ i CLLR wh, 2a ‘a gers _ f a “Je i ee \\\\ DK Toe arn. Atl   A ibho <A \"eae                                                                         141 |                                                                                                     142                                                                                         143                                                                         144                                 5 \\\\ — 7 Nh gr Ay A 7 De = \\\\ _ fe LI a. ia iN = Co TOY A                                     “aL BO 2 Ae B= GD) zen)! ty OE Ae Clif a (eratilte ! Ar | aes Be DF ; Nf | _ Sy te : p — ! _ 4 i     145     dhe, Y Ne igs) cate (. v7 JN           c y     ¢         AtKS a 7 Spe LX a ther mao he                                         + a } 7 -— Ae feo 5A W Ce v4 Ne La N HX ok                         - TY ’ Ne am 7 7 (pgp : an x ) i - Xe oT eas EEA a “ jf ~ re ra 0 f f) - Np merge ot —< oe         oe ae 2 CAL a s APR nes = Tye IONE } “er mo =p . my 6                                                                       147   es ey ee “A x bb -f AeC xR             | oft wy AL Pee —> Zara : it \\\\ A ee xD) [ € 4 ofF-       iffy ) ea Tale     to Ya A wr Pa a <7 LL) —— as Go", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cfr4.wf5u~wi7n", "00000000-0000-0000-16E8-5F0D9570EEF1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on nematodes and chemotaxis", "101584910X201", null, "1967", "[10-22 June 1967]", "In these notes, Nirenberg outlines experiments he plans to perform to study neurobiology.  These notes are focused on the study of nematodes and on the chemotaxis of bacteria.", "Laboratory notes", "Chemotaxis,Neurobiology", "Neuroblastoma Research, 1967-1976", "10", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p6fr.s8u6.4n78", "00000000-0000-0000-5C28-0FB483B26AF4", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on nematode experiments", "101584910X202", null, "1966", "26 September 1966", "While studying the properties of the neuroblastoma system, Nirenberg also investigated nematodes, small roundworms, with the help of post-doctoral fellow Ruth Pertel.  These notes list some of the other scientists studying nematodes in neurobiology.  Sydney Brenner's name appears first in the top left corner.", "Laboratory notes", null, "Neuroblastoma Research, 1967-1976", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-acya.zrs6-n3zr", "00000000-0000-0000-769C-47C61666B0DA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "In Vitro Ideas", "101584910X203", null, "1967", "6 November 1967", "Marshall Nirenberg studied the neuroblastoma system by growing tissue cultures in order to investigate the cells in vitro.  These notes list Nirenberg's early ideas about how to utilize the in vitro method to study neuroblastoma.", "Laboratory notes", "Neurobiology,Research Design", "Neuroblastoma Research, 1967-1976", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "ea       pee", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bsfd_6hxq.gv7q", "00000000-0000-0000-18FF-8EA05B5BCE80", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Very Good Research Problems", "101584910X204", null, "1968", "26 January 1968", "In these pages, Nirenberg lists over 30 different research problems he wishes to study experimentally.  The problems he found most important are marked with \"**\" next to the numbers.", "Laboratory notes", "Research Design,Neurobiology", "Neuroblastoma Research, 1967-1976", "10", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "voowsn NI QaH:                             LL             asia   AZ ISAM NOSIGQY--‘v'S'h NI                 lavad “DNi", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6pdr_ywdf-xgei", "00000000-0000-0000-CB16-B136D920DB6E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Nelson", "101584910X205", null, "1967", "April 1967", "These pages list five features of neurobiology that Nirenberg planned to study with NIH colleague Phillip Nelson: neuromuscular junctions, tissue cultures, nerve growth factor, synapse formation, and contact inhibition.", "Laboratory notes", "Neurobiology", "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "ToT Top _— i 1 a “ _ = a be a i i t an . iL ! : ee nent rl I", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5wqh~95wc-7snw", "00000000-0000-0000-B215-1D7A1D125E55", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Questions for Phillip [Nelson]", "101584910X206", null, "1967", "13 June 1967", "These notes list a series of questions Marshall Nirenberg was to ask Philip Nelson about their work on neuroblastoma.  The topic is specifically focused on the synapses that Nirenberg and Nelson attempted to form between nerve and muscle cells.", "Laboratory notes", "Neurobiology,Neuromuscular Junction", "Neuroblastoma Research, 1967-1976", "4", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "y BS Ptah TS, (Boe GL   Yeo a9 2. / piakef y) rs 2 Ma A~% , Vin ar ava             ety, VL Be ey SS oie i (Ve th FR om ee ‘ Sore ace |   pre PATS “3 yon aL... 3) Bok eke   “yee ep LL, tor Ay oe > ff ely cel / [om nenenats", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-npn6-zrgt_h8nc", "00000000-0000-0000-F9DC-1AC0D4DA867A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francois Jacob to Marshall W. Nirenberg", "101584910X207", null, "1970", "30 January 1970", "By 1970, Marshall Nirenberg had turned his attention to neurobiology and developed valuable microassays for enzyme study.  In this letter, Jacob requests information from Nirenberg on techniques and protocols for enzyme and transmitter assays in mouse nervous system and mouse cell culture research.  He informs Nirenberg that he has left his own work on nematodes and asks what is new with Nirenberg's own nematode or neuroblastoma research.", "Letters (correspondence)", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Text", "English", "Reproduced with permission of Francois Jacob.", "Copyright may apply", null, null, "30 janvier 1970 Dear Marshall, I understand from the paper of Gordon Sato in PNAS that you have developed a series of elegant micro-assays for the enzymes of catecholamine, acetylcholine, serotonin and GABA biosynthesis and catabolism. I have left the nematodes which do not allow me to do what I would like.  I am turning now to work on mouse nervous system and mouse cell cultures.  I have of course no experience in the field, but I would very much like to have in hand the techniques for the study of the transmitters.  If you have no objection, I would be very grateful for a detailed protocol for these enzyme and transmitter assays. What is new with nematodes with mouse neuro-blastoma and other cells in your lab?  Is there any chance to see you around Paris in the near future? With very best wishes, Yours sincerely, Francois Jacob", "Jacob, Francois, 1920-2013", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jaib~r6x3~awvq", "00000000-0000-0000-8597-F88F48C25DF1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Regulation of Acetylcholinesterase in Neuroblastoma Cells", "101584910X208", null, "1970", "October 1970", "This article is one of the first published reports of Marshall Nirenberg's work during his transitional move toward neurobiological investigation.  Experimental findings in the article show that when tumor cells shift from the dividing to the non-dividing state, they are influenced by the expression of a set of genetic characteristics required for neuron differentiation.  As progeny cells remained committed to a program of neuron differentiation, the authors anticipated implications for the study of healthy neurons--the \"possibility\" that \"normal sympathetic neuroblasts are also inversely committed to a pattern of neuronal differentiation.\"", "Articles", "Neuroblastoma,Cell Transformation, Neoplastic,Enzyme Activation,Acetylcholinesterase", "Neuroblastoma Research, 1967-1976", "7", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proceedings of the National Academy of Sciences Vol. 67, No. 2, pp. 786-792, October 1970 Regulation of Acetylcholinesterase in Neuroblastoma Cells A. Blume}, F. Gilbert, S. Wilson, J. Farber, R. Rosenberg, and M. Nirenberg LABORATORY OF BIOCHEMICAL GENETICS, NATIONAL HEART AND LUNG INSTITUTE, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND 20014 Communicated July 31, 1970 Abstract. The specific activity of mouse neuroblastoma acetylcholinesterase (EC 3.1.1.7) increased 25-fold when the rate of cell division was restricted. The results show that acetylcholinesterase activity is regulated in neuroblastoma cells and that the regulatory mechanism is inversely related to the rate of cell division. Under the same conditions the specific activity of catechol-O-methy] transferase (EC 2.1.1.6) did not change significantly. Mouse neuroblastoma C-1300 has been transplanted from animal to animal approximately 1000 times over a period of 30 years; nevertheless, cells, after 5000-8000 cell generations, continue to follow a program of neuron differentia- tion. Cloned cells contain acetylcholinesterase (AChE), choline acetyltrans- ferase (EC 2.3.1.6), and enzymes for norepinephrine synthesis.’? Cells also are capable of extending processes, several millimeters in length, that contain microtubules,** neurofilaments, and dense core vesicles? and possess membranes capable of generating action potentials in response to electrical stimulation’ * or acetylcholine. Burkhalter e al.2 and Goodwin and Sizer\" have shown that, the acetylcholines- terase activity of cultured chick embryo intestine and skeletal muscle can be regulated. In this report the activities of mouse neuroblastoma acetylcholines- terase and catechol-O-methyl transferase were investigated.11 We wish to report that neuroblastoma acetylcholinesterase activity is regulated and that the regulatory process is coupled to the rate of cell division. Materials and Methods. Cells: Experiments were performed with mouse neuroblastoma C-1300, clone N-18,° passage 43-60. Cells were grown in Dulbecco’s modi- fication of Eagle’s medium supplemented with: 50 units of penicillin G and 10 yg of streptomycin sulfate/ml, 10% fetal calf serum, and 10% horse serum (except where noted) in 10% CO.-90% air. Cells were kept in logarithmic growth in monolayer cul- ture (ie., cells grown attached to the surface of a Petridish) for 7~10 generations before each experiment. For experiments, cells were grown in 150-mm Petri dishes (Falcon Co.) containing 20 ml of growth medium. The medium was changed every other day. Cells in logarithmic phase growth were dissociated by incubation without Ca?+ or Mg?t for 10-15 min in saline D,,!* supplemented with 5.6 mM glucose and 59 mM sucrose (modi- fied D,). Cells in stationary phase growth were firmly attached to the growth surface, and their subculture required an additional incubation with 0.05% “trypsin” (Difco 1:250) in modified D, for 10-15 min at 37°C. Homogenates: The medium from each dish was transferred to a centrifuge tube and the cell monolayer was washed 3 times with modified D,. Washes and growth medium were 786 Vou. 67, 1970 NEUROBLASTOMA ACETYLCHOLINESTERASE 787 pooled and centrifuged (250 X g, 10 min, 3°C); the cell pellet was suspended in modi- fied D,, then washed twice more in the same manner. Dishes were drained, and cell monolayers recovered by scraping. Dishes were then washed twice with small amounts of a solution containing 50 mM potassium phosphate buffer, pH 6.8, and 1 mM EDTA. The washes, scraped cells, and cell pellet were combined and sonicated. Homogenates were stored in small portions at —196°C. No effect of storage on AChE or catechol-O- methyl transferase activities was detected. Assays: Cell concentrations were determined in duplicate with a hemocytometer, cell viability was determined by the nigrosin method,\" and protein was determined by a modification of the method of Lowry ef al.\\\\4 Catechol-O-methyl transferase activity was determined by a modification of the method of Nikodejevic, Senoh, Daly, and Creveling.\"* Each reaction contained the fol- lowing, in a final volume of 0.05 ml: 5 mM potassium phosphate buffer, pH 6.8; 0.8 mM EDTA; 5 mM MgCl; 1 mM dihydroxybenzoic acid; 1.1 mM [!C]methyl-S- adenosyl-L-methionine iodide (4.6 »Ci/umol); and homogenate protein. Reactions were incubated at 37°C for 20 min. The rate of reaction was proportional to enzyme concentrations. Each reaction mixture was acidified and then extracted with toluene. AChE was measured by a modification of the method of Reed et al.,!” to be described in detail elsewhere (S. Wilson, ef al., in preparation). In brief, 1-[}4C]acetylcholine is incubated with homogenate; then 1-[!*C]acetate is separated from the substrate and counted. Each reaction contained the following components, in a final volume of 0.05 ml, except where noted: 0.05 M potassium phosphate buffer, pH 6.8; 0.2 M NaCl; 1 mM EDTA; 0.5% Triton X-100 (Packard Instrument Co.); 3.3 mM 1-[4Clacetyl- choline iodide (0.39 »Ci/umol); and 0-0.05 mg homogenate protein. Reactions were incubated for 10 min at 37°C except where noted, and terminated by the addition of 1.5 ml of a solution, at 3°C, containing 2 »M 1,5-bis-(4-allyldimethylammoniumphenyl) pentane-1,3-dibromide (BW284C51, Burroughs-Wellcome Co.), an inhibitor of AChE.\" In all cases, the rate of reaction was proportional to homogenate concentration. The diluted reaction mixture, and a subsequent 1.5 ml wash containing the AChE inhibitor, were passed through a disposable 0.5 X 5 cm column of BioRad AG50X8 (Nat-form, 100-200 mesh) washed with water. The eluates were collected in a scintillation vial, 10 ml scintillation fluid (1000 g Triton X-100-2000 ml toluene-165 ml Liquifluor [New England Nuclear]) was added and the radioactivity was determined. Most values re- ported represent the average of triplicate homogenates; each homogenate was assayed at four concentrations. The reproducibility of AChE and catechol-O-methyl trans- ferase specific activity determinations is +15%. One unit of AChE or catechol-O-methy] transferase activity is defined as 1.0 nmol [44C]-product formed per minute. Specific activity of the enzymes is expressed as units of enzyme per mg protein. Enzyme activity per dish corresponds to units of enzyme per dish. Results. Enzyme activity as a function of cell division: The relation be- tween AChE and catechol-O-methy] transferase activities and the rate of multi- plication of neuroblastoma cells is illustrated in Fig. 1. Each Petri dish was inoculated with 2 X 10° cells. After a slight lag, cells multiplied rapidly with a generation time of 24 hr. The maximum cell concentration (28 X 108 cells) was attained on approximately the 6th day of incubation. The specific activity of AChE did not change appreciably during the period of rapid cell division. However, during the stationary phase of growth, the 6th through the 17th day of incubation, the specific activity of AChE increased 25-fold. To investigate the effect of logarithmic growth upon AChE specific activity, stationary phase cells were subcultured at lower cell concentrations on the 13th day of incubation and then maintained in logarithmic growth. A 50% decrease 788 BIOCHEMISTRY: BLUME ET AL. Proc. N. A. S.       40 [a” WEES, Praren T 5 25 a 40 & ~ ~ \"5 20 /s SUBCULTURE i 30 Zz % 15st fe * o 5 o wolff || IP & aa a t a a a 5t te oh iy 1 '0 2 Fig. 1. Neuroblastoma cells in logarithmic oe ae 0 growth were subcultured without trypsin. On soe ACE TYLCHOLINESTERASE 16000 the 13th and 17th days of incubation, some of 2 Perry the cells were dissociated with trypsin (indicated wi IS) petiwry\\\\, v4 9000 | by the vertical dotted lines). Closed and open 2 oor 14000 2 — symbols represent the values obtained before and es 75 ji \\\\ | 43000 3 after subculture, respectively. In panel A, cells/ = sof | J2000 = dish (@) and mg protein/dish (4) are shown. Zz sen} : a More than 85% of the cells were viable throughout 2 25 DISH + loo0 & . : < vA 2 the experiment. In panel B, AChE specific wy of ia Oo S activity (@) and AChE activity/dish (A) are & O18 [C caNsreRAse, | © )6©6 shown. In panel C, catechol-O-methyl trans- * Qg . - woe . © O15 Tf  speemic 15 2 ferase specific activity (@) and activity/dish (a) PER wetnt 8 o.l2 ren 40 4 © are presented. 2 0.09} Te 13 9 pos, fk fy 2 3 8 0.03 + x pa | | c < 4 Q , 9 36 9 i215 182i 7 DAYS in AChE specific activity was observed within 40 hr; after 6 days the specific activity returned to the basal level. The amount of protein per average cell doubled, from 0.6 to 1.2 ng after cells shifted from logarithmic to stationary growth. Over the same period, AChE activity per dish increased greatly, resulting in a 50-fold rise in AChE/cell. When stationary phase cells were shifted to logarithmic growth, corresponding decreases in protein and AChE per cell were found. No significant change in the specific activity of catechol-O-methyl transferase was noted throughout the course of the experiment. The possibility that subculturing procedures affected AChE activity was also investigated. Incubation of stationary phase cells with trypsin resulted in a 30% decrease in cell protein and in AChE activity; thus the specific activity of AChE remained constant. In other experiments not shown here, stationary phase cells were dissociated with trypsin and then plated at the original cell con- centration and incubated for 2 days. No change in AChE specific activity was detected throughout the period of incubation. In addition, two cell populations were maintained separately in logarithmic growth by subculturing every other day for 10 days. One population was subcultured by the trypsin procedure, the ‘other with modified D,. Cells then were incubated for 17 days. Values ob- tained with cells treated with trypsin did not differ significantly from those obtained with cells passaged with modified D, shown in Fig. 1. The results suggests that AChE, but not catechol-O-methyl-transferase, responds to a regula- tory mechanism that is coupled to the rate of cell division. Neuroblastoma cells stop dividing in the absence of serum but continue to Vou. 67, 1970 NEUROBLASTOMA ACETYLCHOLINESTERASE 789 synthesize protein.’ If AChE activity and cell division are inversely related, then removal of serum from the growth medium should result in an increase in AChE activity. The effect of serum concentration on AChE activity and cell growth is shown in ]‘ig. 2. In the presence of serum, cell growth was logarithmic and AChE specific activity remained constant (Fig. 2A). In the absence of serum, the cell population quadrupled within 2 days and then remained constant. When cell division was restricted, a marked increase in AChE specific activity was observed (Fig. 2B). In this experiment, the cell concentration at the time that AChE specific activity increased was 31,000 cells/cm? compared with 200,000 in the experiment illustrated in Fig. 1. Thus regulation of AChE activity is not a function of cell concentration under these conditions.               2 io T T T T T T T T T t r T T T T xr 5 A SERUM B NO SERUM C SERUM ON 3rd DAY ai e t 30 + 600 & 100440 r a } AChE AChE > SP. ACT. = S eot32 = - 13 PROTEIN Z 941 4802 g ce « 2 a = a a ceLts w N ae = g 8 iI & 18 +3600 W 60724 4 + ia 9 i ~ oO tu o a PROTEIN © iG 4 a 4 + 124+240£ # 407 16 a CELLS ACHE Q us AChE g ° 1 S 20+ 8 ACHE 6+ 120M a AChE ra wn 4 —@SP ACT cELLs SP ACT. a a yg O\",__PROTEWN | o 2 2 , es 4 5°70 1 @¢3 4 5 °°3 456 7 c DAYS Fic. 2. The rate of cell division was regulated by adjusting the serum concentration. Neuroblastoma cells were incubated for 24 hr prior to sero time in 150-mm dishes containing the medium described, except that horse serum was omitted. At zero time the medium was removed and cells were washed once with growth medium; then fresh medium with or without 10% fetal calf serum was added as specified in panel A and B, respectively. Some cells were incubated for 3 days without serum, as shown in panel B; nondividing cells were then shifted up to the rapidly dividing state by the addition of 10% fetal calf serum (panel C). The me- dium was changed and fresh medium containing 10% fetal calf serum was added at 4 and 5.5 days. Symbols correspond to the following: @, AChE specific activity; O, AChE ac- tivity/dish; A, cells (X10-*)/dish; 0, mg protein/dish. Cells were incubated without serum for 3 days (Fig. 2B); serum was added on the 8rd day to shift cells from stationary to logarithmic growth without subculturing them (Fig. 2C). A decrease in AChE specific activity resulted. The results again show that regulation of AChE activity is coupled to cell division and is not a consequence of subculturing procedures. Cell division was also restricted by incubating cells with 2 mM thymidine in the presence of serum and growth medium. An increase in AChE specific activity was found (data not shown). The possibility that the changes in AChE activity were due to activation or inhibition of the enzyme was investigated by mixing homogenates prepared from cells in the logarithmic and stationary phase of growth. Acetylcholinesterase activity was additive; thus no enzyme effector was detected. The relation between protein synthesis and AChE regulation was studied by determining the effect of cycloheximide upon AChE activity, cell viability, and 790 BIOCHEMISTRY: BLUME ET AL. Proc. N. A. S.       Z T T T T ka 5 sofA ACh & q!20 2 a 5 fi0oo § = = ra 480 wi < ‘= 3 4 60 3 Fie. 3. Neuroblastoma cells were incubated — —. for 2 days without serum, as described in the +? 440 +° legend to Fig. 2, to restrict cell division and in- ars “z  erease AChE activity. Symbols denote the Ww j20 “ following: Open symbols, no cycloheximide; 2 S closed symbols, 3.5 x 10-§ M cycloheximide © oh} +-++4+ o * added at zero time (48 hr after removal of B CELL VIABILITY serum). In panel A, AChE specific activity and AChE: activity/dish are shown both in the presence and absence of cycloheximide. In panel B the percent of cells that were viable and 1 mg of protein/dish are shown, both in the presence and absence of cycloheximide. Each dish contained 4.0 « 108 cells, denoted on the ordinate as 100%. The number of cells/dish did not change significantly during the course of the experiment. %NIABLE CELLS     C PROTEIN   2 -CH 4 +CH   MG PROTEIN/DISH Oo 6 (2 18 24 HOURS protein (Fig. 34, B, and C respectively). Cells were incubated without serum for 48 hr before cycloheximide was added. In the absence of cycloheximide, AChE specific activity and units/dish increased 2.5-fold within 24 hr; in the presence of the drug, AChE specific activity and units decreased. The amount of protein and number of viable cells per dish also declined in the presence of cycloheximide. The results suggest that the increase in AChE activity is dependent upon protein synthesis but further data are needed to substantiate this possibility. Properties of neuroblastoma AChE: A number of enzymes catalyze acetyl- choline hydrolysis, including AChE, cholinesterase (EC 3.1.1.8), and acetyl- esterase (EC 3.1.1.6). The enzymes differ from one another in relative activity towards various substrates and in sensitivity to inhibitors. The effects of selec- tive esterase inhibitors on AChE from stationary phase cells are shown in Fig. 4. Acetylcholine hydrolysis was inhibited 50% in the presence of the following compounds: 1 X 10-8 M diisopropyl fluorophosphate, an inhibitor of AChE, cholinesterase, and carboxylesterase (EC 3.1.1.1), but not arylesterase (EC 3.1.1.2); 6 X 10-§ M 1,5-bis-(4-allyidimethylammoniumpheny]) pentane-1,3- dibromide (BW284C51), a more potent inhibitor of AChE than cholinesterase; 7 X 10-7 M neostigmine sulfate, a more potent inhibitor of AChE or cholines- terase than arylesterase or carboxylesterase; and 4 X 10~‘ M tetramonoisopropy] Vou. 67, 1970 NEUROBLASTOMA ACETYLCHOLINESTERASE 791 Fic. 4. Effects of esterase inhibitors on AChE 1 Ne from stationary phase neuroblastoma cells. The INHIBITION OF AChE dotted lines correspond to reactions with a final volume of 0.1 ml, containing the components de- scribed and 12.3 pg homogenate protein/reaction, that were incubated at 37°C for 15 min. Abbrevia- tions are as follows: DFP, diisopropylfluorophos- phate; BW, = 1,5-bis(4-allyldimethylammonium- pheny!)pentane-!,3-dibromide; NS, neostigmine sulfate; DPDA, tetramoncisopropyl pyrophosphor- tetramide; NEM, N-ethylmaleimide. The solid lines correspond to reactions incubated   180 150 120 30 60 430     % MAXIMAL VELOCITY     ae 6   in two stages. Stage 1 reactions contained the OW\" FS dae . : : owoKn eo ow oe components described in Materials and Methods MOLARITY NMOLES ACETATE FORMED/MIN/MG PROTEIN minus [4C]acetylcholine, the esterase inhibitors indicated at 5 times the molarity shown on the abscissa, and 12.3 ug of homogenate protein in a final volume of 20 ul. Stage 1 reactions were incubated for 10 min at 37°C, then placed in an ice bath. Stage 2 reactions were prepared by the addition of 4.13 mM [\"C]acetylcholine iodide and other reaction components de- scribed so that the final volume of each reaction was 0.1 ml. The final molarity of esterase inhibitors corresponded to that shown on the abscissa. Stage 2 reactions were incubated 15 min at 37°C, pyrophosphortetramide, a more potent inhibitor of cholinesterase than AChE. N-Ethylmaleimide had relatively little effect on the hydrolysis of acetylcholine even at 1 X 10-2 M. In addition, the relative rates of hydrolysis of [*C]- acetylcholine and [!C]butyrylcholine were determined; butyrylcholine hydroly- sis was 1% that of acetylcholine. Although definitive characterization awaits the analysis of purified enzyme fractions, the neuroblastoma enzyme appears to be AChE (EC 3.1.1.7). Discussion. The results show that neuroblastoma acetylcholinesterase ac- tivity is regulated and that the regulatory mechanism is inversely related to the rate of cell division. Acetylcholinesterase specific activity increased about 25- fold when the rate of cell division was restricted. Under the same conditions, the specific activity of catechol-O-methyl transferase remained relatively con- stant. Acetylcholinesterase activity in stationary phase neuroblastoma cells was approximately twice that of mammalian brain or erythrocytes, both known to contain relatively high levels of the enzyme. Studies with neuroblastoma cells revealed that the shift from the dividing to the nondividing state influences the expression of a set of characteristics required for neuron differentiation. Thus far, five properties have been found to increase upon restriction of neuroblastoma cell division: acetylcholinesterase activity, protein per cell, axon-dendrite development, formation of electrically active membranes, and synthesis of acetylcholine receptors. Neuroblastoma cells are still committed to a program of neuron differentiation even though thousands of cell generations have elapsed since the tumor orig- inated. Commitment to the program may be thought of as the initiating event in the process of differentiation. Once initiated, the progeny apparently do not terminate such a program. Although the mechanism of initiation is not known, the stability of the phe- nomenon suggests that it is not fully reversible, It should then be relatively 792 BIOCHEMISTRY: BLUME ET AL. Proc. N. A. S. easy to explore the possibility that normal sympathetic neuroblasts are also irreversibly committed to a pattern of neuronal differentiation. We acknowledge the invaluable contributions of Drs. A. Gilman and T. Amano and the help of Mrs. H. Carpenter in the preparation of this manuscript. Abbreviation: AChE, acetylcholinesterase. + NIH Postdoctoral fellow (HE 29,138-02). 1 Augusti-Tocco, G., and G. Sato, Proc. Nat. Acad. Sci. USA, 64, 311 (1969). 2 Schubert, D., 8S. Humphreys, C. Baroni, and M. Cohn, Proc. Nat. Acad, Set. USA, 64, 31 (1969). 3 Olmsted, J. B., K. Carlson, R. Klebe, F. Ruddle, and J. Rosenbaum, Proc. Nat. Acad. Sct. USA, 65, 129 (1970). ‘Nelson, P., W. Ruffner, and M. Nirenberg, Proc. Nat. Acad. Sci. USA, 64, 1004 (1969). 5 Seeds, N. W., A. G. Gilman, T. Amano and M. W. Nirenberg, Proc. Nat. Acad. Sct. USA, 66, 160 (1970). ‘ Harris, A. J.. and M. J. Dennis, Science, 167, 1253 (1970). 7 Nelson, P. G., J. H. Peacock, T. Amano, and J. Minna, in preparation. 8 Nelson, P., J. H. Peacock, and T. Amano, in preparation. ® Burkhalter, A., R. M. Featherstone, F. W. Schueler, and M. Jones, J. Pharmacol., 120, 285 (1958). Goodwin, B. C., and I. W. Sizer, Develop. Biol., 11, 136 (1965). 11 A preliminary report of these findings has been published. Blume, A. J., and F. Gilbert, Fed. Proc., 29, 861 (1970). 2 Ham, G. R., and T. T. Puck, in Methods in Enzymology, eds. 8. P. Colowick and N. O. Kaplan (New York: Academic Press, 1962), vol. 5, pp. 90-119. 13 Phillips, H. J., and J. E. Terryberry, Hap. Cell Res., 13, 341 (1957). “Lowry, O. H., N. F. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 6 Nikodejevic, B., S. Senoh, J. W. Daly, and C. R. Creveling, J. Pharmacol., in press. 6 Austin, L., and W. K. Berry, Biochem. J., 54, 695 (1953). ” Reed, D. J., K. Goto, and C. H. Wang, Anal. Biochem., 16, 59 (1966).", "Blume, Arthur J. ; Gilbert, F. ; Nirenberg, Marshall W. ; Wilson, S. ; Farber, John L. ; Rosenberg, Roger N.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy Press (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-pcsg.fgnn~mr7v", "00000000-0000-0000-9ECB-2BF47247CE66", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Genes for Neuronal Properties Expressed in Neuroblastoma x L Cell Hybrids", "101584910X209", null, "1971", "January 1971", "As part of Marshall Nirenberg's early work in neurobiology, this early article uses neuroblastoma systems to explore steps in the maturation process as well as aspects of neural function.  Somatic cell hybrids are shown to possess the \"neuronal property of electrically excitable membranes.\"  The authors hope to apply these findings to promote neuronal differentiation in normal neuroblasts.  The article provides evidence that \"at least part of the genetic information for neuron differentiation can be functionally expressed in\" hybrid cells.", "Articles", "Neuroblastoma,Hybridization, Genetic,Cell Differentiation,Membrane Potentials,Genes,L Cells (Cell Line)", "Neuroblastoma Research, 1967-1976", "6", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proceedings of the National Academy of Sciences Vol. 68, No. 1, pp. 234-239, January 1971 Genes for Neuronal Properties Expressed in Neuroblastoma x L Cell Hybrids JOHN MINNA, PHILLIP NELSON, JOHN PEACOCK, DEVERA GLAZER, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart and Lung Institute, and the Behavioral Biology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Communicated November 6, 1970 ABSTRACT Neuroblastoma cells with electrically ex- citable membranes were fused with electrically passive L cells having a hitherto undescribed electrical marker. Hybrid cells, examined 10-40 generations after fusion, were found to be electrically excitable. The results show that at least a part of the genetic information for neuron differentiation can be functionally expressed in N X L hybrid cells, Evidence for the regulation of action poten- tial components was also found.   Slonal lines of mouse neuroblastoma cells thus far have been shown to possess ten properties characteristic of differentiated neurons including electrically excitable membranes and ace- tylcholine receptors (1-9). Some of the properties also are responsive to regulatory influences (4, 5, 9). When the number of neuronal functions expressed is considered, it is likely that the cells follow a genetic program for neuron differentiation. The neuroblastoma system thus may be useful in exploring steps in the maturation process as well as aspects of neural function. The techniques of somatic cell hybridization have been used to probe the expression of the differentiated state with various cell types. Questions of dominance, complementation, and gene segregation can be studied (10-18,24,25). Also, the gencrality of control mechanisms can be tested by comparing intra- with interspecific hybrids. In this report, somatic cell hybrids of mouse neuroblastoma and L cells are shown to possess the neuronal property of electrically excitable membrancs. MATERIALS AND METHODS Cell lines Mouse (C3H/AN) L cell mutant clones B82 and A9 were the gift of Dr. J. Littlefield (10/20/69). B82 lacks thymidine kinase (I8C 2.7.1.21); A9 Jacks hypoxanthine phosphoribosyl- transferase (EC 2.4.2.8) (15). We have detected no revertants aud none have been reported in the literature. Mouse (A/J) neuroblastoma C-1300, clone N4, was derived by Dr. T. Amano from a single cell isolated from cells adapted to tissue culture. Mutant production Neuroblastoma N4, passage 36, was treated with 5 x 107? M ethyl methane sulfonate (Eastman Kodak Co.) for 2 br to kill 60% of the cells, grown for five generations, and then ex- posed to 10-§ M 6-thioguanine (6-SGua) (Sigma Chemical Co.). Survivor frequency was approximately 2 X 10~%. The 234 survivors were pooled, grown up, and then exposed to 5 x 10-§ M 6SGua. The frequency of resistant colonies was approximately 4 X 10-*. Cells were pooled and then cloned in 10-4 M 6-SGua. One clone, N4TG1, selected for the present study, was maintained in 10-4 M 6-SGua, but was also shown to be resistant to 6-SGua after growth for several weeks in its absence. At the time of fusion, N4TG1 was approximately 80-100 generations removed from neuroblastoma N4. The reversion frequency was determined at the time of each fusion experiment by incubating 2 x 10° cells/60-mm plate and then subjecting the cells to the standard fusion procedure (see Table 1). Media Parental cells were cultured in medium D: Dulbecco’s modification of Eagle’s medium; 10% fetal calf serum, sodium penicillin G (50 units/m!), and streptomycin sulfate (10 »g/ ml) in Falcon flasks or Petri dishes at 37°C in an atmosphere of 10% CO: 90% air, 100% humidity. Hybrid cells were grown in HAT medium: medium D, supplemented with 1 X 10-4 M hypoxanthine, 1 X 10-5 M aminopterin (a gift of Lederle Pharmaceutical Co.), and 1.6 x 10-' M thymidine. Glycine (4 X 10-4M) is present in medium D. Cell fusion An inoculum of Sendai virus, obtained from Dr. A. Rabson, was grown in embryonated chicken eggs, harvested, inactiva- ted with 6-propiolactone, and titered for hemagglutinating activity against guinea pig red blood cells (17, 18). Cell lines to be fused were mixed and immediately poured into a 60-mm Falcon Petri dish (2 X 108 total cells/5-ml medium D per dish). After 18-20 hr of incubation, cells were fused with 200- 500 hemagglutinating units (HAU) of inactivated Sendai virus per dish according to the method of Ephrussi and David- son (17). Plates were incubated for 15-20 days in HAT medium. Colonies containing approximately 1000 cells were counted, the medium was removed, and well-isolated large colonies were picked with the aid of disposable Pasteur pipets. Electrical studies The cells were maintained in medium D supplemented as follows: 1X 10-4M 6-SGua for N4TGI1 and A9; 1 K 10~* M BrdU (Sigma) for B82; HAT medium for hybrid cell lines. Cells from 50-100% confluent cultures were dissociated with 0.05% trypsin, centrifuged, resuspended, and then inoculated into 60-mm Faleon Petri dishes (1 X 10 cells/5 ml per dish) Vol. 68, 1971 Neuroblastoma Cell Hybrids 235     Tasty 1. Cell tines Glucose phosphate Base analog isomerase phenotypet resistance Reversion Cell lines* Fusion ratio a h b (10-4 M) frequency Neuroblastoma, N4TG1 + _ - 6-thioguanine ~1L-4 & 1077 L-cell, AQ - - + 6-thioguanine <5 x 107° L-cell, B82 =~ _ + 5-bromodeoxyuridine <5 X 107° Neuroblastoma x L-cell (N4TG1 X B82) NL-1 hybrid 1:20 + 4+- + NL-2 hybrid 1:200 + + + NL-3 hybrid 1:20 + + + NL-4 hybrid 1:20 + + + NL-5 hybrid 1:0.7 + + + NL-6 hybrid 1:0.7 + + + NL-7 hybrid* 1:200 L-cell X L-cell (A9 & B82) LL-1 hybrid* 1:20 - - + LL-2 hybrid* 1:20   * Cell lines are clones except NL-7 (13 clones pooled); LL-1 (15 clones pooled); LL-2 (14 clones pooled). The probability of a revertant occurring in NL-7 is ~0.005. Designations in our laboratory for the cell lines are: N4TG1 (N16604B); NL-1 (23406B), NL-2 (23407C ) NL-3 (23406C), NL-4 (23406D), NL-5 (22014A), NL-6 (22016A), NL-7 (23409). + Starch gel electrophoresis of glucose phosphate isomerase. Bands a, h, and 6 migrated 1.2, 1.8, and 2.3 cm from the origin toward the cathode. Band nomenclature (19) is as follows: GPI-1A = a band alone; GPI-1B = 6 band alone; GPI-1AB = a, h, b, bands. without 6-SGua or BrdU. After 24 hr, the medium was re- moved, plates were washed once with medium minus serum, and 5 ml of the following was added per plate: medium D minus serum for N4TG1, B82, A9; or HAT medium minus serum for hybrid cells. Multiplication of neuroblastoma cells is dependent upon serum (4). Cells were incubated without serum to restrict cell division and shift the cells to a more differentiated state; ie., an increase in specific activity of acetylcholinesterase and axon extension (4, 5). After 2-4 days incubation without serum, cells were used for electrical studies. In preliminary experiments neuroblastoma cells were found to be electrically active after incubation without serum. The methods for studying cells with intracellular micro- electrodes are described elsewhere (2,6). Use of an intercellular electrode in a bridge circuit allows measurement of cell mem- brane potentials and stimulation of the cell with intracellu- larly applied current. ‘Transmembrane voltages and stimulat- ing currents were digitized and stored with a Digital Equip- ment Corp. PDP-12 computer along with calculated values of the first derivative of membrane potential with respect to time. These parameters were then used to compute active and passive cell membrane properties. Glucose phosphate isomerase phenotypes Glucose phosphate isomerase (EC 5.3.1.9) phenotypes were determined by starch gel electrophoresis (19). Glucose-6- phosphate dehydrogenase (crystallized once) and fructose-6- phosphate (contaminated with less than 2% glucose-6-phos- phate) were obtained from Sigma Chemical Co. Homogenates were prepared as previously described (5). RESULTS Formation of hybrid cells In the presence of aminopterin, an inhibitor of nucleoside synthesis, cell growth depends upon the availability of pre- formed bases and the ability to synthesize enzymes required for base utilization. A 6-SGua resistant neuroblastoma mutant (N4TG1) unable to utilize hypoxanthine was obtained and Neuroblastoma X L cell hybrids containing genetic informa- tion from both parents within a common nucleus were se- lected by growth in HAT medium. The frequency of putative neuroblastoma Xx L cell (NL) hybrids per input neuro- blastoma parental cell depended on the initial ratio of the parental cells; i.e., approximately 3 X 10-5 for 1:1 (B82: N4TG1); 5 X 10-4 for 20:1; 1.5 X 10-3 for 200:1, Neuroblastoma and L cell lines are derived from A and C3H mouse strains, respectively, which express different glucose phosphate isomerase isozyme phenotypes at the Gm-! locus (19). As shown in Table 1, each NL hybrid clone expresses the Gpt-1 isozymes of both neuroblastoma and L cell parents and at least one additional isozyme as previously described in F, animals heterozygous for this locus (19). A mixture of non- hybrid N4TG1 and B82 cells, grown in the same vessel for 1 week, contained only the two parental bands (a and 5), not the intermediate band (h). These results show that the clones are indeed hybrids of neuroblastoma and L cells. No revertants of parental L cell lines have been found. Hence, growth in HAT medium of fused L cells (B82 x A9) indicates that these cells are also hybrid (14,16). Electrical properties The electrophysiological properties of single cells were studied by inserting the tip of a microelectrode within a cell and mea- suring the voltage difference between this intracellular elec- trode and a second electrode immersed in the extracellular fluid. Pulses of current were then passed through the elec- trodes and the change in voltage across the cell membrane was measured as a function of time. These responses can be used to characterize both the active (excitable) and the passive properties of the cells (6). Pulses of current which decrease the transmembrane volt- age (depolarizing stimuli) provide a test for electrically active 236 Biochemistry: Minna ed al. Proc. Nat. Acad. Sci. USA TaBLE 2. Response of cells to electrical stimulation       Responses AtB- Post fusion A-B- A-Bt+ A+Bt Total C- ct Total Cell lines Generations Days (P) (DR) (AR) cells (HA) cells Number of cells Parents Neuroblastoma, N4TG1 52 8 8 68 36 0 36 L-cell, B82 20 0 0 20 2 8 10 T-cell, AQ 16 0 0 16 0 4 4 NL hybrids NI-1 25-40 50-76 7 3 11 21 11 5 16 NI-2 25-35 50-76 7 13 3 23 16 5 21 N1L-3 25 51 5 0 3 8 5 2 7 NI-4 25 50 9 1 0 10 2 1 3 NL-5 30 65 1 1 3 5 2 1 3 NL-6 30 65 1 3 5 9 4 2 6 NL-7 20 42 18 3 3 24 4 4 8 NL plate-1 10 20 12 1 15 28 NL plate-2 10 26 1 4 6 1 4 1 5 NL plate-3 10 23 9 4 5 18 7 1 8 LL hybrids LL-1 25 30 11 0 0 11 2 9 ll LL-2 25 30 10 0 0 10 0 6 6 LL plate-1 10 15 10 0 0 10 0 Total Total % of total cells % of total cells Totals Neuroblastoma 76 12 12 68 100 0 36 L-cells 100 0 0 36 14 86 14 NL hybrids 10 39 16 45 57 8d 1d 13 NI hybr ds 20-40 48 24 28 100 69 31 64 LIL hybr'ds 10-25 100 0 0 31 12 88 17   Responses were scored as described in the results and Figs. 1 and 2. The abbreviations in parentheses are: P, passive; DR, delayed rectification; AR, active response; HA, hyperpolarizing activation. Hybrid cells assayed 10 generations after fusion were studied on the fusion plate. The number of colonies tested on each plate and the probability that a colony was a revertant were: NL plate-1, 3 colonies, 0.017; NL plate-2, 4 colonies, 0.012; NL plate-3, 2 colonies, 0.0008. The revertant probabilily was calculated by multiplying the reversion frequency of N4TG1 in HAT medium (obtained at. the time of fusion) by the number of N4TG1 cells per fusion reaction, divided by the number of putative hybrid colonies found. Similar responses were obtained with different NL colonies on the same plate. Data for each plate are pooled. Each colony contained cells with 4~8+ response; A+ response was found in 8 of the 9 colonies. Some L cells and LL cells were Lested with medium plus serum, but still were A~B~. cell membranes. Each cell was tested for active responses at the resting membrane voltage (about 20-40 mV) and with the membrane voltage adjusted to a standard level (about 80 mV). This standard voltage was optimal for eliciting maximal responses and necessary for comparing cell types (2,6). ‘Three types of response to electrical depolarizing stimuli are shown in Fig. 1, A-D. The response from a cell that was uot excitable, termed passive response (A~B7-), is shown in Fig. 14, A pulse of current elicited a smoothly rising unin- flected change in membrane voltage. The B response (A ~B*), termed delayed rectification, is scored when a negative in- flection is present late in the response (Arrow B in Fig. 1B). Response A(A+B7- or A+B*) is scored when a positive inflec- tion (an increase in rate of voltage change) occurs early in the response (Arrow A in Fig. 1C and D). The two A responses (Fig. 1C versus 1D) differ quantitatively; the A response of Fig. 1D corresponds to an action potential, while that of Fig. 1C corresponds to a local response. The amount of A activity per cell varied over a wide range. Thus, quantitative as well as qualitative differences were observed. A type of response not previously described was found with L cells but not with neuroblastoma cells (Fig. 2A and B). When a large (5-50 NA) 100-msec pulse of current, opposite in direction to that used in Fig. 1 A~D, was passed across the membrane a large (200-400 mV) increase in membrane po- tential resulted. After the current was turned off, the mem- brane potential returned to its initia] value, and then under- went a second, prolonged (10-20 sec) increase, with subsequent return to the resting level (Fig. 2A). The voltage change was accompanied by a large decrease in cell resistance. We have termed this response the C response (hyperpolarization acti- vation). The threshold for cliciting the C response was rela- tively sharp, and some increase in the threshold was secn after a C response; that is, cells were refractory. The responses of neuroblastoma, L cell, and hybrid cell lines to electrical stimulation are shown in Table 2. The maximum responses of individual cells are shown. Cells from the parent neuroblastoma clone were found that either exhibited no response (A ~B7), the B response (A~B*), or the A response (A+B~ or A+B+). The parent, therefore, Vol. 68, 1971 resembles the wild type neuroblastoma in that it expresses electrically active membranes, However, the incidence of passive cells (A~B-) was much greater than previously noted (6). None of the parental neuroblastoma cells gave a C response. No L cell tested showed A or B responses; all were passive. However, nearly all the L cells exhibit the C response. Thus, A and B responses are neuroblastoma markers, whereas the C response is an L cell marker. Six clonal lines of neuroblastoma X L cell hybrids and one uncloned NL hybrid line were tested 20-40 generations after fusion, Cells with excitable membranes were found with every NL hybrid line. With only one exception (NL-4), the inci- dence of expression of A or B responses found with NL hy- brid lines equaled or exceeded that found with the neuro- blastoma parent. With most NL hybrid lines passive cells, B, and A responding cells were found. The C response was also found in all N ¥ L hybrid clones. Nine colonies of putative NL hybrids were studied 10 generations after fusion directly on the fusion plate. The inci- A CLASS I Posswe ol a BO i fo . \\\\ “5 pe f N , 1 oN ore | i | “100- Bg CLASS T Delayed > ATB* Rectification Q & pen / . / \\\\ “100 a ted   gs C; CLASS DW Actue pA Response | so - MILLIVOLIS MUMBRANL PCILATIAL ee Di — ave | iResponse oO i AMA of 50° “160 80 200 mSFC Fic. 1. Responses to electrical stimulation. A, B, C, and D are examples of the different categories of response to stimu- lating current, discussed in the text. The cell membrane potential is shown in each case as a function of time. A voltage calibration pulse and the response to a current calibration pulse are shown at the left of each trace, and the response to a long pulse of current occurs in the middle of each trace. Onset and cessation of current are indicated by ON and OFF in Fig. 14, and the time course of the current is shown in the lower part of Fig. 1D. A) Passive response with no inflections (4~B-). B) Delayed rectification; a negative deflection occurs at the arrow, and in no other place, defining the B property (A~B+). C) and D) Active responses; a positive inflection occurs on the rising phase of the response, this defines the A property (A+). Inflections may occur elsewhere. The response in Fig. 1C corresponds to a local or partial response ; Fig. 1D represents an action potential. 2, F, G,and H show cells under the conditions of electrical study, (medium minus serum for 3 days) with a microelectrode in place. Cell lines represented are: B82 A and £; NL-2 hybrid, B and F; N4TG1, C and G; NL-1\\\\ Hybrid, D and H.   S3UIdVONYN IN3NYND 03) dv Neuroblastoma Cell Hybrids 237                       A; CLASS IW: 8 Hperpol ac a ctivation tH yperpciarizalen C Response No C Response 20r Powe na ane eo RK 2 L Cell 3 7 , ra we C J Zz Seed | z= oa 108 = tn ER -50 00 & x2 Hie FO OB 4 mSe> ao S ay + - 2 ~100 5 ) aN Consigrt | . aed ON feos sok Stim. Slim, 15 “A gee - 9 boi jh 1 i 4 | | S25 0 3 © M0 W 60 90 Oe By SECONOS mel (Left) Fic. 2. Hyperpolarization activation. Tracings of penwriter records show A) the presence of hyperpolarization activation or C response in an L cell, and B) the absence of a C response in a neuroblastoma cell. (Right) Fic. 3. (A) Passive change in membrane voltage (upper irace) in response to hyperpolarizing current (lower trace). (B) Semilogarithmic plot obtained by evaluating the expression —In (Vj — V/V»), where V, is the maximum voltage change developed in A, and V is the voltage occurring at the membrane. Time constant is equal to the time required for (Vo — V)/Va to decrease by 1/e. dence of the B and A responses were again comparable to the neuroblastoma parent. Thirty-one LL hybrid cells were tested 10-25 generations after fusion; the C response was found, but not A or B re- sponses. The overall incidence of expression of A and B responses was approximately twice as great in NL hybrids as in the neuroblastoma parent. When a pulse of current is passed across the cell membrane so as to increase the potential difference across it, the voltage changes can be used to determine passive membrane proper- ties such as resistance, time constant, and capacitance. The cell resistance is equal to the change in voltage divided by the amount of current (Fig. 3A); the membrane capacity is equal to the time constant (Fig. 3B) divided by the membrane resistance. A measure of the intensity of the A response is provided by the amount of current generated by a cell during the response, termed the action potential current (this is approximately equal to the product of the membrane capacity and the maximum rate of change of membrane voltage during the response). The average action potential current of the NL hybrid cells scored with A responses is compared to that found with neuroblastoma cells (Table 3). Neuroblastoma cells with A responses had an average action potential eurrent of 13 pA/ cm?; the most active cell was 25 pA/cm?. In 3 of the 6 NL hybrid clones 20-40 generations after fusion, the maximum activity was higher (peak value, 85 »A/cm*) than that found with the neuroblastoma parent, and the average action potential current was 22 »A/cm?. Thus, the A response of hybrids equaled or exceeded that of parent neuroblastoma cells. Neuroblastoma cells displayed low specific resistivity and high specific capacitance compared to L cells and LL hybrids. High capacitance suggests either that the surface area of cells is greater than that estimated or that cells are coupled electrically (7). Some correlation between electrical properties and the morphology of the cells in lines NL-1 and NL-2 was noted. 238 Biochemistry: Minna et al. Proc. Nat. Acad. Sct. USA TasLe 3. Membrane properties       Action Maximum potential § Specific Specific Membrane resting Surface current resistivity capacitance time constant C response potential area Total Cell line (wA/em?) (ohm cm?) (uF /em?*) (msec. ) (mV) (mV) (um?) cells Neuroblastoma, N4TG1 13 1600 7.3 7.3 0 24 15,400 14 L-cell, B82, A9 (average) 0 5100 2.9 8.8 30 47 12,480 15 NL-1 hybrid 19 3500 3.7 11.5 4 27 7,900 20 NL-2 hybrid 3.8 5500 3.5 15.5 3 a4 13,900 23 LL-1,2 hybrids (average) 0 5200 4.1 11,2 20 42 12,770 21   Membrane properties of neuroblastoma and L-cell parents, 2 lines of NL hybrids, and 2 L-I hybrids. Action potential current refers to the average value for all cells in each line which showed the A * response. Surface area was determined by measuring cell body and process dimensions on photographs such as those shown in Fig. lZ-H. The average action potential current for all A * cells in NL hybrids 1-7 was 19 wA/em?. Hybrid NL-1, which had extensive processes (Fig. 1H), also had low membrane resistivity and action potential current as high or higher than the neuroblastoma parent. Hybrid NL-2 consisted of large flat cells with no processes (Fig. 1 E/B & fF), This line has high membrane resistivity as do the L cell lines. While the level of A* activity is low, the NL-2 line still had evidence of neuroblastoma function, as shown by the presence of the B+ response (Table 2). The C response was found with every NL hybrid clone, but less frequently than in L cells or LL hybrids. The average amplitude of the C response of NL hybrids was 10-20% that of L cells. DISCUSSION Neuroblastoma cells with electrically excitable membranes were fused with electrically passive L cells, and the resulting hybrid clones were selected by the procedure of Littlefield (15). Hybrid cells were examined 10-40 generations after fusion and found to be electrically excitable. The results show at least part of a genetic program for neuron differentiation can be functionally expressed in N  L hybrid cells. No evi- dence for a repressor terminating the neuron differentiation program was observed. Since each assay for electrical activity is performed with a single cell, variation within a clone as well as variation be- tween clones was studied. One disadvantage of this procedure is that cells are not randomly assayed since large cells can be studied more easily than small ones. Two properties of electrically excitable membranes were assayed: a positive inflection on the rising phase and a nega- tive inflection on the falling portion of the membrane-voltage curve, termed A and B, respectively. These correspond to the rising phase of the action potential and the descending phase of the action potential or delayed rectification. Three general categories of cells were found; cells without electrical activity (A~B7), cells exhibiting only B activity (A~B*), and cells with A responses (no attempt was made to distinguish be- tween A*B~ and A*B* cells). The three types of response were found with cells from almost every NL hybrid line, cloned and uncloned. A quanti- tative difference in the intensity of the A response also was found with cells from each of the 7 neuroblastoma clones that have been studied thus far, and with every N X Lhybrid line studied. The simplest explanation of both quantitative and qualitative variation is that A and B activities are reg- ulated and that 33 can be active independently of the A re- sponse. Some chromosomes probably are lost by NL hybrids during the course of growth (12,13,16,23); however, no definitive evidence for gene segregation was found. Chromosomes are lost preferentially from the genome of the parent with the longer generation time (13). Neuroblastoma and L cell genera- tion times are similar; thus, part of the genome of either parent may be lost from NL hybrids. Cell lines defective in neuronal properties would be useful both in elucidating steps that per- tain to neuron maturation and in defining neural functions. The finding that NL hybrids often are more active electrically than the parental neuroblastoma line could be due to activa- tion of L cell genes for action potential components, comple- mentation, or other forms of regulation. Transmission of an action potential by excitable cell mem- branes probably involves a series of reactions initiated sequen- tially by reactions of neighboring molecules. The available information on the nature of the action potential indicates that the A response results, at least in part, from Nat entry into the cell, and the B response from the exit of K+ from the cell (20). The cations are transported with specificity, probably without a requirement for ATP; hence, transport by facilita- ted diffusion seems likely. Although the components required for the action potential have not been identified, it is possible that the A response is dependent upon a Nat entry permease which is converted reversibly from an inactive to an active form when cell membrane potential is decreased, and is in- hibited by tetradotoxin, whereas the B response may require a K+ exit permease inhibited by tetraethylammonium ions. It seems likely that other steps also are required. An alternate model for the production of the action potential has been proposed (22). The relatively low frequency and amplitude of the C re- sponse found with NL hybrid cells may be due to repression of the L cell marker. Relatively few cells were found with C+tAt, C+B*, or C+A*B* responses. The mechanism of the C response possibly involves an increase in membrane con- ductance to potassium. The techniques of somatic cell hybridization applied to normal neuroblasts may well provide a relatively simple means of establishing clonal lines of cells derived from differ- ent types of neurons. The results obtained with neuroblastoma cells show that rapidly dividing cells still retain the ability to express neuronal function and that some neuronal genes Vol. 68, 1971 remain active in somatic cell hybrids. The possibility that neurons may be capable of initiating a program for neuron differentiation in recipient cells also deserves consideration. We are grateful for discussions with Drs. A. G. Gilman, 8. Wilson, H. Epstein, H. Coon, and T. Amano. 1. Augusti-Tocco, G., and G. Sato, Proc. Nat. Acad. Sei. USA, 64, 311 (1969). 2. Nelson, P., B. W. Ruffner, and M. Nirenberg, Proc. Nat. Acad. Sci. USA, 64, 1004 (1969). 3. Schubert, D., S. Humphreys, C. Baroni, and M. Cohn, Proc. Nat. Acad, Sci. USA, 64, 316 (1969). 4. Seeds, N. W., A. G. Gilman, T. Amano, and M. Niren- berg, Proc. Nat. Acad. Sci. USA, 66, 160 (1970). 5. Blume, A., F. Gilbert, 8. Wilson, J. Farber, R. Rosenberg, and M. Nirenberg, Proc. Nat. Acad. Sci. USA, 67, 786 (1970). 6. Nelson, P., J. Peacock, T. Amano, and J. Minna, J. Cell. Physiol. (in press). 7. Harris, A. J., and M. J. Dennis, Science, 167, 1253 (1970). 8. Olmsted, J. B., K. Carlson, 8. Klebe, F. Ruddle, and J. Rosenbaum, Proc. Nat. Acad. Sct. USA, 65, 129 (1970). 9. Schubert, D., and F. Jacob, Proc. Nat. Acad. Set. USA, 67, 247 (1970). 10. Finch, B. W., and B. Ephrussi, Proc. Nat. Acad. Sct. USA, 57, 615 (1967). Neuroblastoma Cell Hybrids 239 11. Kao, F. T., L. Chasin, and T. T. Puck, Proc. Nat. Acad. Sci. USA, 64, 1284 (1969). 12. Weiss, M. C., and H. Green, Proc. Nat. Acad. Sci. USA, 58, 1104 (1967). 13. Littlefield, J. W., Science, 145, 709 (1964). 14. Davidson, R. L., B. Ephrussi, and K. Yamamoto, Proc. Nat. Acad. Sci. USA, 56, 1437 (1966). 15. Littlefield, J. W., Exp. Cell Res., 41, 190 (1966). 16. Kao, F. T., and T. T. Puck, Nature, 228, 329 (1970). 17. Davidson, R. L., Exp. Cell Res., 55, 424 (1969). 18. Harris, H., J. F. Watkins, C. E. Ford, and G. I. Schoefl, J. Cell. Sci., 1, 1 (1966). 19. DeLorenzo, R. J., and F. H. Ruddle, Biochem. Genet., 3, 151 (1969). 20. Hodgkin, A. L., and A. F. Huxley, J. Physiol. (London), 117, 500 (1952). 21. Cole, K. S., Membranes, Ions, and Impulses, U. of Cal. Press, Berkeley, Calif. (1968). 22. Tasaki, I., Nerve Excitation, C. Thomas, Springfield, Tllinois (1968). 23. Engle, E., B. J. McGee, and H. Harris, Nature, 223, 152 (1969). 24. DidZerega, G., and J. Morrow, Exp. Neurology 28, 206 (1970). 25. Jacobson, C., Exp. Cell Res., 53, 316 (1968).", "Peacock, John ; Nirenberg, Marshall W. ; Minna, John, 1941- ; Nelson, Phillip G. ; Glazer, Devera", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy Press (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4kuf-275d-42ej", "00000000-0000-0000-38D4-298ECEFD030A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Dual Regulation of Adenylate Cyclase by Endogenous Opiate Peptides", "101584910X212", null, "1976", "1976", "This monograph expands on the relationship between morphine and other narcotics and the enzyme adenylate cyclase, arguing that endogenous opiate peptides act as dual regulators of adenylate cyclase exactly as morphine and other conventional narcotics.  As potent, receptor mediated, inhibitors of adenylate cyclase activity in homogenates of hybrid cells, these peptides also have \"addiction-like\" qualities.", "Monographs, Excerpts, Proceedings", "Adenylyl Cyclases,Receptors, Opioid ; Opioid Peptides", "Neuroblastoma Research, 1967-1976", "4", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "opiates and Endogenous Opioid Peptides > 1976, Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands 153 DUAL REGULATION OF ADENYLATE CYCLASE BY ENDOGENOUS OPIATE PEPTIDES Werner A. Klee, Arthur Lampert and Marshall Nirenberg Laboratory of General and Comparative Biochemistry, NIMH and Laboratory of Biochemical Genetics, NHLI National Institutes of Health Bethesda, Maryland 20014 Morphine and related narcotics affect adenylate cyclase in two ways, both mediated by the opiate receptor. The first process is the in- hibition of adenylate cyclase activity (1-3). The second phenomenon is a delayed increase in the specific activity of the enzyme, which compensates for the inhibitory action of the narcotics, but requires hours of exposure to the drugs before being expressed (4,5). This dual regulation of adenylate cyclase has been proposed to account for oniate tolerance and dependence, as well as for their acute effects (4). The discovery and characterization of endogenous opiate peptides (6) prompted us to ask whether these substances also inhibit adenylate cyclase and evoke a delayed increase in enzyme activity. Our studies have shown that methionine enkephalin (Tyr-Gly-Gly-Phe-Met) , and other endogenous opiate peptides, are potent. receptor mediated, inhibitors or adenylate cyclase activity in homogenates of neuroblastoma x glioma NG108-15 hybrid cells. Furthermore, upon prolonged incubation of NG108-15 cells with Met-enkephalin, the specific activity of adenylate cyclase is increased. Thus, Met-enkephalin causes both the immediate inhibition and the delayed increase in adenylate cyclase activity characteristic of dual regulation. The high potency of Met-enkephalin as an inhibitor of basal and PGE] stimulated adenylate cyclase in homogenates of NG108-15 cells is shown in Fig. 1. The concentrations of Met-enkephalin required to inhibit both basal and PGE, stimulated adenylate cyclase half- 154       z= 5 220 : : = 30 3s of a iz eo < & = 2 3 z a 4 20 = wi = a a =z uw = ao 8 io Ly-— ; . 0 9 8 7 6 —LOG MET—ENKEPHALIN MOLARITY Fig. 1. Inhibition of basal —\"O° —:; and PGE, (10 uM) stimulated —e —, adenylate cyclase activity of homogenates (97 pe protein/ tube of neuroblastoma x glioma NG108-15 cells by Met-enkephalin. Assays were performed as described by Sharma et al (2) except that incubations were for three minutes. Materials used in this work are as described elsewhere (8). maximally, along with similar data for Leu-enkephalin, morphine, and etorphine (2), are shown in Table 1. Clearly, the enkephalins are of very high potency in this assay, as they also are in the mouse vas deferens (6). Indeed, in the adenylate cyclase assay, Met-enkephalin is comparable to etorphine in activity. Inhibition of adenylate cyclase by Met-enkephalin is receptor mediated since it is reversed by naloxone, as shown in Fig. 2. As expected for a competitive interaction at the opiate receptor, the concentration of naloxone required to reverse the inhibitory action of Met-enkephalin increases with increasing peptide concentration. The dissociation constant for naloxone, Ke, calculated from these data by the dose-ratio method (7), is 30 nM, in ,good agreement with the value of 20 nM calculated from similar’ experiments with morphine (8), instead of enkephalin, or obtained by direct measurement of fialoxone binding affinity for the opiate receptor (2).   155 TABLE 1 ACTIVITY OF PEPTID. E AND OTHER N. OF AD ARCOTICS A ENYLATE CYCLASE IN NG108-15 HOMOGENATES (2).       Compound x, °” (nM) Basal ( PGE, 6°) Tyr-Gly-Gly~Phe-Met 1 (met-enkephalin) 12 20 Tyr-Gly-Gly-Phe-Leu (leu-enkephalin) 40, 100 Morphine 150 0 Etorphine 1500 10   (a) Assays (ey ys performed as described previously (2). Concentration of i {c) 10 uM in assays. nhibitor required for 50% of maximal effect CAMP (pmoles/mg/min)     L I l 10° 10-4 1023 | to or\" 107 10-* NALOXONE (Molarity} Fig. 2. The relati betwe of aden on en naloxone con ylate cyclase inhibition in homogenates (90 ug protes Weakest ein/tube) of NG108-1 mney oes AS ak SeheMGBDMann tb mt {SUUPTUME concentrations The inhibitory action of Met-enkephalin on NG1O8-15 adenylate “yetase is short lived, typically only 15 minutes or so (Fig. 3) Note that morphine continues to inhibit the activity of the enzyne for at least 90 minutes. The short duration of action of Met- Lao         Jf L Zz f T | a | T 9 2.0 a = N a _ a 06 = Oo 0.4 7 ” wi 4 a 02 = 2 | l l 1} I 0 io 20 30 60 90 MINUTES Fi 3 Adenylate cyclase activity of a homogenate (111 e protein/ tube) of NG108-15 cells, as a function of time. Additions to the standard assay mixture were as follows: none —o—; 0.16 nM —A—; morphine, 20 uM —O—. enkephalin is due to its destruction during the incubation rather hl than to desensitization since the enzyme is inhibited by a freshly added portion of methionine enkephalin after 25 minutes of pre- incubation with the peptide (Table 2). The relative lability of ee- Met-enkephalin activity compared with that of morphine is in agr t ment with results of analgesic assays of these materials in intac animals (9-11), and with tests performed with the guinea pig ileum preparation (6). - Incubation of cultures of NG1O8-15 cells for 12 or more hours Ww fic methionine enkephalin results in a marked increase in the speci f{ the activity of adenylate cyclase as shown in Table 3. In view o d lability of Met-enkephalin, the experiments shown were performe h with a 100 fold excess of peptide over that required to saturate the dz . Control experi- receptors, and the medium was changed twice. aily n ments showed that approximately 90% of the enkephalin activity he such cultures is destroyed in 12 hours of incubation, and so t   157 TABLE 2 ADENYLATE CYCLASE ACTIVITY OF HOMOGENATES OF NG108-15 CELLS PREINCUBATED WITH AND WITHOUT MET-~ENKEPHALIN   Adenylate cyclase activity - pmoles cAMP/min/mg protein   Preincubation Additions to assay Control Enkephalin Water 16.4 16.5 6 Naloxone (10 1M) 16.4 17.2 Met-enkephalin 11.5 11.5 (0.16 uM)   Homogenates of NG1O8-15 cells (111 ug protein/tube) were pre- incubated at 37° in the standard adenylate cyclase assay mixture, omitting only radioactive ATP, for 25 min, with and without 0.16 uM Met-enkephalin. Control experiments showed that adenylate cyclase was inhibitgg by enkephalin for less than 20 minutes. After 25 min, P 1 uCi of [a ] ATP was added to each tube along with other additions as shown in column 1 of the table. for an additional 5 min and [2p] ca was determined in triplicate. Incubations at 37° were continued MP formed during this interval amount of enkephalin present is in excess throughout the experiment (12). Note that the extent of the increase in adenylate cyclase activity elicited by enkephalin is similar to that found with etor- phine; the time dependence is also similar. The experiments described here demonstrate that an endogenous opiate peptide acts as a dual regulator of adenylate cyclase exactly as morphine and other conventional narcotics. Thus, these neuro- hormones, or transmitters, can be expected to inhibit adenylate cyclase activity of neurons with opiate receptors and thereby suppress the effects of other neurohormones or transmitters which activate adenylate cyclase. As a result of the delayed increase in adenylate cyclase activity caused by the endogenous opiates, some neurons may be rendered supersensitive to the effects of agents which stimulate the enzyme. Shifts in the levels of endogenous oplate peptides may increase or decrease the flow of information TABLE 3 ADENYLATE CYCLASE ACTIVITY IN HOMOGENATES OF NG108-15 CELLS CULTURED FOR THE TIMES SHOWN WITH MET-ENKEPHALIN OR ETORPHINE   Adenylate cyclase activity Ho.vs of treatment : pmoles cAMP/min/mg protein   Control Met-enkephalin Etorphine 0 7 - - 12 9 17 19 25 11 19 22 48 15 23 31 97 17 38 43   Logarithmically growing cultures of NG108-15 cells were maintained with additions to the culture media as follows: Met~enkephalin, 10 uM, and etorphine, 1 uM. Media were changed twice daily, and cells were harvested, homogenized, and assayed for basal adenylate cyclase activity in the presence of 100 1M naloxone. PGE stimulated adenylate cyclase activities also were increased, although to a smaller extent. (12). through specific neural circuits, and also regulate the responses of adenylate cyclase to other kinds of receptor mediated reactions. Cellular tolerance and dependence induced by the endogenous opiate peptides may play a normal role in switching certain neural pathways on or off. REFERENCES 1. Collier, H. 0. J. and Roy, A. C. (1974) Nature 248, 24-27. 2. Sharma, S. K., Nirenberg, M. and Klee, W. A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 590-594. 3. Traber, J., Fischer, K., Latzin, S. and Hamprecht, B. (1975) Nature 253, 120-122. 4. Sharma, S. K., Klee, W. A. and Nirenberg, M. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3092-3096.   10. ll. 12, Loy Traber, J., Gullis, R. and Hamprecht, B. (1975) Life Sci. 16, 1863-1868. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. and Morris, H. R. (1975) Nature 258, 577-579. Kosterlitz, H. W. and Watt, A. J. (1968) Brit. J. Pharmacol. 33, 266-276. Klee, W. A. and Nirenberg, M., in preparation. Belluzzi, J. D., Grant, N., Garsky, V., Sagantakis, D., Wise, C. D. and Stein, L. (1976) Nature 260, 625-626. Buscher, H. H., Hill, R. C., Romer, D., Cardinaux, F., Closse, A., Hauser, D. and Pless, J. (1976) Nature 261, 423-425. Pert, A., Simantov, R. and Snyder, 8. H. (1976) Proc. Natl. Acad. Sci. U.S., in press. Lampert, A., Klee, W. A. and Nirenberg, M., in preparation.", "Nirenberg, Marshall W. ; Lampert, Arthur ; Klee, Werner A.", null, null, "Elsevier/North-Holland Biomedical Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fayj.tf2c~xh8c", "00000000-0000-0000-C9B2-646BCCDE5F93", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Coding of Neural Information by Neuroblastoma Cells", "101584910X213", null, "1975", "1975", "This monograph reports on the results of Marshall Nirenberg's work with neuroblastoma clonal cells in the period before 1976, including discussions of gene expression, hybridization, transmitter synthesis, and morphine receptors.", "Monographs, Excerpts", "Action Potentials,Neuroblastoma,Hybrid Cells,Neurotransmitter Agents", "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Reproduced with permission of Lippincott, Williams, & Wilkins.", "Copyright may apply", null, null, "Regulation of Growth and Ditferentiated Function in Eukaryote Cells, edited by G. P. Talwar. Raven Press, New York, © 1975. Coding of Neural Information by Neuroblastoma Celis Marshall W. Nirenberg Laboratory of Biochemical Genetics, National Heart arf Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Numerous neuroblastoma clones originating from mouse tumor C1300 were characterized with respect to neuronal properties such as neuro- transmitter metabolism, cell receptors and response to transmitters, and action potential reactions. Three types of clones were found with respect to enzymes for transmitter synthesis: clones with choline acetyltransferase activity, which synthesize acetylcholine; clones with tyrosine hydroxylase activity, which synthesize catechols; and clones without these enzyme activities. Cells from each type of clone were recloned approximately 200 cell generations after the initial clones were isolated, and enzyme activities of the sublines were determined. Most subclones resembled the parental type with respect to transmitter synthesis. No stem cells or conversion of cholinergic to adrenergic cells were detected. These results show that genes determining neurotransmitter species can be expressed in dividing cells, that the parental programs of gene expression are inherited, and that dividing cells can be programmed with respect to their ability to communi- cate with other cells. Characterization of receptors and cell responses to putative transmitters also revealed different phenotypes which were in- herited in a clonal fashion. Two types of action potentials are elicited by electrical stimulation: Na’ spikes that are inhibited by tetrodotoxin, and Ca*+ spikes that are in- sensitive to this toxin. Three types of action potential “ionophores” can be distinguished: ‘ionophores” for Na*, Ca*+, or K*. Defective cell lines were obtained which lack one or more action potential ionophores. Many neural properties are expressed conditionally. Populations of cells can be shifted from a poorly differentiated, neuroblast-like state to a well- differentiated, neuron-like state by selecting for nondividing cells, decreasing the rate of cell division, or increasing intracellular levels of cyclic AMP. The specific activities of tyrosine hydroxylase, choline acetyltransferase, and acetylcholinesterase can be increased 100-, 15-, and 50-fold, respec- tively. Populations of electrically passive cells also can be shifted in syn- chrony to the electrically excitable state. Additional cell lines with new neural phenotypes were generated, and questions of dominance of gene expression and complementation were explored by fusing cells and obtaining hybrid cell lines. The expression of genes for neural properties was found to be dominant with most matings. Approximately 25% of neuroblastoma x L cell hybrid clones expressed the neural phenotype of the neuroblastoma parent 25 to 50 cell generations after fusion. Most of the remaining hybrid clones had specific neural defects. Cell lines with defects in transmitter synthesis, storage, and catabolism; response to neurotransmitters; action potential reactions; and so forth, were generated in abundance. ’ With other crosses new neural properties were acquired by hybrid prog- eny. For example, fusion of mouse neuroblastoma cells with rat glioma cells, both lacking choline acetyltransferase, yielded hybrid cell lines with high caoline acetyltransferase activity. Some but not all of the cholinergic hybrid progeny also store acetylcholine and have clear vesicles identical in appearance to those found at synaptic junctions. Fusion of mouse neuro- blastoma cells which do not synthesize catecholamines with normal sympa- thetic ganglion cells from mouse embryos yielded a hybrid cell line that synthesizes dopamine and has muscarinic excitatory acetylcholine receptors and both small and large dense-core vesicles. These and other results show that fusion of neuroblastoma cells with cells from the normal nervous system generates hybrid cells with new neural properties not detected with neuroblastoma cells. The new neural phenotypes are inherited and thus are perpetuated in a fairly stable fashion for more than 100 cell generations. A selection procedure was devised for neurons and related cells that depends on the ability of the cells to synthesize certain species of trans- mitters. The rationale for selection derives from the fact that tyrosine has long been known to be an essential amino acid for mammals, and that three mammalian enzymes are known to catalyze in vitro the synthesis of tyrosine from phenylalanine, i.e., phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. The two latter enzymes are found predomi- nantly in neurons in related cells that synthesize either dopamine, norepi- nephrine, epinephrine, serotonin, or melatonin. Only one of 70,000 uncloned mouse neuroblastoma cells grew well in the absence of tyrosine. No colonies were found with cell lines which lack tyrosine hydroxylase, such as cholin- ergic neuroblastoma cells, glioma cells, or L-cells. Approximately 50% of the neuroblastoma cell lines that grew well in the absence of tyrosine had tyrosiie hydroxylase activity. Some adrenergic cell lines obtained by selec- tion also possessed storage mechanisms for catecholamines. This method of selection coupled with somatic cell hybridization affords a simple, versatile means of generating and selecting cells of-ntural origin on the basis of cell specificity in synthesizing neurotransmiitters. In collaboration with Dr. Werner Klee, neuroblastoma and various hybrid cell lines were assayed for morphine receptors. One cell line, a neuroblastoma x glioma hybrid, was found which synthesizes stereo- specific, high-affinity morphine receptors. The average cell has approximately 3 X 10° narcotic receptors. In contrast, the neuroblastoma parent cell line has few morphine receptors, and the receptors were not detected with the glioma parent. Additional studies in collaboration with Drs. Shail Sharma and W. Klee show that morphine inhibits adenylate cyclase activity of cells with morphine receptors but has no effect on adenylate cyclase of cells without these receptors. Thus two forms of adenylate cyclase can be dis- tinguished; one form is sensitive and the other is insensitive to narcotics. Questions pertaining to the mechanism of narcotic addiction and dependence are currently being explored with this system.", "Nirenberg, Marshall W.", null, null, "Raven Press", null, null, null, null, "Talwar, G. P.", null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-czm3_5ia8~in2x", "00000000-0000-0000-9422-795B02618E91", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Opiate Receptors as Regulators of Adenylate Cyclase", "101584910X214", null, "1975", "24 May 1975", "This monograph reports on the progress of Nirenberg's neuroblastoma research.  The authors find that morphine inhibits enzymatic activity and enzyme production levels.  Additionally, hybrid cells show that the number of opiate receptors mediates inhibition of morphine.  Results suggest that because drugs may act as enzyme inducers, recovery from the addicted state requires return of adenylate cyclase activity to normal levels.", "Monographs, Excerpts", "Adenylyl Cyclases ; Receptors, Opioid, mu", "Neuroblastoma Research, 1967-1976", "6", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "OPIATE RECEPTORS AS REGULATORS OF ADINYLATE CYCLASE Werner A. Klee, Shail K. Sharma* and Marshall Nirenberg Laboratory of General and Comparative Biochemistry, NIMH, and the Laboratory of Biochemical Genetics, NHLI, NIH, Bethesda, Maryland 20014 (Received in final form May 24, 1975) We have reported the presence of opiate receptors in some neuroblastoma derived cell lines cultured in vitro and that a neuroblastoma x glioma hybrid cell line, NG108=-15, contains a particularly large number of morphine receptors (1), Table 1 shows that whereas the hybrid cell line has opiate receptors   Table I RECEPTOR BINDING li-dihydromorphine H naloxone HYBRIDS totes 7g protein NG1098-15 17 37 (19) PARENTS “NISTG=2 0 11 (6) C6BU-1 1 1 (1) The concentration of radioactive narcotic was 1 nM in each case, In neither case is this close to a saturating amount, naloxone one has twice the affinity of dihydromorphine and so to be comparable the -naloxone data should be divided by 2 (numbers in parenthesis), NG108-15 (also called 108CC15) was obtained by 8B. Hamprecht, T. Amano and M. Nirenberg (in preparation), N18TG-2 by Minna et al. (2), C6BU-1 by Amano et al. (3). which are readily demonstrated by both radioactive ligands those of N18TG2 were only detected with? H-naloxone binding whereas the C6éBli-l line does not have a detectable number of opiate receptors by either assay method. This group of cell lines with no, few and an abundance of opiate receptors has provided us with material with which to study the biochemical consequences of the interaction of morphine with its receptor. Collier and Roy reported that morphine and related drugs inhibit the PGEz stimulated conversion of 37H ATP into cAMP by rat brain homogenates in a way that correlates with agonist potency and receptor affinity (4,5). These experiments prompted us to examine the effect of morphine on adenylate cyclase activity and on the cAMP levels of NG108-15 hybrid cells and their parental cell lines (6). We found that morphine inhibits the adenylate cevclase activity of NG108-15 cells and lowers cellular cAMP levels in the presence and in the absence of added PGE, (fig 1). Fogarty International Fellow, on leave from the Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, ndia. 117           woo, & EUS 1200) = se a & je fi :“ z ~ 3 wn i : = j 20 ? wre ened ° mates   Figure 1, Inhibition by morphine (105m) of the rate of cAMP accumulation in intact NG108-15 hybrid cells(part A) and of adeny- late cyclase activity in homogenates (part B). Basal and PGEz, (10°°M) stimulated results are shown (6). . There is so a dramatic reduction in e adenosine stimulated rise in cellular cAMP levels in the presence of morphine (17). Thus, in this cell, morphine inhibits both stimulated . and unstimulated adenylate cyclase. ‘ a Morphine inhibits the adenylate cyclase of the neuro- blastoma parent somewhat, but does not affect the activity the enzyme found in the glioma parent (Table II). Thus, the de-   Table Ti Effect of morphine on adenylate cyclase activity of neuroblastoma and glioma parents N18TG=-2 C6BU-1 Addition* pmole/min/mg protein None 6 20 Morphine 4 19 Naloxone 5 20 Morphine + naloxone 5 20 PGEs. 75 24 PGEz, + morphine 63 25 PGEz + naloxone 72 24 PGE, morphine + naloxone 70 23 *1Qum of each component   gree of inhibition of adenylate cyclase by morphine is correlated with the number of opiate receptors. There are a number of other properties of the enzyme of NG108-15 cells which show that the inhibition by opiates is mediated by their receptors. Thus, naloxone, an apparently pure antagonist of narcotic drugs reverses morphine inhibition of adenylate cyclase (6). Furthermore, the inhibition is sterospecific in that levorphanol, but not its inactive isomer, dextrorphan, inhibits adenylate cyclase (6). Traber et al. (7-9) have also reported that morphine reduces PGE elevation of cAMP levels and Blosser et al. (10) have reported that morphine inhibits the PGFy, dependent activation of adeny late cylase in neuroblastoma or hybrid cell lines derived from neuro- blastoma cells. There is a good correlation between the concentrations at which opiate agonists displace 3 Il=-naloxone from the receptors and and those required for inhibition of adenylate cyclase. This agreement is readily apparent in the data presented in Table III. 118 Table III Comparison of narcotic affinity for the opiate receptor and ability to inhibit adenylate cyclase Ka Ki Narcotic receptor Adenylate cyclase Narcotic nM nM Etorphine 5 10 Levorphanol 200 200 Morphine 4,000 2,000 3-Allylprodine 10,000 50,000 Dextrorphan 10,000 -_ Naloxone 20 -   However, we found that the opiate binding and enzyme inhibition curves not superimposible (6). Enzyme inhibition takes place over a much narrower range of drug concentrations than does dis- placement of 3li-naloxone from the receptors. This behavior im- plies cooperativity among liganded receptors in their interaction with the adenylate cyclase complex. Analysis of the data by means of Hill plots shows that the slope for narcotic binding (reactionl of scheme 1) is close to 1 indicating little or no cooperativity a 3 (1) M + receptor ===> receptors M===Aenzynesreceptor. H in the formation of fharcotic-receptoy) complex but that the maximum slopes of the curves for adenylate cyclase inhibition by narcotics (reaction 2) are between 2 and 3, indicating strong positive cooperativity in the reactions that couple {narcotic ~ receptor] complexes with adenylate cyclase. Coupling of opiate-receptor complexes to adenylate cyclase may occur by any of 3 general mechanisms: 1) Direct interaction of receptor and enzyme, by analogy with enzyme systems composed of catalytic and regulatory subunits, or linkage via a modulator (15). 2) Opiate-receptor complexes may elicit the production of chem- ical messages, suggested as a rather unlikely possibility by H.0.J. Collier (personal communication). 3) Indirect coupling mediated by conformational transitions of the membrane. The membrane conformation may reflect either the proportions of receptors in states A and B or may change in re- ponse to the process of transition of the conformation of the receptors between states A and B induced by the association and dissociation of agonists (but not of antagonists, since Na* main- tains receptors in the B state). The indirect coupling mechanisms, which involve membrane changes, may allow opiate receptors to express their interaction with narcotics in more than one way. Thus, the inhibition of adenylate cyclase and the electro- physiologocal effects of narcotics on NG108-15 cells found by Traber et al. (9), Myers and Livengood (16) and in our own laboratories can be different manifestations of the same fundamental effects on membrane structure. An important property of many of the narcotic analgesics is that of mixed agonist-antagonist behavior. Perhaps the best studied example of such a compound is nalorphine which is a potent antagonist of morphine, but also is a good analgesic in its own right (11). tow may this dualism of action be understood 119 in the context of opiate action as an inhibitor of adenylate cyclase? Figure 2 shows the effects of nalorphine upon adenylate         HaLORPRNE HOLARITY Figure 2, Effects of nalorphine on the adenylate cyclase activity of homogenates of NG108-15 cells. The curves, reading from top to bottom, represent experiments performed at the following concentrations of morphine: none, 2 x 10 M, 1o~tM, and 2x 10” The data have been normalized so that uninhibited adenylate cyclase activity is constant. ~   cyclase activity at several Goncentrations Of Morphine. In the absence of morphine, nalorphine inhibits the enzyme but only partially when compared with the degree of inhibition produced by morphine. In the presence of morphine, on the other hand, the effect of nalorphine is to reverse the inhibition produced by morphine. The reversal of morphine inhibition by nalorphine is also not complete but only restores enzyme activity to the level seen in the presence of nalorphine alone. There is evidence that opiate receptors exist in two confor- mational states (12, 13) as shown below: Nat Receptor A ¢-——==} Receptor B [asonsat~neckiéor >] [ ntidbnsees naceptor a] inhibition of adenylate cyclase uncoupled Receptor form A has a high affinity for agonists and form B for antagonists as shown. Binding of agonists to the receptor will shi the equilibrium to the left and convert most receptors to the [agonists Receptor Al complex which will result in inhibition of . adenylate cyclase. Conversely, when receptors are in the form of the [Antagoniste Receptor complex, as the result of interaction with a pure antagonist, adenylate cyclase is not inhibited. Mixed agonist-antagonists, such as nalorphine, may have a comparable af- finity for receptors in both states. The interaction of opiate receptors with agonist-antagonist narcotics will then resuit in the receptor complexes being partitioned between states A and B in comparable amounts. Inhibition of adenylate cyclase will thus be only partial as is observed. When NG108-15 cells are cultured in the presenge of morphine for a number of days, the level of adenylate cyclase activity in- creases by approximately 50-100%. An experiment which demon~ strates this phenomenon is shown in fig 3. The cells after 2 or more days of exposure to morphine are tolerant in the sense that adenylate cyclase activity is nearly normal when assayed in the 120 adenylate cyclase activity is nearlv normal when assayed in the                    z T T T T t T T 2 w BASAL PGE] STIMULATED 2 40 FADENYLATE CYCLASE ACTIVITY NYLATE CYCLASE activity 7200 g ro] 2 30 NONE, 7 (50 2 = 3 ~ ~ a 8 rey owen: = 20 PHONE 100 ¢ a MORPHINE = q10 + 1504 a a F 4 ° 4. 1 4 4 0 g z 0 6 2 3 4 0 Il 2 3 DAYS CELLS TREATED WITH MORPHINE Figure 3, Basal and PGhy stinulated adenylate cyclase activity of homogenates of NG108-15 cells cultured in the presence of 10 M morphine for the times shown (17).   presence of morphine. They are dependent upon it in the sense that adenylate cyclase activity measured in its absence is abnormally high, The dependence phenonenon is dranatically seen when cAMP levels of cells cultured in the presence of morphine for 48 hours are measured after a brief exposure to naloxone.   Table IV cAMP Tevels of normal and addicted cells (17) CELLS Conditions of Assay B_of Normal pnoles cAMP/mq protein basal 20 23 113 naloxone 21 37 175 PGE 264 81 31 PGE + naloxone 241 1183 491 adenosine ' 103 65 63 adenosine + naloxone 72 217 301 naloxone   Addicted celIs show as much as a 4 to 5 fold increase in cAMP levels over the control, in the presence but not in the absence of naloxone precipitated withdrawal. There is no change in the number of opiate receptors in tolerant cells (17). Figure 4 summarizes the general conclusions which we have reached concerning tolerance and dependence on the basis of these and other (17), related, experiments, We find that morphine inhibits adenylate cyclase activity and thus de- creases cAMP levels, On continued exposure to morphine the cells adapt by an increase in adenylate cyclase activity which results in tolerance and dependence. The fully toler- ant cells have cAMP levels close to normal in the presence of morphine, When the opiate is withdrawn on addition of an antagonist, cAMP levels rise to abnormally high values. This abrupt increase in cAMP indicates that the cells are depen- lal   ado WITHDRAW MORPHINE MOR PHINE 2oot =f { ]          ISOF   NORMAL   ADDICTED 100 oo 50 % NORMAL cAMP LEVEL       0 TIME Figure 4. dent upon morphine and is the biochemical counterpart of the abstinence syndrome. Recovery of the cells from the’ addicted state requires the return of adenylate cyclase activity to its normal levels. These results support the suggestions of Goldstein and Goldstein (14) and Shuster (19), made many years ago, that drugs may act as enzyme inducers. Increases in cAMP levels in the abstinence syndrome of animals has recently been demonstrated by Collier and Francis (18). REFERENCIS 1, W.A. KLEE, M. NIRENBERG, Proc. Nat. Acad. Sci. U.S.A., 71, 3474-3477 (1974). 2. od. MINNA, D. GLAZER, M. NIRENBERG, Nature New Biol., 235 225-231 (1972). OT 3. T. AMANO, B. HAMPRECHT, W. KEMPFR, Exp. Cell Res., 85 399-408 (1974). —_™ 4. H.O.J. COLLIER, A.C. ROY, Nature, Lond., 248 24-27 (1974). 5. H.0.J,. COLLIER, A.C. ROY, Prostaglandins, 7 361- -376 (1974). 6. S.K. SHARMA, M. NIRENBERG, W.A. ah, proc. Nat. Acad. Sci., U.S.A., 72 590-594 (1975). TT 7. J. TRABER, K. FISCHER, S. LATZIN, B. HAMPRICHT, FBS Letts., 49 260-263 (1974). eT 8. J. TRADER, K. FISCHER, S. LATZIN, B. HAMPRECHT, Nature, Lond., 253 120-122 (1975). 9, J. TRABER, G. REISER, K. FISCHER, B. HAMPRECHT, FEBS Letts., 52 327-332 (1975). a 10. J.C. BLOSSER, J.R. ABBOTT, W. SNAIN, Fed. Proc., 34 713 (1975). 11. R.I. TABER, D.D. GREENHOUSE, J.K. RENDEEE, 5. IRS J. Pharmacol. Exp. Ther., 169 29-38 (1969). 12. G.B. PERT, G. PASTERNAK, S.H. SNYDER, Science, 182 1359-1361 (1973). TT 13. E.J. SIMON, J.M. HILLER, J. GROTH, I. EDELMAN, J. Pharmacol. « Ther., in press (1975). 14. D.B. GOLDSTEIN, A. GOLDSTEIN, Biochem. Pharmacol., 8 48 (1961). 15. M. RODBELL, M.C. LIN, Y. SALOMON, ©. LONDOS, J.P. HARWOON, D.R. MARTIN, M. RENDELL, M. BERMAN, Adv. in Cyclic Nucleotide Research, 5 3-29 (1975). 16, BP. MYERS, D. LIVENGOOD, Fed. Proc., 43 359 (1975). 17. S.K. SHARMA, W.A. KLFF, Ml NYRENBERG, in preparation. 18. H.0O.3. COLLIER, D.L. FRANCIS, Nature Lond., 255 159-162 (1975). 19, L. SHUSTER, Nature, Lond., 189 61). 122", "Nirenberg, Marshall W. ; Sharma, S. K. (Shail K.) ; Klee, Werner A.", null, null, "Pergamon Press, Inc.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-r8da~vb6t~xfc5", "00000000-0000-0000-0A16-EABD065CBE6D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Morphine Receptors as Regulators of Adenylate Cyclase Activity", "101584910X215", "101584910X216", "1975", "February 1975", "This article is an extension of earlier published work describing the neuroblastoma hybrid cell line and examining the effects of morphine and other narcotics upon enzyme activity.  The authors find that morphine and other narcotics inhibit enzyme activity and that the inhibition is dependent upon morphine receptors.  This leads to a hypothesis concerning the molecular basis for narcotic addiction and tolerance at the end of the article.", "Articles", "Adenylyl Cyclases,Tumor Cells, Cultured ; Receptors, Opioid, mu", "Neuroblastoma Research, 1967-1976", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Nat. Acad. Set. USA Vol. 72, No. 2, pp. 590-594, February 1975 Morphine Receptors as Regulators of Adenylate Cyclase Activity (neuroblastoma X glioma hybrid/prostaglandin E,/narcotic dependence) SHAIL K. SHARMA*t, MARSHALL NIRENBERG*, AND WERNER A. KLEEt * Laboratory of Biochemical Genetics, National Heart and Lung Institute, and the ¢ Laboratory of General and Compurative Biochemistry, National Institute of Mental Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, November 11, 1974 ABSTRACT Morphine inhibits adenylate cyclase (EC 4.6.1.1) activity of neuroblastoma X glioma hybrid cells. The inhibition is stereospecific and is reversed by the antagonist, naloxone. The relative affinities of narcotics for the opiate receptor agree well with their effectiveness as inhibitors of adenylate cyclase. Morphine-sensitive and -insensitive cell lines were found, and the degree of sensi- tivity was shown to be dependent upon the abundance of narcotic receptors. Thus, morphine receptors are func- tionally coupled to adenylate cyclase. A molecular mech- anism for narcotic addiction and tolerance is proposed.   A neuroblastoma xX glioma hybrid cell line with morphine receptors has been described (1). The receptors are stereo- specific with respect to narcotic binding, and receptor affinities for narcotics closely resemble those of rat brain (2-4) and correlate well with their pharmacologic potencies. Collier and Roy have reported that prostaglandin E de- pendent cAMP synthesis is inhibited by narcotics in rat brain homogenates (5, 6). In this communication the effects of morphine and other narcotics upon adenylate cyclase (EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity of neuroblastoma X glioma hybrids and parental cells are examined. We find that morphine and other narcotics inhibit adenylate cyclase activity and that the inhibition is dependent upon morphine receptors. MATERIALS AND METHODS Materials. [G-?H]Naloxone (26.3 Ci/mmol) and [(G-3H]- cAMP (22.1 Ci/mmol) were from New England Nuclear; [a-PJATP (6-10 Ci/mmol) was from ICN; and {U-*4C]- cAMP (0.295 Ci/mmol) was from Amersham/Searle. Mor- phine sulfate was from Merck; naloxone-HCl was a gift of Endo Labs; etorphine was from Dr. R. Willette, NIDA; other narcotics were from Dr. Everette May; PGE, was from Dr. John Pike, Upjohn Co.; Ro20-1724 [4-(3-butoxy-4- methoxy benzy])-2-imidazolidinone was from Hoffman La- Roche; IBMX (3-isobutyl-l-methylxanthine) was from Aldrich; Gpp(NH)p (5’-guanylylimidodiphosphate) was from ICN; cAMP, ereatine phosphate, and creatine kinase (155 U/mg of protein) were from Sigma. Cell Lines. Neuroblastoma X glioma hybrid NG108-15 was obtained§ by fusion of mouse neuroblastoma clone N18TG-2   Abbreviations: Ro20-1724, 4-(3-butoxy-4-methoxybenzy])-2-imid- azolidinone; IBMX,  3-isobutyl-l-methylxanthine; Gpp- (NH)p, 5’-guanylylimidodiphosphate; PGE,, prostaglandin Ey. t Fogarty International Fellow, on leave from the Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India. § B. Hamprecht, T. Amano, and M. Nirenberg, in preparation. 590 (7) and rat glioma clone C6BU-1 (8), derived from C6 (9). Cells were grown as described (1). Assay of cAMP of Intact Cells. Confluent cells (8-4 my of protein 60 min dish) were washed 3 times with 5 m] of medium A [Dulbecco’s modified Eagle’s medium with 25 mM Hepes (N-2-hydroxyethylpiperazine-N‘-2-ethanesulfonie acid) pH 7.4, instead of NaHCO, adjusted to 340 mosmol/liter with 1.1 g of NaCl/liter] and incubated with medium A plus 0.5 mM Ro20-1724 and 0.6 mM 13MX for 30 min at 37°. Reactions were initiated by the addition of 30 ul of narcotic in water, 30 ul of PGE, in ethanol, or solvent. Ethanol (0.5% with Ro20-1724 present or 1% when Ro20-1724 and PGE, were present) had no effect upon cAMP formation. After incubation the medium was discarded and 3 ml] of 5% trichloroacetic acid with 5 pmol of [4C]cAMP (3000 cpm) at 3° were added. The extract and 2 washes (each 1.5 ml of 5% trichloroacetic acid) were combined and centrifuged and the supernatant fluids applied to 0.8 * 8-cm columns of AG 50W-X4 resin, 200-400 mesh, H+ form (BioRad). The 3 ml of eluate following a 6-ml water wash was assayed for cAMP by the method of Gilman (10). Values reported are for duplicate dishes and are corrected to 100% recovery of cAMP. Adenylate Cyclase Assay. Cells that had been washed three times were homogenized in 0.32 M sucrose, 10 mM Tris-HCl, pH 7.4 (15 mg of protein per ml) with 10-15 strokes, by hand, of a ground-glass homogenizer. Enzyme activity was <le- termined by a modification of method C of Salomon et al. (11). Each tube contained: 45 mM Tris-HCl, pH 7.4; 5 mM MgCl; 160 mM sucrose; 20 mM creatine phosphate; 10 U of creatine kinase; 1 mM cAMP; 0.5 mM Ro20-1724 (0.5% ethanol, final concentration); 1 mM [a-@P]ATP (3 to 5 & 10° cpm); and 100-300 ug of homogenate protein in a final volume of 100 yl. The reaction was terminated by addition of 50 yl of 15% trichloroacetic acid. For product characterization, the 32P product purified through the alumina column step (11) was subjected to Dowex-1 formate column chromatography. The 2 M formic acid eluate was lyophilized, and the radioac- tivity was characterized by paper or thin-layer chromatog- raphy in: (A) isopropanol-NH,OH-0.1 M boric acid (7:1:2); (B) 1 M ammonium acetate-95% ethanol (3:7); (C) isobutyric acid-2 M NH,OH (2:1); and (D) H,O-washed polyethylene- imine-cellulose thin-layer plates developed sequentially with HO and 0.25 M LiCl. Greater than 90% of the radioactivity recovered from the alumina columns was cAMP. Opiate Binding. The assay (1) was modified so that incuba- tion mixtures contained the components of the adenylate cyclase assay and 875 ug of homogenate protein; & X 10-§ M (*J]I]naloxone (4150 cpm); and unlabeled narcotic in a final volume of 200 ul. Under these conditions, narcotic affinities Proc, Nat. Acad. Sci. USA 72 (1978)   A CELLS 1400 1200 1000 800 600 400 200F PMOL cAMP /MG PROTEIN             205 9 1 1OF 9 MORPHINE + L oa 1 L e = 4 é a 6 MINUTES Morphine Receptors and Adenylate Cyclase 591   T T T T T T T T B HOMOGENATE           2:00 f % 3a 3 1000 PMOL cAMP/MG PROTEIN 500       0 4 2 6 20 8 ' MINUTES Fic. 1. Inhibition by morphine of the rate of cAMP accumulation in intact neuroblastoma X glioma hybrid NG108-15 cells and of adenylate cyclase activity in homogenates. The effect of morphine on basal and PGE,-stimulated cAMP levels in intact cells is shown in part A. In B, the rate of adenylate cyclase activity in NG108-15 homogenates is shown. Concentrations of PGE, and morphine were 10 «M. for the receptor are lower than those as measured (1) by the following factors: naloxone, 2; etorphine, 13; dextrorphan, 67; levorphanol, 667; morphine, 200; and 3-allylprodine, 100. RESULTS The effects of morphine upon basal and PGE,-stimulated cAMP levels in intact NG108-15 hybrid cells and on adenylate cyclase activity in cell-free homogenates are shown in Fig. 1A and B, respectively. The addition of PGE, to intact cells results in a 40-fold increase in intracellular cAMP levels. The addition of morphine markedly reduces cAMP levels, both in the presence and absence of PGE. As shown in panel B, the addition of PGE; to homogenates similarly results in a 10-fold increase in adenylate cyclase activity (5- to 12-fold increase in other experiments), Morphine inhibits basal and PGEi-dependent adenylate cyclase activity about 45% (30- 60% inhibition in other experiments). The rate of cAMP formation was lincar for about 8 min; therefore, in all sub- sequent experiments reaction mixtures were incubated for 5 min. The effect of morphine upon adenylate cyclase activity was verified in the following ways: (1) the [#?P]product of the re- action was shown to have the chromatographic mobility of authentic cAMP in five systems (see Materials and Methods) ; (ii) the concentration of substrate, ATP, did not change appreciably during incubation by direct measurement with the luciferase assay (12); and. (itt) the specific activity of ATP changed less than 5% during the incubation. The effect of naloxone, an antagonist that displaces mor- phine from the opiate receptor but has no narcotic activity, is shown in Table 1. Naloxone reverses the inhibitory effect of morphine upon adenylate cyclase activity both in the presence and absence of PGE,, but naloxone alone has little or no effect upon basal or PGE;-stimulated adenylate cyclase activity. The relation between molarity of PGE, and the specific activity of adenylate cyclase is shown in Fig. 2. Adenylate cyclase is stimulated by concentrations of PGE, greater than 10-8 M, A discontinuity in the concentration curve is observed at 5 X 10-77M PGE. The effects of different concentrations of naloxone, in the presence and absence of morphine, on adenylate cyclase are shown in Fig. 3. The concentration of morphine was 10-5 M, which inhibits adenylate cyclase activity maximally. Naloxone completely reverses the inhibitory effect of morphine on adenylate cyclase activity at 2 X 1075 M; naloxone at higher concentrations stimulates adenylate cyclase activity 15-20%. Stimulation has not been observed in all experiments, and further experiments are needed to clarify this observation. In Fig. 4 the decrease in binding of [*H]naloxone to the narcotic receptor, as a function of narcotic concentration, is compared with the effectiveness of the narcotics as inhibitors of adenylate cyclase activity. Naloxone, a pure antagonist, binds with high affinity to the opiate receptor but does not inhibit adenylate cyclase activity (panel A). Etorphine, levorphanol, morphine, and 3-allylprodine, each a potent narcotic, inhibit adenylate cyclasc at essentially the same TaB.e 1. Reversal of morphine inhibition of adenylate cyclase by naloxone in cell-free preparations of NG108-16   Membranet Homogenatet fraction (pmol of cAMP formed/   Addition* min per mg of protein) None 17 24 Morphine 8 17 Naloxone 17 22 Morphine + naloxone 17 23 PGE; 113 124 PGE, + morphine 68 | 101 PGE, + naloxone 110 PGE, + morphine + naloxone 100 122   * 10 uM, each component. { Unfractionated homogenate. t The homogenate was centrifuged at 1500 X g for 10 min and the supernatant fraction at 17,000 x g for 30 min. The 17,000 x g pellet was washed and resuspended in the homogenizing me- dium (membrane fraction). 592 Biochemistry: Sharma eé al.   f T T T     z 100 & g 80 ‘E = 60 = = £40 $ g 20 Ol : rere poo i? wo? 168 =o ®t?\" PGE, MOLARITY Fic. 2. Activation of adenylate cyclase in homogenates of NG108-15 by PGF, as a function of PGE, concentration. concentrations as those needed to displace [\"H ]naloxone from the opiate receptor (panels B, C, E, and F). In contrast to levorphanol, the analgesically inactive enantiomer dex- trorphan is without effect on adenylate cyclase activity and has a much lower affinity for the opiate receptor (panel D). Apparent dissociation constants for [narcotic- opiate re- ceptor] complexes are shown in Table 2, along with apparent K, values for adenylate cyclase calculated from the data of Fig. 4. A striking similarity is observed between the dissocia- tion constants and the concentrations of narcotics required for 50% inhibition of adenylate cyclase activity. Narcotic binding and enzyme inhibition curves shown in Fig. 4 are not superimposable, even though the narcotic concentrations for half-maximal inhibition of binding and enzyme activity agree well. With each of the four narcotics, enzyme activity changes over a relatively small range of narcotic concentration compared to narcotic binding. This relationship is seen more clearly in Fig. 5A and B, which shows Hill plots of narcotic binding and adenylate cyclase inhibition, respectively. Each slope for narcotic binding is approximately 1, whereas the maximum slopes for adenylate cyclase inhibi- tion are 2.3. These results suggest that the binding of nar- cotics to receptors is not a cooperative process, whereas the effects of narcotics on adenylate cyclase activity exhibit a cooperative character. The relation between the molarity of Gpp(NH)p, a rela tively stable analog of GTP, and adenylate cyclase activity, in the presence and absence of morphine, is shown in Fig. 6. TasBLe 2. Comparison of narcotic affinity for the opiate receptor and ability to inhibit adenylate cyclase     Ka* Kit Narcotic Adenylate receptor cyclase Narcotic nM nM Etorphine 5 10 Levorphanol 200 200 Morphine 4,000 2,000 3-Allylprodine 10,000 50,000 Dextrorphan 10,000 _ Naloxone 20 _—   * Apparent dissociation constant of the narcotic-recéptor com- plex. t Narcotic concentration required for 50% of maximal in- hibition of adenylate cyclase. —, no inhibition. Proc. Nat. Acad. Sei. USA 72 (1976) - —     10) f Pus MORPHINE | | _7 PMOL cAMP/MIN PER MG PROTEIN ° L 10° 10° 107 10% 10° 10% 10° NALOXONE MOLARITY Fig. 3. The effect of naloxone upon adenylate cyclase activity of an NG108-15 homogenate in the presence and absence of 10 uM morphine. The GTP analog activates adenylate cyclase of NG108-15 homogenates, similar to the effects reported with other systems (13). Complete reversal of morphine inhibition of adenylate cyclase was observed in the presence of 1 X 10-5 M                                     (3 42 700% 4H lO 600} . soo | NALOXONE ee, 410 15 700 a cd, 5 600 joo ® 500 7° 8 w LEVORPHANOL z § 400} ~ Js 2 a 3 32 2 2% = 0 = a a a S00/ DEXTRORPHAN m0 400 o- s 18 700k\" * ‘ E 6 (2 600 16 8 300 MORPHINE oad & 400 4 POOK a eeceeecceeeeeeeeeeg owe 600 500 3-ALLYL PRODINE 400 : . L   pet te gt el tg\" oem oe & io ow oO NARCOTIC MOLARITY Fic. 4. The effectiveness of various narcotics as inhibitors of adenylate cyclase activity of NG108-15 homogenates is com- pared with their ability to displace [*H]naloxone from the opiate receptor. Symbols represent the following: ©, [*H]naloxone bound to receptor; 4, [#7P]cAMP formed per 5 min per tube (250 ug of protein per tube). Proc. Nat, Acad. Set. USA 72 (1978)       */o } . a oft a a ° ‘ oe Ld 10° 10\" 107 10* 10* 107 NARCOTIC MOLARITY Fie. 5. (A) Hill plots of narcotic binding to receptor; and (B) inhibition of adenylate cyclase activity by narcotics. The sym- bols represent the following: O, etorphine; m, naloxone; @, levor- phanol; 4, morphine; and 4, 3-allylprodine. In panel A the symbol n in the ordinate represents the maximum amount of [*H]- naloxone displaced at saturating concentrations of unlabeled narcotic and » is the amount of [*H]naloxone displaced at non- saturating concentrations of narcotic. In panel B, n represents the maximum inhibition of adenylate cyclase observed at satu- rating narcotic concentrations and » is the inhibition observed at each nonsaturating concentration of narcotic. Gpp(NH)p. In the absence of morphine, <1 X 10-° M Gpp- (NH)p has little effect upon adenylate cyclase activity. Similar results were obtained with partially purified mem- brane preparations (not shown), The effects of morphine upon adenylate cyclase activity of intact neuroblastoma N18TG-2 and glioma C6BU-1 cells, the parents of hybrid NG108-15, are shown in Fig. 7A and B, respectively. Morphine reduces cAMP levels of neuro- blastoma Ni8TG-2 cells slightly but does not affect cAMP levels of glioma C6BU-1 cells. The effects of morphine and naloxone upon adenylate cyclase activity of homogenates prepared from parent neuroblastoma N18TG-2 and gliorna C6BU-1 cells are shown in Table 3. Morphine inhibits adenylate cyclase activity of N18TG-2 slightly, and the inhibition is reversed by naloxone; however, these compounds do not affect adenylate cyclase activity of C6BU-1. Thus, the neuroblastoma parent, which has few morphine receptors,‘ is slightly sensitive to morphine whereas the glioma parent, which lacks these receptors, is insensitive to morphine. DISCUSSION ‘The results show that morphine reduces basal and PGE,- stimulated cAMP levels of intact neuroblastoma x glioma hybrid cells and inhibits adenylate cyclase activity of homoge- nates. The effects of morphine are blocked by the narcotic antagonist, naloxone, which competes with narcotics for sites” on the opiate receptor. The inhibition of adenylate cyclase activity by narcotics is specific for the levorotatory, pharma- cologically active, stereoisomer. The relative affinities of narcotics for the opiate receptor agree well with their effective- ness as inhibitors of adenylate cyclase and with their pharma- cologic potency. Morphine-sensitive and -insensitive cell lines   1 Neuroblastoma clone NI8TG-2 can be shown to have a rela- tively small number of morphine receptors when binding experi- ments are performed with {*H]naloxone in place of the [#H]- dihydromorphine used previously (1). Glioma clone C6BU-1 shows no specific binding of either naloxone or dihydromorphine. Morphine Receptors and Adenylate Cyclase 593 Taste 3. Effect of morphine on adenylate cyclase activity of neuroblastoma and glioma parents     Homogenate Neuro- . blastoma Glioma N18TG-2 C6BU-1 , (pmol of cAMP formed/ Addition * min per mg of protein) None 6 20 Morphine 4 19 Naloxone 5 20 Morphine + naloxone 5 20 PGE, 75 24 PGE, + morphine 63 25 PGE, + naloxone 72 24 PGE, + morphine + naloxone 70 23   * 10 »M, each component. were found, and the degree of sensitivity to morphine is dependent upon the abundance of narcotic receptors. Thus, two kinds of adenylate cyclase complex were de- tected, one sensitive, the other insensitive, to narcotics. Sensitivity to narcotics is dependent upon functional inter- action of the {morphine-receptor] complex with adenylate cyclase. The partial inhibition of adenylate cyclase observed in the preseice of saturating concentrations of narcotics may be due, at least in part, to the presence of narcotic-sensitive and -insensitive adenylate cyclase molecules within cells and, possibly, heterogeneity in the cell population with respect to the number of narcotic receptors per cell. Interactions that couple morphine receptors with adenylate cyclase exhibit positive cooperativity; however, the inter- action of narcotic with receptor is not a cooperative process. The Hill coefficient of 2.3 suggests that multiple interactions between [narcotic-receptor] arid the adenylate cyclase com- plex may be required for inhibition. Alternate mechanisnis for’ apparent cooperativity may be envisaged involving threshold effects and spare receptors. The cooperative activation of adenylate cyclase has been reported for thyroid stimulating hormone (14), but glucagon (15) and oxytocin (16) do not exhibit cooperativity. The available information suggests that one molecule of   —- - - NN N- Bo 7O” B. ao   PMOL cAMP/MIN PER MG PROTEIN © ry     6 q| PLUS MORPHINE Qe Lyp. 9 io\" 16° rou 67 Gop(NH}p MOLARITY Fic. 6. Effect of Gpp(NH)p on adenylate cyclase activity of an NG108-15 homogenate, in the presence and absence of 10 uM morphine. 594 Biochemistry: Sharma et al.             A N18 TG2 B cé6 BU PGE 3000 pce, 1 600} PGE, z + wi 500 MORPHINE. 5 o wp 2000+ PGE, { 400 a 4 = + g MORPHINE] 4 a 3 8 1000} 200 z NONE MORPHINE t NONE '90 1 MORPHINE ° 7 4 6 8 © 0 a MINUTES Fig. 7. Cyclic AMP accumulation, as a function of time, in intact parental cells. Cells used were neuroblastoma Ni8TG-2 in panel A and glioma C6BU-1 in panel B. Concentrations of mor- phine and PGE, were 10 »M. adenylate cyclase can respond to different kinds of hormones, depending upon the species of receptors present, but that only one kind of hormone is translated at a time. Usually, hor- mones activate adenylate cyclase but enzyme inhibition mediated by some a- or prostaglandin receptors has been reported (17, 18). Morphine thus resembles an inhibitory hormone. In essence, three kinds of mechanism have been proposed for the regulation of adenylate cyclase activity by hormones and other effector molecules: (¢) Hormones and GTP are allosteric effectors that activate the enzyme by reducing its affinity for free ATP, a competitive inhibitor of the substrate {[ATP:Mg] (19, 20). Gt) Mg++ or Ca++ bind to other sites and thereby regulate enzyme activity (21, 22). (iz) Adenylate cyclase is inactivated by phosphorylation, catalyzed by a protein kinase, and activated by dephosphorylation, catalyzed by a phosphatase (23). Reactions that terminate reading of messages by adenylate cyclase regulate the duration of response of the enzyme to each message. GTP may be involved both in initiation and termination of reading of hormonal messages by adenylate cyclase-in ways that may be analogous to the roles of GTP in aminoacyl-tRNA bindirig to ribosomes and translocation reactions in protein synthesis. The GTP analog, Gpp(NH)p, activates adenylate cyclase, and morphine reduces the threshold for activation 10-fold. Thus morphine and Gpp(NH)p act in a concerted fashion, which suggests that both are physically coupled to the adeny- late cyclase complex. The K,, for glucagon stimulation of adenylate cyclase is reduced 10-fold by GTP (24). The possi- bility that GTP is required for morphine action, as it is for hormone action, should bé considered. Collier and Roy have presented cogent arguments support- ing the thesis that PGE-stimulated adenylate cyclase is the primary site of morphine action (5, 6). Our results support their view, although we find that morphine inhibits adenylate cyclase both in the presence and absence of added PGE. The inhibition of adenylate cyclase should have many secon- Proc. Nat. Acad. Sci. USA 72 (1978) dary consequences which might account for the varied pharma- cologic effects of morphine. We wish to propose the following hypothesis concerning the molecular basis for narcotic addiction and tolerance. Inhibition of adenylate cyclase by morphine reduces intra- cellular levels of cAMP. This may lead to a compensatory shift in enzyme synthesis, degradation, or activity which restores the normal level of cAMP. The cell then is dependent upon narcotic because the level of cAMP is normal in the presence of the drug and abnormally high upon withdrawal. Our working hypothesis is that the number of adenylate cyclase molecules increases as cells become addicted to nar- cotics. This also leads to tolerance since, at a given narcotic concentration, the amount of uninhibited enzyme is greater in addicted than in normal cells. We thank Doyle Mullinex for growing cells and Richard Streaty for his help. 1. Klee, W. A. & Nirenberg, M. (1974) Proc. Nat. Acad. Sct USA 71, 3474-3477. Pert, C. B. & Snyder, S. H. (1973) Sczence 179, 1011-1014. Terenius, L. (1973) Acta Pharmacol. Toxicol. 32, 317-320. Simon, E. J., Hiller, J. M. & Edelman, I. (1973) Proc. Nas. Acad. Sct. USA 70, 1947-1949. Collier, H. O. J. & Roy, A. C. (1974) Nature 248, 24~27. Collier, H. O. J. & Roy, A. C. (1974) Prostaglandins 7, 361- 376. Minna, J., Glazer, D. & Nirenberg, M. (1972) Nature New Biol, 235, 225-231. Amano, T., Hamprecht, B. & Kemper, W. (1974) Exp. Cell Res. 85, 399-408. 9. Benda, P., Lightbody, J.; Sato, G., Levine, L. & Sweet, W. (1968) Science 161, 370-371. 10. Gilman, A. G. (1970) Proc. Nat. Acad. Sci. USA 67, 305- 312. 11. Salomon, Y., Londos, C. & Rodbell, M. (1974) Anal, Bio- chem. 58, 541-548. 12. Klofat, W., Picciolo, G., Chappelle, Iu. W. & Freese, I. (1969) J. Biol. Chem. 244, 3270-3276. 13. Londos, C., Salomon, Y¥., Lin, M. C., Harwood, J. P., Schramm, M., Wolff, J. & Rodbell, M. (1974) Proc. Nat. Acad. Sci. USA 71, 3087-3090. : 14. Pochet, R., Boeynaems, J. M. & Dumont, J. Is. (1974) Biochem. Biophys. Res. Commun. 58, 446-453. 15. Pohl, S. L., Birnbaumer, L. & Rodbell, M. (1971) J. Biol. Chem. 246, 1849-1856. 16. Bockaert, J., Roy, C. & Jard, S. (1972) J. Biol. Chem. 247, 7073-7081. 17. Robison, G. A., Butcher, R. W. & Sutherland, E. W. (1971) Cyclic AMP (Academic Press, N. Y.), pp. 145-231. 18. Kantor, H. S., Tao, P. & Kiefer, H. C. (1974) Proc. Nat. Acad. Sci. USA 71, 1317-1821. 19. deHaen, C. (1974) J. Biol. Chem. 249, 2756-2762. 20. Rendell, M., Lin, M., Salomon, Y., Rodbell, M. & Berman, M. (1974) Abstracts, Second International Conference on Cyclic AMP (University of British Columbia, Vancouver, B.C.), p. 38. 21. Birnbaumer, L., Pohl, 8. L. & Rodbell, M. (1969) J. Biol. Chem. 244, 3468-3476. 22. Severson, LD. L., Drummond, G. I. & Sulakhe P. V. (1972) J. Biol. Chem, 247, 2949-2958. 23. Constantopoulos, A. & Najjar, V. A. (1973) Biochem. Bio- phys. Res. Commun. 53, 794-799. 24, lRodbell, M., Lin, M. C. & Salomon, Y. (1974) J. Biol. Chem, 249, 59-65. moe ot er N", "Nirenberg, Marshall W. ; Sharma, S. K. (Shail K.) ; Klee, Werner A.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy Press (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-upm8_aypf_e44e", "00000000-0000-0000-619D-FF927FFF2E94", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Dual Regulation of Adenylate Cyclase Accounts for Narcotic Dependence and Tolerance", "101584910X216", "101584910X215", "1975", "August 1975", "This article is an extension of the article titled, \"Morphine Receptors as Regulators of Adenylate Cyclase Activity.\"  The dependence and tolerance phenomenon involving enzymes and morphine receptors is explained as follows: narcotics affect enzymes in two opposing ways, each mediated by the opiate receptor.  One process is responsible for the inhibition of the enzyme by narcotics, the other is a compensatory increase in enzyme activity, which is delayed in onset and relatively stable.  Narcotics dependence and tolerance is the result of the positive regulation of the enzyme counteracting the inhibitory influence of the morphine.  Furthermore, the inhibitory and positive regulatory mechanisms provide a means for \"activating and deactivating neural circuits hours after the initial event and thus may play a role in memory process.\"", "Articles", "Adenylyl Cyclases,Narcotics,Receptors, Cell Surface", "Neuroblastoma Research, 1967-1976", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Nat. Acad. Sci, USA Vol. 72, No. 8, pp. 3092-3096, August 1975 Biochemistry Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance (neuroblastoma X glioma hybrid cells /cell culture /memory /synaptic transmission/opiate receptors) SualL K. SHARMA*?, WERNER A. KLEE', AND MARSHALL NIRENBERG* * Laboratory of Biochemical Genetics, National Heart and Lung Institute, Bethesda, Maryland 20014; and t Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, June 10, 1975 ABSTRACT Narcotics affect adenylate cyclase [ATP py- rophosphate-lyase (cyclizing), EC 4.6.1.1] in two opposing ways, both mediated by the opiate receptor. The first process is the readily reversible inhibition of the enzyme by narcot- ics; the second is a compensatory increase in enzyme activity which is delayed in onset and relatively stable. Late positive regulation of the enzyme counteracts the inhibitory influ- ence of morphine and is responsible for narcotic dependence and tolerance. The coupled inhibitory and positive regulato- ry mechanisms for adenylate cyclase provide a means of acti- vating and deactivating neural circuits hours after the initial event and thus may play a role in a memory process.   Recent observations with neuroblastoma X glioma hybrid cells indicate that the binding of morphine and other opiates to narcotic receptors results in an inhibition of adenylate cy- clase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] ac- tivity (1-3) and a decrease in cAMP levels in intact cells (1, 2, 4, 5). Similar observations have been made with brain (6, 9) but the heterogeneity of cell types present and apparent lability of the brain enzyme may be responsible for conflict- ing observations (8-10). Both dependence upon opiates and tolerance to these compounds were hypothesized to result. from either an increase in the number of molecules of ade- nylate cyclase or a long-lived factor which affects the rates of adenylate cyclase activity or turnover (1). In this report, we describe the results of experiments which were designed to test the hypothesis illustrated diagramatically in Fig. 1. The addition of morphine results in the rapid inhibition of adenylate cyclase activity and a resultant decrease in intra- cellular cAMP levels. Further incubation reveals a second regulatory process which involves a compensatory increase in adenylate cyclase activity termed late positive regulation. The increase in adenylate cyclase activity counteracts the in- hibition of enzyme activity by morphine and cAMP levels are restored to the normal value. Cells now are tolerant to morphine and are also dependent upon the narcotic, since withdrawal of the drug or the addition of a specific narcotic antagonist will raise cAMP levels to abnormally high values and secondarily produce a gradual return to the normal level of adenylate cyclase activity. In this communication we report data which demonstrate a rapid inhibition and a late positive regulation of adenylate cyclase which are dependent upon narcotics and account for the phenomena of narcotic dependence and tolerance. METHODS AND MATERIALS The source of each chemical and the medium and growth conditions for neuroblastoma < glioma hybrid NG108-15 were described previously (1).   Abbreviations: Ro 20-1724, 4-(3-butoxy-4-methoxybenzyl)-2-imida- zolidinone; PGEj, prostaglandin E, + On leave from the Department of Biochemistry, All India Insti- tute of Medical Sciences, New Delhi, India. 3092 Assay of cAMP in Intact Cells and Medium. Growth medium was changed 12 hr before assay. Narcotic was added to the growth medium [Dulbecco's modification of Eagle’s medium (DMEM), hypoxanthine-aminopterin- thymidine (HAT), and 10% fetal bovine serum] and plates were incubated in a humidified atmosphere of 90% air-10% COz. At appropriate times the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzy])-2-imidazolidinone (Ro 20-1724), was added (0.5 mM, final concentration) and incubation was continued for 15 min. Reactions were initiat- ed by the addition of narcotic and/or adenosine in H2O or prostaglandin E, (PGE)) in ethanol. Ethanol (0.5% with Ro 20-1724 present, or 1.0% when both Ro 20-1724 and PGE were present) had no effect upon cAMP formation. Reac- tions were terminated by the addition of 1.0 ml of trichloro- acetic acid at 0° (final concentration, 5%). A solution con- taining 5 pmol of [!4C]eAMP (3000 cpm) was added, and the suspension and two washes (each 1 ml) of 5% trichloro- acetic acid were combined and centrifuged. Each superna- tant fraction was applied to an 0.8 X 8 cm column of AG 50W-X4 resin, 200-400 mesh, H* form (Bio-Rad) washed with H2O. Each column was washed with 6 ml of HzO and cAMP was eluted with an additional 3 ml of HO, and ap- plied to a 0.8 X 2.5 cm column of AG 1-X8 resin, 200-400   (Q) app MORPHINE           WITHDRAW 400+ 4 a be 4 $ 300 aq ad uo = 200F 4 = DEPENDENCE g NORMAL TOLERANCE] NORMAL = 100+ 4 5 bE a 0 uJ Ow ADD MORPHINE WITHDRAW fig pepenoencet @ 4 400- 4 > TOLERANCE oO W 300+ 4 aft al Fa 3 200+ 4 a = RMA NORMAL loop NO 4 9 TIME Fic. 1. A model of the role of adenylate cyclase regulation in the development of morphine tolerance and dependence. Part A shows the effects of morphine upon cAMP levels, and part B, the effects of the opiate upon adenylate cyclase activity as a function of time. ‘ Biochemistry: Sharma et al.   soot INTACT CELLS |      200F |0O0F pMOL CAMP/ MG PROTEIN PGE, + MORPHINE     wl. 1 at 1 L 1 1 2 4 HOURS Fic. 2. Levels of cAMP in intact NG108-15 hybrid cells and the medium measured as a function of time in the presence of 10 uM PGE, or 10 uM PGE, and 10 uM morphine sulfate. No phos- phodiesterase inhibitor was present. The average 60 mm petri dish contained 3.5 mg of cell protein. mesh, formate form (Bio-Rad) equilibrated with water. The eluate and a subsequent 10 ml HgO wash were discarded; cAMP was eluted with 4 N HCOOH and lyophilized. Each dried sample was taken up in 0.5 ml of HgO and assayed for cAMP by the method of Gilman (11). Values reported are average values obtained with two to four replicate plates and.are corrected to 100% recovery of cAMP. At each time of incubation 6 to 8 dishes were washed free of serum (1) and the cells were transferred quantitatively to a tube. After centrifugation the pellets were dissolved in 1 N NaOH and protein was estimated by a modification of the method of Lowry et al. (12). The average value of protein at each time point was used to calculate the specific activity of cAMP. The adenylate cyclase assay and preparation of homoge- nates have been described previously (1), except that Tris- ac was 30 mM and pH 7.5 in the previous as well as this study. Opiate Receptor Assays. Washed cells from two 100 mm culture dishes were homogenized in 10 ml of medium D1 (13), centrifuged for 10 min at 20,000 rpm (48,000 X g), and the pellet was resuspended in the original volume of D1 whose pH had been adjusted to 8.0 with 2 M Tris (free base). Specific binding of (8H]naloxone (5 X 107° M, 68,500 cpm/ml) was measured as described (13). Results are ex- pressed in fmol of specifically bound naloxone per mg of protein in the whole homogenate. RESULTS The effect of morphine on PGE,-stimulated cAMP accumu- lation in neuroblastoma X glioma hybrid cells, (NG108-15), and cAMP excreted into the medium are shown in Fig. 2 as a function of time of incubation. The results show that mor- phine inhibited PGE,-dependent accumulation of cAMP by more that 90% throughout the 4 hr period of incubation. Since a phosphodiesterase inhibitor was not present in this experiment, at least part of the PGE)-dependent increase in cAMP specific activity may represent extracellular cAMP. No evidence for cellular tolerance to morphine was observed during the 4 hr incubation period. The effect of morphine upon adenosine-dependent eleva- tion of cAMP levels and basal cAMP levels is shown in Fig. Proc. Nat. Acad. Sci. USA 72 (1975) 3098                    5000 ACENOSINE STIMULATED cAMP BASAL cAMP 50 z z wu z 5 4000 {405 & a a Z 3000 4303 a a = = J 2000 4204 wl al 6 = 1000 MORPHINE. 410 2 °     5 3045 600.1530 45 60 MINUTES Fic. 3. Basal and adenosine-stimulated levels of cAMP in in- tact NG108-15 cells measured as a function of time in the presence or absence of morphine sulfate. The concentrations used were 100 uM adenosine and 10 uM morphine sulfate. Each 100 mm petri dish also contained 0.1 mM Ro 20-1724. The average dish con- tained 16.9 mg of protein. 8A_and B, respectively. Cells were preincubated with a phosphodiesterase inhibitor, Ro 20-1724, prior to the addi- tion of adenosine and/or morphine. The addition of adeno- sine evoked a large increase in cAMP and morphine inhibit- ed adenosine-dependent cAMP formation. Morphine also re- duced the basal level of cAMP (Fig. 3B). The effect of culturing cells in the presence of morphine for 0-4 days upon the specific activity of basal or PGE)- stimulated adenylate cyclase activity is shown in Fig. 4A and B, respectively. Both basal and PGEj-stimulated adenylate cyclase specific activities increased gradually during the pe- riod that cells were exposed to morphine. After 2-3 days of incubation, the specific activity of adenylate cyclase, as- sayed in the presence of morphine, equaled values found at zero time in the absence of morphine. Thus, culturing cells with morphine results in tolerance to the narcotic. However, a marked increase in adenylate cyclase specific activity was observed when enzyme activity was determined in the ab- sence of morphine. Thus, morphine still inhibits adenylate cyclase activity but the inhibition is masked by a gradual, compensatory increase in the specific activity of the en- zyme. Similar results were obtained in other experiments (not shown) with cells in the logarithmic phase of growth and with confluent cultures of cells, which multiply at a greatly reduced rate. One experiment of this type is shown in Fig. 5. Homogenates prepared from cells cultured with or          ———+ PGEI STIMULATED 40 LADENYLATE CYCLASE ACTIVITY TADENYLATE CYCLASE ACTIVITY 720° Y Pcel | “| a 4150 a o PGEI+ MORPHINE | [OO a °o   i) ° 4 MORPHINE 3     1. 1 1 4 i i 4 1 oO 2 3 4 0 1 2 3 DAYS CELLS TREATED WITH MORPHINE Fic. 4, Basal and PGE,-stimulated adenylate cyclase activity of homogenates of NG108-15 cells cultured for the times shown in the presence of morphine. Confluent cultures (30-50%) in 100 mm petri dishes were divided into four groups; 10 1M morphine sulfate was first added to one group of cultures on day 0, to a second group on day 1, and to a third group on day 2, Media were changed and fresh morphine added daily. Cells were harvested, washed, homog- enized, and assayed on day 4. : .   PMOL cAMP FORMED /MIN PER MG PROTEIN PMOL CAMP FORMED /MIN PER MG PROTEIN o Proc. Nat. Acad. Sci. USA 72 (1975)   3094 Biochemistry: Sharma et al. TTT TdT TT at ® BASAL PGEI STIMULATED 30F 4 _.._ ADDICTED. _.._.- © 25+ - a“ MINUS MORPHINE. nN oO NORMAL pMOL cAMP/MIN PER MG PROTEIN _                  L n a a °o t. 8 PMOL cAMP/MIN PER MG PROTEIN     Sp Ober etee 475 PLUS MORPHINE 10} 4 50 Sr 425 pop ld dd pot ad 41 Oo v l2 24 36 480 l2 24 36 48 HOURS Fic. 5. Basal and PGE,-stimulated adenylate cyclase activity of homogenates of NG108-15 cells cultured and assayed in the presence or absence of 10 4M morphine sulfate and/or PGE; as indicated. Cells 90% confluent in 100 mm petri dishes were cultured for the times indi- cated and were harvested, washed, and assayed immediately. Protein values ranged from 8.2 to 9.2 mg per dish. Filled symbols, addicted cells; empty, normal; triangles, plus morphine; circles, minus morphine. without morphine for 0-48 hr were assayed in the presence or absence of morphine and/or PGE, as indicated in the fig- ure, Some variation in the specific activity of adenylate cy- clase was observed both with normal and addicted cells, The specific activities of adenylate cyclase of normal and addict- ed cells did not differ appreciably during the first 12 hr of incubation, whereas exposure of cells to morphine for 24-48 hr resulted in an increase in adenylate cyclase activity. Thus the delayed increase in adenylate cyclase specific activity requires more than 12 hr of exposure to the drug. The effects of withdrawal, a narcotic antagonist, and sterecisomeric narcotics on positive regulation of adenylate cyclase are shown in Table 1. Cells were cultured with mor- Table 1. Effects of withdrawal, a narcotic antagonist, and stereoisomeric narcotics on positive regulation of adenylate cyclase     pmol cAMP formed/ min Additions during min per mg protein” cell culture H,0 PGE, Cells cultured 4 days H,O 18 95 Morphine 27 115 3 Days Morphine 1 Day Withdrawal 21 98 Morphine + Naloxone 18 79 Cells cultured 3 days H,0 18 100 Naloxone 21 90 Levorphanol 30 _ Dextrorphan 21 -   NG108-15 hybrid cells were cultured in 100 mm petri dishes for 3 or 4 days in the presence or absence of 10 nM narcotic as indicated. Cultures were 50% confluent when narcotic was first added. The medium. was replaced each day. For the withdrawal experiment cells were cultured 3 days with 10 «4M morphine sulfate, then plates were washed three times with growth medium and then cultured for an additional 24 hr in growth medium devoid of morphine. The average protein per dish was 7.2 mg after 3 days and 8.2 mg after 4 days. Adenylate cyclase activity was assayed in the presence of 10 uM naloxone. The final concentration of PGE; was 10 uM. The activities found with morphine and levorphanol in the presence of naloxone were higher than those observed in the absence of nalox- one; however, in all other cases little or no difference was observed in the absence of naloxone (data not shown). phine for 3 days and then for an additional day with or without morphine to test the effects of withdrawal. The re- sults show that the morphine-dependent increase in adenyl- ate cyclase activity was reversed, almost completely, 24 hr after withdrawal of the drug. In another experiment, not shown here, withdrawal for 30 min did not restore adenylate cyclase activity to normal levels. Thus the increase in ade- nylate cyclase activity due to narcotic-dependent positive regulation is stable > 80 min.   Table 2. cAMP levels of hybrid cells grown in the presence or absence of morphine pmol cAMP/mg protein Hours Control Addicted cell cells, cells, Exp. cul- Additions to test cell no morphine no. ture responses morphine present   1. Tested without phosphodiesterase inhibitor 48 H,O 21 23 48 Naloxone 21 37 48 PGE, 264 81 48 PGE, + Naloxone 241 1183 48 Adenosine 103 65 48 Adenosine + Naloxone 72 217 2. Tested with phosphodiesterase inhibitor 0 H,O 89 - 12 H,0 54 40 12 Naloxone 61 125 24 H,0 61 40 24 Naloxone 60 285 48 H,0 63 38 48 Adenosine 2000 - 859 48 Adenosine + Naloxone 1430 3020   NG108-15 cells were cultured in 60 mm petri dishes until 50% con- fluent. Then 10 .M morphine sulfate or HzO was added and incu- bation was continued as indicated in the table. In Exps. 1 and 2 at appropriate times cells were incubated for 15 min in the presence or absence of 0.5 mM Ro 20-1724 (final concentration). To test cell responses, 10 #M naloxone and/or PGE; 100 4M adenosine, or H20 were added and incubation was continued for an additional 10 min, Reactions were terminated as described under Methods and Materials. Average mg of protein per dish at 0, 12, 24, and 48 hr were 2.6, 2.8, 3.5, and 4.4, respectively. Biochemistry: Sharma et al. Table 3. Opiate receptors of hybrid cells cultured with or without morphine   fmol of [H]naloxone specifically bound/mg   Cell culture conditions of protein Control cells 78 (67-92) Cells cultured with morphine 72 (63-80)   Cells were cultured for 26 hr in the presence or absence of 10-5 M morphine. Homogenates of washed cells were assayed for opiate re- ceptors as described in the Methods and Materials section. The values are based on duplicate determinations and are the average of four sets of duplicate dishes. Numbers in parentheses represent the range found. Naloxone, a specific opiate antagonist with high affinity for the narcotic receptor, when present together with mor- phine reduced adenylate cyclase activity to levels below those of the control cells. Naloxone also reduced the PGE;- dependent adenylate cy¢lase activity when cells were cul- tured with this compound. These results possibly suggest ei- ther that the cells synthesize or serum contains a morphine- like factor. Levorphanol, a potent narcotic, increased adenylate cy- clase activity of cells cultured in its presence for 3 days, whereas under the same conditions dextrorphan, the phar- macologically inactive stereoisomer, had little or no effect on basal adenylate cyclase activity. In Table 2, cAMP levels of cells grown in the presence and absence of morphine and tested for 10 min with nalox- one and/or activators of adenylate cyclase are shown. Cell response was tested in the absence of a phosphodiesterase in- hibitor in Exp. 1. The data show that cells cultured in the presence of morphine have normal basal cAMP levels. In the presence of added naloxone, however, basal, PGE;-, and adenosine-stimulated cAMP levels were considerably higher than those found with control cells. These data demonstrate morphine-dependent positive regulation of cAMP levels in intact cells, which agrees well with the increased adenylate cyclase activity found with homogenates. The data also show that cells grown in the presence of morphine are tolerant to morphine since their basal cAMP level is similar to that of control cells. Note that the cAMP assays were performed with cells still in the presence of growth medium and in the case of the addicted cells with morphine. The increase in cAMP levels found after short exposure of the cells to nalox- one indicates that the cells have become dependent upon the presence of morphine for the preservation of normal cAMP levels. This phenomenon is, we believe, the biochemical counterpart of the abstinence syndrome seen with animals and man upon administration of antagonists to dependent individuals. In Exp. 2, the phosphodiesterase inhibitor Ro 20-1724 was present when cell responses were tested. Cells grown in the presence of morphine for 12 hr had higher cAMP levels than control cells in the presence of naloxone. The difference was greater after 24 hr of growth with morphine. However, complete tolerance to morphine was not achieved under the conditions used even after 48 hr of culture with morphine. In the presence of naloxone, adenosine-stimulated cAMP levels of addicted cells were higher than those of control cells, similar to the observations made in the absence of Ro 20-1724. In Fig. 6, morphine is shown to have no effect upon the rate of protein synthesis during 9 days of growth. The Proc. Nat. Acad. Sci. USA 72 (1975) 3095   30- MG PROTEIN /DISH | ] 20F MORPHINE MG PROTEIN / PETRI DISH       Po td 03, ° 4 DAYS Fic. 6. Lack of effect of morphine on cell growth. Replicate cultures, each with 1.5 X 105 NG108-15 cells per 150 mm petri dish, were maintained in the presence or absence of 10 uM mor- phine sulfate. Cella were washed three times and the amount of protein per dish was determined. amount of protein synthesized per dish is used as a measure of cell growth and replication. Thus, the effects of morphine upon adenylate cyclase activity of cells made tolerant to the narcotic are specific ones and are not due.to an increase in total protein synthesis. Another way in which the cells might have adapted to the presence of morphine is by changing the number or proper- ties of opiate receptors. The data shown in Table 3 indicate that such changes have not taken place. The number of mor- phine receptors measured in homogenates prepared from well-washed hybrid cells grown in the presence of morphine is, within experimental error, unchanged from those of the control cells. The number of opiate receptors found in mor- phine-dependent rats also is similar to that of control ani- mals (14). DISCUSSION Narcotics affect adenylate cyclase in two ways, both mediat- ed by the opiate receptor. The first process is a readily re- versible inhibition of the enzyme by narcotics observed with homogenates and, indirectly, with intact cells. The second phenomenon, late positive regulation, results in an increase in adenylate cyclase specific activity which is dependent upon incubation of cells with narcotics for 12 or more hours, The effects of narcotics on both inhibition and positive regu- lation of adenylate cyclase are stereospecific and are re- versed by naloxone. After culture with morphine for 1-2 days cells are tolerant to the narcotic, since the increase in adenylate cyclase activity due to positive regulation com- pensates for the inhibition of enzyme activity observed in the presence of the narcotic. Cells are also dependent upon morphine, since withdrawal, or displacement of morphine from the opiate receptor by the antagonist, naloxone, in- creases basal, PGE,-, and adenosine-stimulated adenylate cyclase activities, which results in an overproduction of cAMP. This phenomenon can be likened to the abstinence syndrome in animals, Thus, NG108-15 cells become depen- dent upon morphine in 12-24 hr. The mechanism of positive regulation of adenylate cy- clase is not known. The long incubation in the presence of 3096 Biochemistry: Sharma et al. narcotics required to increase adenylate cyclase activity, the fact that increases in basal, adenosine-, and PGE)-stimulated adenylate cyclase activities are observed, and the relative stability of the elevated activity suggests that positive regula- tion represents an increase in the number of molecules of adenylate cyclase. The PGE)- and adenosine-dependent ele- vations of cAMP levels in NG108-15 cells are not additive; thus adenylate cyclase molecules rather than PGE, or aden- osine receptors probably are limiting in these cells. We do not rule out the possibility that the enzyme activity is in- creased by the formation of a relatively long-lived modula- tor; however, no evidence for the formation of a diffusable activator or inhibitor of adenylate cyclase was detected when homogenates prepared from normal and narcotic-de- pendent cells were combined and assayed for adenylate cy- clase activity. Goldstein and Goldstein (15) as well as Shuster (16) in 1961 proposed that enzyme induction might be a mechanism for drug tolerance and dependence. Positive regulation of adenylate cyclase represents a new type of receptor-mediated control of adenylate cyclase ac- tivity. Inhibition of adenylate cyclase activity and late posi- tive regulation are coupled, since both are initiated by inter- actions of narcotic with the opiate receptor. The inhibition of adenylate cyclase activity and concomitant reduction in cAMP levels may be required for positive regulation; that is, the concentration of cAMP or the activity of adenylate cy- clase may regulate the number of adenylate cyclase mole- cules in a sequential feedback fashion. If so, each transmit- ter, hormone, or effector of adenylate cyclase is, in fact, a dual regulator, for transient stimulation or inhibition of ade- nylate cyclase may evoke a regulatory process in the oppo- site direction hours after the initial event. The slowly expressed, relatively stable, positive regulatory process is a form of memory. Shifts in the number of mole- cules of adenylate cyclase or a long-lived modulator of en- zyme activity would alter the sensitivity of cell responses to other activators or inhibitors of adenylate cyclase, and thus would profoundly affect the efficiency of trans-synaptic communication.   § H. Matsuzawa and M. Nirenberg, in preparation. Proc. Nat. Acad. Sci. USA 72 (1975) We think it likely that the endogenous morphine-like fac- tor termed enkaphaline, a newly discovered peptide or farn- ily of peptides found in the nervous system which mimic the effects of opiates (17), will evoke both transient inhibition and late positive regulation of adenylate cyclase and, thus, may be involved in a memory process. Certainly any reac- tion affecting either the number of molecules needed for sending or receiving neural information or molecules that regulate their activities provides a potential for activating or deactivating neural circuits and thus may play a role in a memory process. We thank Doyle Mullinax, Deborah Carper, and Linda Lee for their excellent assistance, and Mary Ellen Miller for typing the manuscript. S.K.S. is a Fogarty International Fellow at the National Institutes of Health. 1. Sharma, S. K. Nirenberg, M. & Klee, W. A. (1975) Proc. Nat. Acad. Sci. USA 72, 590-594. 2. Klee, W. A, Sharma, S. K. & Nirenberg, M. (1975) Life Sct., in press. 3. Blosser, J. C., Abbott, J. R. & Shain, W. (1975) Fed. Prac. 34, 718. . 4, Traber, J., Fischer, K., Latzin, S. & Hamprecht, B. (1975) Na- ture 253, 120-122, 5. Traber, J., Reiser, G., Fischer, K. & Hamprecht, B. (1975) FEBS Lett. 52, 327-332. 6. Collier, H. O. J. & Roy, A. C. (1974) Nature 248, 24-27. 7. Collier, H. O. J. & Roy, A. C. (1974) Prostaglandins 7, 861- 376. 8. Chou, W. S., Ho, A. K. S. & Loh, H. H. (1971) Proc, West. Pharmacol. Soc. 14, 42-46. 9. Puri, S. K., Cochin, J. & Volicer, L. (1975) Life Sci. 18, 759- 768, 10. Tell, G. P., Pasternak, G. W. & Cuatrecasas, P. (1975) FEBS Lett. 51, 249-948, 11. Gilman, A. G. (1970) Proc. Nat. Acad. Sct. USA 67, 305-312. 12. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. Klee, W. A. & Nirenberg, M. (1974) Proc. Nat. Acad. Sci. USA 71, 3474-3477, 14. Klee, W. A. & Streaty, R. A. (1974) Nature 248, 61-66. 15. Goldstein, D. B. & Goldstein, A. (1961) Biochem. Pharmacol. 8, 48. 16. Shuster, L. (1961) Nature 189, 314-315. 17. Hughes, J. (1975) Brain Res. 88, 295-308.", "Nirenberg, Marshall W. ; Sharma, S. K. (Shail K.) ; Klee, Werner A.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy Press (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-479n_n2te~29w6", "00000000-0000-0000-09BA-1CFA27DE2FF0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Tolerance and Dependence Evoked by an Endogenous Opiate Peptide", "101584910X217", null, "1976", "September 1976", "As part of an ongoing series of articles devoted to the topic, this article provides experimental evidence that the dual regulation of enzyme production by narcotics accounts for the phenomenon of narcotic dependence and tolerance.  Additionally, results show that cells become tolerant and dependent on certain peptides, suggesting that \"tolerance and dependence are normal responses which regulate transynoptic communication\" coupled to enzyme activity.", "Articles", "Adenylyl Cyclases,Receptors, Opioid ; Opioid Peptides", "Neuroblastoma Research, 1967-1976", "3", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci, USA Vol. 73, No. 9, pp. 3165-3167, September 1976 Biochemistry Tolerance and dependence evoked by an endogenous opiate peptide (adenylate cyclase /methionine-enkephalin/narcotic dependence/neuroblastoma X glioma hybrid cells) ARTHUR LAMPERT*, MARSHALL NIRENBERG*, AND WERNER A. KLEE “Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health; and Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, July 9, 1976 ABSTRACT - Incubation of neuroblastoma X glioma hybrid cells for 12-97 hr with methionine-enkephalin results in an in- crease in adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] that is mediated by the opiate receptor. The results show that cells become tolerant to, and dependent upon, enkephalin.   The members of a recently discovered family of peptides, which includes methionine-enkephalin (Tyr-Gly-Gly-Phe-Met) and leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu), behave as opiates in pharmacologic tests (1-7). The enkephalinst, like morphine and other narcotics, are potential inhibitors of ade- nylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in cells that possess opiate receptors (8-11). As pre- viously reported, exposure of neuroblastoma X glioma hybrid cells to morphine for 12-48 hr also results in a compensatory increase in adenylate cyclase activity (11, 12). Cells then have normal 3’:5’-cAMP levels and appear tolerant to morphine because the increase in adenylate cyclase activity is approxi- mately equal to the inhibition. The cells also are dependent upon morphine to maintain normal cAMP levels because withdrawal of morphine, or displacement of the narcotic from the opiate receptor by the antagonist naloxone, reverses the inhibition and results in the synthesis of abnormally high levels of cAMP. Thus, the dual regulation of adenylate cyclase by. narcotics accounts for the phenomena of narcotic dependence and tolerance. We report here that incubation of NG108-15 hybrid cells for 12-97 hr with methionine-enkephalin results in an increase in adenylate cyclase activity that is mediated by the-opiate re- ceptor. The results show that cells become tolerant to, and de- pendent upon, enkephalin and suggest that tolerance and de- pendence are normal responses which regulate transynaptic communication coupled to adenylate cyclase activity. MATERIALS AND METHODS The source of each chemical and the medium and growth conditions for neuroblastoma X glioma hybrid NG108-15 cells were described previously (11), except that the medium con- tained 3% fetal bovine serum. Adenylate cyclase assays were performed as described (10) with the following modifications: washed cell suspensions were stored at ~191°; immediately before use frozen cell suspensions were thawed and homogenized in a motor-driven Potter-El- vejhem homogenizer at 1400 rpm for 40 sec. RESULTS AND DISCUSSION As shown in Fig. 1, exposure of cells to enkaphalin for 12-97 hr results in an increase in the specific activity of adenylate   Abbreviations: cAMP, adenosine 3/:5’-cyclic monophosphate; PGE), prostaglandin E}. + W. A. Klee and M. Nirenberg, submitted for publication. 3165 cyclase. The basal activity of the enzyme is increased in a par- allel manner in the presence of either 10 7M enkephalin or 1 uM etorphine. Neither enkephalin nor etorphine affects the growth rate of the cells (data not shown). Adenylate cyclase activity stimulated by prostaglandin E; also is increased by incubation of cells in the presence of enkephalin or etorphine. The basal specific activity of adenylate cyclase continues to rise for 97 hr in the presence of enkaphalin or etorphine, whereas prostaglandin-E}-stimulated activity ceases to increase after 12-25 hr. The relative increase in specific activity of basal adenylate cyclase is greater than that observed in the presence of prostaglandin E;. Thus, the effects of methionine-enkephalin closely resemble those found with morphine (11). The increase in adenylate cyclase activity shows that NG108-15 cells exposed to methionine-enkephalin for 12 hr acquire tolerance to this compound as defined by the model of Sharma et al. (11). In view of the short duration of action of enkephalin in many systems (13, 15), which may be due to rapid degradation, the experiments described in Fig. 1 were performed with 10 uM enkephalin, 100 times the concentration required to achieve maximum inhibition of adenylate cyclase, and the medium was changed twice daily. This regimen maintained saturating concentrations of enkephalin throughout the course of the ex- periment. As shown in Fig. 2, incubation of confluent cultures of NG108-15 with 10 uM methionine-enkephalin resulted in the rapid disappearance of methionine-enkephalin (50% loss in 90 min). The enkephalin concentration remaining after 12 hr of incubation was still 10 times the amount needed for ‘maximum inhibition of adenylate cyclase. Naloxone (50 uM) prevents the inhibition of adenylate cyclase by 1 »M enkephalin?. This concentration of naloxone also prevenits the increase in adenylate cyclase activity produced by exposure of cells to 1 4M methionine-enkephalin for 24 hr as shown in Table 1. Naloxone is a pure narcotic antagonist which binds to the opiate receptor but does not affect adenylate cyclase activity. Blockade by naloxone shows that the en- kephalin-dependent increase in adenylate cyclase activity is mediated by the opiate receptor. In cells which become tolerant to a narcotic, basal cAMP levels are normal or approach the normal level in the presence of the narcotic (11). The level of cAMP would be lowered by increasing the concentration of an opiate peptide, or elevated by decreasing the opiate concentration. The elevation in cAMP levels in tolerant cells due to a reduction in opiate peptide concentration would resemble the effect of an activator of adenylate cyclase because cAMP levels would rise above the basal value. Thus, the opiate peptides that are inhibitors of adenylate cyclase can also mimic the effects of activators of this enzyme. Several other peptides with opisite activity have been isolated (3, 5-7). Those whose structures are known contain the amino acid sequence of methionine-enkephalin at the amino terminus.                                     8166 Biochemistry: Lampert et al. Prac. Natl. Acad. Sct. USA 78 (1976) T qT t T qT qT T t T T “Tv T T F T + J T T ~T T T A BASAL B PGE,-STIMULATED L T 4 Zz L 4 4 UD fi 40 r 200. 3 oO ETORPHINE | ETORPHINE J oO > e € Mr 4 4150 ~ . ENKEPHALIN ENKEPHALIN 3 a | | & é | i g 2 CONTROL 7 CONTROL 100 2 3 $ | é = m a 10 + 70 2 L ao 4 0 1 1 pd L 1 1 1 l 1 tL Jd 1 i 1 i 1 L +. 0 24 48 72 9 120 0 24 48 72 9 120 HOURS Fic. 1. Basal (A) and prostaglandin E; (PGE,)-stimulated (B) adenylate cyclase activity of homogenates of NG108-15 cells cultured for the times shown in the presence of 10 nM methionine-enkephalin (A), 1 »M etorphine (0), or in the absence of these compounds (0). NG108-15 cells were grown in 60 mm petri dishes (20 cm? surface area) as described (11), except that 3% fetal bovine serum was used. The medium was changed twice daily. The cells in the early logarithmic growth phase (0.4 mg of protein per plate at zero hours and 2.4 mg of protein at 97 hr) were harvested at the times shown. Each of these peptides, including methionine-enkephalin, corresponds to a portion of the carboxyl-terminal sequence of B-lipotropin (5-7). Some have a much longer duration of action ENKEPHALIN ACTIVITY (%)       i 1 1 1 6 8 HOURS Fic. 2. Methionine-enkephalin activity remaining in filtrates of NG1i08-15 cultures exposed to 10 uM enkephalin for the times shown. Quadruplicate confluent cultures (60 mm petri dishes) were used for each time point. The media were removed and 2.5 mi portions were passed through Millipore filters (GS 0.22 um pores, 25 mm di- ameter). After dilution with 9 volumes of water, 5 ul aliquots were assayed in standard 100 ul reaction mixtures for inhibition of NG108-15 adenylate cyclase activity in the presence and absence of 100 uM naloxone. Methionine-enkephalin concentration was pro- portional to naloxone-reversible inhibition of adenylate cyclase ac- tivity within the range of 2-50 nM.   4 10 12 than enkephalin, and a long-acting opiate peptide should be more effective in producing cellular tolerance and dependence. Using the method of Teschemacher et al. (3), we have prepared from crude adrenocorticotropic hormone a peptide with a molecular weight of 3000-4000 by gel filtration. This peptide, when added to cultures of NG108-15 hybrid cells for 4 days, elicits an increase in adenylate cyclase activity. The increase in activity is blocked by naloxone in the manner demonstrated with enkephalin and other opiates. The finding that at least two opiate peptides induce cellular tolerance and dependence suggests that these phenomena represent normal mechanisms which enable opiate peptides to regulate the efficiency of communication across certain synapses. Shifts in the levels of endogenous opiate peptides may increase or decrease the ac- tivity of specific neural circuits by regulating the responses of adenylate cyclase to different kinds of receptor-mediated ac- tivators of the enzyme. Table 1. Blockade by naloxone of methionine-enkephalin- induced increase in adenylate cyclase activity   Adenylate cyclase activity, pmol cAMP/ min per mg of protein   PGE,- Additions Basal stimulated Control 14.2 158 Met-enkephalin 20.2 203 Met-enkephalin + naloxone 16.3 149   NG108-15 cells were cultured for 24 hr where indicated with 1 .M enkephalin, 50 uM naloxone, or water. Each value represents the average specific activity of six homogenates, each assayed at three to four protein concentrations. Biochemistry: Lampert et al. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. & Morris, H. R. (1975) Nature 258, 577-579, Terenius, L. & Wahlstrém, A. (1975) Life Sci. 16, 1759-1764, Teschemacher, H., Opheim, K. E., Cox, B. M. & Goldstein, A. (1975) Life Sct. 16, 1771-1776, Pasternak, G. W., Simantov, R. & Snyder, S. H. (1976) Mol, Pharmacol. 12, 504-513. Lazarus, L. H., Ling, N. & Guillemin, R. (1976) Proc. Natl. Acad. Sci. USA 73, 2156-2159. Bradbury, A. F., Smyth, D. G., Snell, C. R., Birdsall, N. J. M. & Hulme, E. C. (1976) Nature 260, 793-795. Cox, B. M., Goldstein, A. & Li, C. H. (1976) Proc. Natl. Acad. Sct. USA 73, 1821-1823, Collier, H. O. J. & Roy, A. C. (1974) Nature 248, 24-27. 10. ll. 12. 18. 14. 15. Proc. Natl. Acad. Sct. USA 73(1976) 8167 Traber, J., Fischer, K., Latzin, S. & Hamprecht, B. (1975) Nature 253, 120-129. Sharma, S. K., Nirenberg, M. & Klee, W. A. (1975) Proc. Natl. Acad, Sci. USA 72, 590-594. Sharma, S. K., Klee, W. A. & Nirenberg, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3092-3096. Traber, J., Gullis, R. & Hamprecht, B. (1975) Life Sci. 16, 1863-1868. Hughes, J. (1975) Brain Res. 88, 295-308. Buscher, H. H., Hill, R. C., Romer, D., Cardinaux, F., Closse, A., Hauser, D. & Pless, J. (1976) Nature 261, 423-425, Belluzzi, J. D., Grant, N., Garsky, V., Sarantakis, D., Wise, C. D. & Stein, L. (1976) Nature 260, 625-626.", "Nirenberg, Marshall W. ; Lampert, Arthur ; Klee, Werner A.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy Press (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-428f-4wes_3mpv", "00000000-0000-0000-A05A-F3385FC29909", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Peter Sonderegger to Marshall W. Nirenberg", "101584910X221", null, "1984", "18 December 1984", "Sonderegger, from the Institute of Biochemistry at the University of Zurich in Switzerland, thanks Nirenberg for cell lines and provides him with an updated summary of his results from antibody staining techniques.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Peter Sonderegger.", "Copyright may apply", null, null, "Zurich, December 18, 1984 Dear Marshall, I just got your A2B5-conditioned medium and thank you very much.  Phil asked me in a recent letter about the antibody staining results I obtained with your antibodies. I have done only one experiment.  A copy of my records is included.  After this, Marie Neale did a few more experiments, the records of which should be in her hands.  As a summary of the positive results I obtained: 14H3: Stained cell body on large DRG.  Many DRG neurons negativ. Questionable? Staining of subclass? 16G6: DRG cell bodies and fine processes. Processes intenser than cell bodies. 18B8: DRG cell somas and axons. Stains subpopulation. Spinal Cord cell bodies and axons. Intense dots on axons: boutons? 97C4: Stains extracellular matrix of DRG culture. A2-B5: DRG cell bodies and axons. Non-neuronal cells. 94C2: Staining of certain flat background cells. These are all the data I have.  If I can be of any further help, please let me know. Thanks again. Best wishes,", "Sonderegger, Peter", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qdeg~cbqz.phtd", "00000000-0000-0000-ED51-5A08B05EC7C3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X223", "101584910X222", "1976", "September 1976", "Summary of work for this project as indicated on the report: \"Long range objectives are to define reactions which are required for synapse formation and for transmission of information from cell to cell.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00009-02 LBG Period Covered:  July 1, 1975 through June 30, 1976 Title of Project: Cell Recognition and Synapse Formation Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI:  Marshall Nirenberg, Chief, Lab. of Biochemical Genetics, LBG NHLI OTHER: Donald Puro, Research Associate, LBG NHLI Hiroyuki Sugiyama, Visiting Associate, LBG NHLI Phillip Nelson, Chief, Behavioral Biology Branch, BB NICHD Clifford Christian, Special Fellow, BB NICHD Cooperating Units (if any): Behavioral Biology Branch, NICHD Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, MD 20014 Total Man Years: 2.2 Professional: 2 Other: .2 Summary of Work: Long range objectives are to define reactions which are required for synapse formation and for transmission of information from cell to cell. Project Description: Major Findings: Since normal neurons do not divide, clonal lines of neuroblastoma cells and somatic cell hybrids derived from neuroblastoma cells were generated and characterized with respect to receptors, neurotransmitters, action potential ionophores, and other properties which are required for synaptic communication.  Fusion of clonal neuroblastoma cells with glioma cells yielded clonal hybrid cell lines which synthesize, store and excrete acetylcholine; properties which are not expressed by the parental cell lines.  During the past year cells from one hybrid line were found to form synapses with cultured striated muscle cells.  Synapses between hybrid cells and muscle cells closely resemble the synapses between normal motor neurons and striated muscle before they are fully developed.  Under appropriate conditions, hybrid cells established synaptic connections with almost every muscle cell tested; thus, synapses are synthesized in abundance.  Marked differences were observed in the efficiency of transmission across different synapses.  Axonal activities which were found to be regulated include choline acetyltransferase, acetylcholinesterase, Na+ action potential ionophore specific activities, and the rate of choline transport into cells. Eight species of receptors have been found thus far with the hybrid cell line which forms synapses.  Receptor mediated shifts in cAMP levels, cGMP levels and membrane potentials have been identified and characterized.  Thus the foundation has been laid for studies on the effects of receptor-mediated reactions on synaptic transmission.  In addition, more than 100 cell lines which synthesize acetylcholine have been obtained and are being studied to determine that some cell lines are defective with respect to synapse formation. Significance to Biomedical Research: This is the first time that clonal cells of neural origin have been shown to form synapses.  Thus the set of genes which are required for synapse formation are expressed in this cell line.  The model system which has been established affords extraordinary opportunities to define synapse formation reactions and properties and correlate biochemical events with developmental and electrophysiological phenomena. Proposed Course: Current studies focus on determining the reactions which are required for synapse formation and factors that regulate these reactions. Publications: 1.  Greene, Lloyd A., Shain, William, Chalazonitis, Alcmene, Breakefield, Xandra, Minna, John, Coon, Hayden G. and Nirenberg, Marshall: Neuronal properties of hybrid neuroblastoma x sympathetic ganglion cells.  Proc. Natl. Acad. Sci. USA 72: 4923-4927, 1975. 2. Chalazonitis, A., Greene, L.A. and Shain W.: Excitability and chemosensitivity properties of a somatic cell hybrid between mouse neuroblastoma and sympathetic ganglion cells.  Exp. Cell Res. 96: 225-238, 1975. 3. Nelson, Phillip, Christian, Clifford and Nirenberg, Marshall: Synapse formation between clonal neuroblastoma x glioma hybrid cells and striated muscle cells.  Proc. Natl. Acad. Sci. USA 73: 123-127, 1976. 4. Nirenberg, Marshall W.: Coding of neural information by neuroblastoma cells.  In: Talwar, G. P. (Ed.): Regulation of Growth and Differentiated Function in Eukaryote Cells. New York, Raven Press, pp. 537-539, 1975 5. Thompson, Edward J., Griffith, James M., Glazer-Schoenberg, Deverera and Nirenberg, Marshall W.: An improved method for extracellular recording of action potentials from single cultured neuroblastoma cells.  Med. Biol. Eng. 13: 104-106, 1975. 6. Hamprecht, Bernd, Kemper, Wayne and Amano, Takehiko: Electrical response of glioma cells to acetylcholine.  Brain Res. 101: 129-135, 1976.", "Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Sugiyama, Hiroyuki ; Nelson, Phillip G. ; Puro, Donald ; Christian, Clifford", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6yf6-9nfn.93ng", "00000000-0000-0000-B740-36810D500CC2", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"The Development of Chick Embryo Retina\"", "101584910X224", "101584910X222", "1976", "September 1976", "Summary of work for this project as indicated on the report: \"Some biochemical aspects of chick embryo retina differentiation were studied: 1) An alternate route for GABA synthesis was characterized in the retina; which depends upon the conversion of putrescine to GABA. 2) Glutamic acid decarboxylase specific activity in the retina, increased during the course of embryonic development, either when measured in ovo or in aggregate cultures. 3) The presence of GABA in the culture medium prevented the development of glutamic acid decarboxylase activity in aggregate cultures. 4) The proposed course of this project is to attempt to correlate the biochemical changes observed, with synaptogenesis in the retina.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00011-02 LBG Period Covered: July 1, 1975 through June 30, 1976. Title of Project: The Development of Chick Embryo Retina Names, Laboratory and Institute Affiliations, and Titles of Principal Investigations and All Other Professional Personnel Engaged on the Project: PI: F.G. DeMello, Visiting Associate, LBG NHLI Marshall Nirenberg, Chief, LBG, LBG NHLI Cooperating Units (if any): None Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20014 Total Man Years: 1.2 Professional: 1.2 Other: 0 Summary of Work:  Some biochemical aspects of chick embryo retina differentiation were studied:  1) An alternate route for GABA synthesis was characterized in the retina; which depends upon the conversion of putrescine to GABA.   2) Glutamic acid decarboxylase specific activity in the retina, increased during the course of embryonic development, either when measured in ovo or in aggregate cultures.  3) The presence of GABA in the culture medium prevented the development of glutamic acid decarboxylase activity in aggregate cultures.   4) The proposed course of this project is to attempt to correlate the biochemical changes observed, with synaptogenesis in the retina. Project Description: Objectives:  The objective of the project is to study the biochemical step required for synaptogenesis in chick embryo retina. Major Findings: Two pathways for gamma-aminobutyric acid synthesis were found in chick embryo retina.  The first pathway depends upon the conversion of putrescine to ornithine decarboxylase and the subsequent conversion of ornithing to gamma-aminobutyric acid.  The second route of synthesis is dependent upon the conversion of glutamic acid to gamma-aminobutyric acid, catalyzed by glutamic acid decarboxylase.  Elevation of cAMP levels in neuroblastoma cells was shown to induce ornithine decarboxplase activity.  Thus, in the developing embryo, neurotransmitters which affect cAMP levels may regulate ornithine decarboxylase activity and thereby control the rate of GABA synthesis from ornithine. Proposed Course:  To determine the effect of retina neurotransmitters and other compounds on retina synaptogenesis. Publications: 1.  DeMello, F. G., Bachrach, U. and Nirenberg, M.: Ornithine and glutamic acid decarboxylase activities in the developing retina.  J. Neurochem. In Press.", "Nirenberg, Marshall W. ; DeMello, F. G. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5956~wdk4_cjze", "00000000-0000-0000-F0F2-C5B4563FCDE7", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Muscarinic Acetylcholine Receptors of Cultured Cell Lines\"", "101584910X225", "101584910X222", "1976", "September 1976", "Summary of work for this project as indicated on the report: \"Our work on muscarinic acetylcholine receptors focuses on two fundamental questions: (1) How is binding of acetylcholine to receptors converted into an intracellular response? (2) What factors regulate receptor concentrations in the membrane? A binding assay employing tritiated quinuclidinyl benzilate permits us to investigate properties of receptor binding sites and to measure receptor levels in cultured neuroblastoma and hybrid cells under systematically varied conditions.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00012-02 LBG Period Covered: July 1, 1975 through June 30, 1976 Title of Project: Muscarinic Acetylcholine Receptors of Cultured Cell Lines Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, Lab. of Biochemical Genetics, LBG NHLI OTHER: William Klein, Postdoctoral Fellow, LBG NHLI Orest Hurko, Staff Fellow, LBG NHLI Wolfang Burgermeister, Guest Worker, LC NIAMDD Bernhard Witkop, Chief, Laboratory of Chemistry, LC NIAMDD Cooperating Units (if any): Laboratory of Chemistry, NIAMDD Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20014 Total Man Years: 2.5 Professional: 2.5 Summary of Work: Our work on muscarinic acetylcholine receptors focuses on two fundamental questions: (1) How is binding of acetylcholine to receptors converted in an intra-cellular response? (2) What factors regulate receptor concentrations in the membrane? A binding assay employing tritiated quinuclidinyl benzilate permits us to investigate properties of receptor binding sites and to measure receptor levels in cultured neuroblastoma and hybrid cells under systematically varied conditions. Project Description: Objectives:  The objectives of our work have been (1) to characterize the nature of ligand interactions with muscarinic receptors, and (2) to assess various factors likely to affect receptor concentrations.  Although most methodology involved has been straightforward, our experiments required the custom labeling and purification of muscarinic ligands as commercially available materials were unsuitable due to low specific activity. Major Findings:  The principal findings are: (1) Excitatory and inhibitory muscarinic receptors for ligands which do not differ pharmacologically. (2) The affinity of receptors for acetylcholine is affected by the ionic composition of the medium. (3) Antagonists and agonists differ strikingly in the way that they interact with the receptor.  Antagonist binding to receptor follows the law of mass action whereas agonist binding does not.  When analyzed on Hill plots, agonist interactions with receptor exhibit negative cooperativity that may be related to receptor desensitization. (4) The receptor concentration is greatly reduced by exposure of cultured cells to carbamylcholine or other receptor activators.  The [receptor-agonist] complex is degraded more rapidly than the free receptor. (5) Muscarinic excitatory and inhibitory acetylcholine receptors have been solubilized and have been characterized partially. Significance to Biomedical Research:  This work with a model system suggests that, in the normal nervous system, receptor levels, which ultimately influence the pattern of behavior of neural nets, are regulated by synaptic transmission.  Some of the events which result from the interaction of acetylcholine with muscarinic acetylcholine receptors have been defined.  Two regulatory processes were found which depend upon acetylcholine.  One regulates receptor activity, the other regulates receptor concentration. Proposed Course:  Further elucidation of QNB binding will be pursued.", "Hurko, Orest ; Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Klein, William L. ; Burgermeister, Wolfgang ; Witkop, Bernhard, 1917-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xtgg-endk~psui", "00000000-0000-0000-EF0C-B6A37703ED47", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Selection for Neuroblastoma Cells that Synthesize Certain Transmitters", "101584910X210", null, "1974", "June 1974", "The genetic-neural code parallel that intrigued Nirenberg is showcased in the opening line of this article, which states: \"It seems likely that a universal code has evolved which enables neurons to communicate with one another by sending and receiving information encoded in the form of neurotransmitters.\"  The potential consequences of this statement are revealed in the article.  By demonstrating the ability to synthesize transmitters, the authors suggest this could lead to a clearer understanding of the entire nervous system.", "Articles", "Neurotransmitter Agents,Tumor Cells, Cultured", "Neuroblastoma Research, 1967-1976", "4", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Nat. Acad. Sci. USA Vol. 71, No. 6, pp. 2530-2533, June 1974 Selection for Neuroblastoma Cells that Synthesize Certain Transmitters (differentiation/tyrosine hydroxylase/catecholamines/cell culture/ genetics) XANDRA O. BREAKEFIELD AND MARSHALL W. NIRENBERG Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall W. Nirenberg, March 28, 1974 ABSTRACT A selection procedure was devised for neurons and related cells that depends upon the ability of the cells to synthesize certain amine neurotransmitters. The rationale for selection is that tyrosine is an essential amino acid for most mammalian cells and that three en- zymes from mammalian sources can catalyze the synthesis of tyrosine: phenylalanine hydroxylase (EC 1.14.16.1), tyrosine hydroxylase (EC 1.14.16.2), and tryptophan hydroxylase (EC 1.14.16.4). Tyrosine hydroxylase i is found predominantly in adrenergie neurons and related cells that synthesize dopamine, norepinephrine, and epineph- rine, and tryptophan hydroxylase in cells synthesizing serotonin or melatonin. Only 1 out of 70,000 uncloned mouse neuroblastoma cells grew well in the absence of tyrosine. Approximately 50% of the cell lines obtained by selection had tyrosine hydroxylase activity. This selection procedure thus provides a simple means of obtaining cell lines of neural origin on the basis of their ability to syn- thesize putative transmitters.   It seems likely that a universal code has evolved which enables neurons to communicate with one another by sending and receiving information encoded in the form of neurotrans- mitters. Many steps in the coding of neural information can be studied with neuroblastoma clones currently available; however, more cell lines with additional neural properties are needed. For this reason we examined various ways of selecting cells of neural origin on the basis of their ability to synthe- size transmitters. In this paper we describe a procedure for selecting adrener- gic neuroblastoma, cells by their ability to grow in medium lacking tyrosine. The selection procedure depends on the following phenomena: (a) Tyrosine is an essential amino acid for most mammalian cell types grown in vitro (1); (b) tyrosine hydroxylase (EC 1.14.16.2; tyrosine 3-monooxygenase) cata- lyzes the conversion of phenylalanine to tyrosine, as well as the conversion of tyrosine to 3,4-dihydroxyphenylalanine (dopa) (2, 3) (see Fig. 1); and (c) tyrosine hydroxylase activity is found predominantly in adrenergic neurons and related chromaffin cells of the adrenal medulla (4). During the course of these studies, we learned that T. Puck has used a similar procedure to select for cells with phenylalanine hy- droxylase (EC 1.14.16.1) (personal communication). MATERIALS AND METHODS Cell Culture. The following cell lines were used: an un- cloned line of mouse neuroblastoma C1300, neuroblastoma clones N-18, NS-20, and N1E-115 (also referred to as N-115) (5), a rat glioma, C-6, derived by Benda, et al. (6) (from the American Type Culture Collection, no. CCL107), and mouse L-cells B82 (7). Cells were grown in monolayer culture in polystyrene petri dishes, 60 and 100 mm (20 and 54 em, 2530 respectively) (Falcon Plastics) at 37° in a humidified atmo- sphere of 10% CO2 and 90% air. Cells were grown routinely in either F-14 medium (8) or the Dulbecco-Vogt modification of Eagle’s medium with 4.5 g of dextrose per liter and no Na pyruvate (GIBCO, Catalogue no. H-21). Both media contain tyrosine and phenylalanine and both were supplemented with 5% fetal-calf serum (Colorado Serum), Cells were fed at 1- to 5- day intervals and passaged as described (9). Cells grown in modified Eagle’s medium were adapted to F-14 by cultivation in this medium for at least 10 generations prior to selection. When eloning efficiencies were being de- termined, cells were trypsinized and triturated to achieve a suspension of single cells and then plated at varying concen- trations in either F-14, F-14 minus tyrosine, or F-14 minus tyrosine and phenylalanine. When tyrosine alone was omitted, the phenylalanine concentration was increased to 0.12 mM. All media were supplemented with 5% fetal-calf serum dia- lyzed for 2 days at 3° against 10 volumes of 0.88% (w/v) NaCl (the NaCl solution was changed after 24 hr). Thirty days after they were plated visible colonies (containing >500 cells) were picked with the aid of porcelain penicylinders (Fisher). Cloning efficiencies were determined in plates con- taining <100 colonies, due to the possibility of cross-feeding. Cell lines originating from selected colonies were further grown in F-14 without tyrosine for 1 month and then in either F-14 or modified Eagle’s medium with tyrosine. Cell lines were free of contamination by pleuropneumonia-like organism (PPLO) as determined by culture in plates and broth, and by autoradiography following incubation with [°H }thymidine. For growth rate experiments, cells were fed daily, dissociated by trypsinization, and counted with the aid of a Coulter counter. , Tyrosine Hydrorylase Assay and Chromatographic Identifica- tion of Dopa. Cells were grown in modified Eagle’s medium COOH NH, ~ cH as ag Fie. 1. Reactions re catalyred ht tyrosine hodrexylase Oxygen and tetrahydrobiopterin are required for both reactions as follows: (1) t-tyrosine + Oz + tetrahydropteridine - HOH + u-3,4-dihydroxyphenylalanine + dihydropteridine; (2) 1L- phenylalanine + ©; + tetrahydropteridine — HOH + t- tyrosine + dihydropteridine. COOH NH, -¢H COOH NH, -¢H Proc. Nat. Acad. Sci. USA 71 (1974) with 5% fetal-calf serum, maintained in stationary phase for 5-10 days, then harvested, homogenized, and assayed for tyrosine hydroxylase by a modification of the methods of Nagatsu, Levitt, and Udenfriend (10) and Shiman, Akino, and Kaufman (8) as described (5, 11). Product identification was performed as follows. Reactions contained 0.25 mM N-(3-hydroxy-benzyl)-N-methylhydra- zine (Smith and Nephew Ltd.), an inhibitor of aromatic acid decarboxylase (EC 4.1.1.28) in addition to the usual compo- nents. Reactions were deproteinized and neutralized by the method of Udenfriend and Zaltzman-Nirenberg (12). Samples then were passed through alumina oxide columns as described by Nagatsu, Levitt, and Udenfriend (10), and the tritiated reaction product was characterized by thin-layer and paper chromatography using four solvent systems (13), as listed in the legend to Table 3. RESULTS The Effect of Tyrosine Deprivation on Cell Growth. In this study we have examined the question “Is the growth of neuro- blastoma cells with and without tyrosine hydroxylase de- pendent upon added tyrosine?’ The ability of clones N-18 and N-115, without and with tyrosine hydroxylase, respec- tively, to multiply in complete medium containing both tyro- sine and phenylalanine, in medium lacking tyrosine, and in medium lacking both tyrosine and phenylalanine, is shown in Fig. 2. N-18 cells grew well in complete medium, but not in medium deficient in one or both amino acids. In the absence of tyrosine, 50% of these cells died within 8 days. However, N-115 cells with high tyrosine hydroxylase activity [980 pmoles of dopa formed per min/mg of protein (5)] did multi- ply in medium lacking tyrosine after a lag period. Cell survival was dependent upon phenylalanine, but not upon tyrosine. An N-115 subline obtained by selection in the absence of tyrosine grew well both in the presence and absence of tyro- sine with population generation times of 24 and 54 hr, re- spectively. Again, cells did not survive in the absence of both phenylalanine and tyrosine. The cloning efficiencies of different cell lines in the presence and absence of tyrosine are shown in Table 1. Neuroblastoma tumor C1300 contains at least three cell types with respect to transmitter synthesis: adrenergic cells, cholinergic cells, and cells with no known transmitter (5). The adrenergic line, N-115, formed colonies with almost equal efficiency in media with or without tyrosine. In contrast, no colonies were ob- served in medium lacking tyrosine with both a cholinergic clone, NS-20, with high choline acetyltransferase (EC 2.3.1.6), but no tyrosine hydroxylase activity, as well as clone N-18, which lacks both enzyme activities. The cloning efficiency of an uncloned line of C1300 with little or no tyrosine hydroxylase activity was 16% in the pres- ence of tyrosine. In the absence of tyrosine approximately 1 in 70,000 cells gave rise to a colony. Two other clonal lines, rat glioma C-6 and L-cell clone B82, also did not form colonies in the absence of tyrosine. When higher concentrations of L-cells were cultivated with neuroblastoma N-115 cells with high tyrosine hydroxyl- ase activity, many colonies were found that contained both neuroblastoma and L-cells; no colonies were found that con- tained only L-cells. These results suggest that I-cells obtain sufficient tyrosine for growth from the neuroblastoma cells when the two cell types are in close proximity. Selection for Neuroblastoma Cells 2531   A. CLONE NIB || B CLONE Nis |[C.” NIS SUBLINE NO TYR HYDROXYLASE | | HIGH TYR HYDROXYLASE | | OBTAINED BY SELECTION 50.0F ‘ af dae COMPLETE 10° CELLS /CM* o- gs 8 wa oO Oo oO           Fae ae es oO 24 6 8         o 2 4 6 8 DAYS Fic, 2. Growth of neuroblastoma cells in the presence and absence of tyrosine. The growth of neuroblastoma cells with and without tyrosine hydroxylase was determined in the following media: (O) F-14 with tyrosine and phenylalanine (complete); (A) F-14 minus tyrosine with 0.12 mM phenylalanine; and (0) F-14 minus tyrosine and phenylalanine, All media contained 5% dialyzed fetal-calf serum. In panel A the growth of neuroblas- toma clone N-18 is shown; in panel B, clone N-115 with high tyrosine hydroxylase activity; and in panel C a subline of clone N-115 (N-T45) derived by selection in medium without tyrosine. Cells were grown in 60-mm Falcon petri dishes (20 em?) and dissociated and counted as described in Methods. Characterization of Neuroblastoma Cells Selected by Tyrosine Deprivation. Colonies obtained from the uncloned C1300 cell line by selection in medium without tyrosine were further propagated in modified Eagle’s medium in the presence of tyrosine. Homogenates were prepared from cells grown to stationary phase and assayed for tyrosine hydroxylase activ- ity. Ten out of the 21 cell lines obtained had tyrosine hy- droxylase activity (Table 2). The specific activity of the enzyme varied considerably, as shown. Homogenates of four of the cell lines and a homogenate prepared from N-115 were used for product characterization. Taste l. The effect of tyrosine on cell cloning efficiency     . % of cells Tyrosine forming colonies hydrox- ——_________ ylase + - Cell lines activity Tyrosine Tyrosine Neuroblastoma C-1300 clones Adrenergic N-115 + 5 1.4 Cholinergic NS-20 - 9 <0.0004 No transmitter N-18 - 16 <0. 0004 Uneloned neuroblastoma - 16 0.0014 Glioma C-6 - 15 <0. 0002 L-cells B82 ad 34 <0.0001   The cloning efficiencies of the following cell lines were determined in F-14 with and without tyrosine plus 5% dialyzed fetal-calf serum: neuroblastoma clone N-115 with high tyrosine hydrox- ylase activity; clone NS-20, with choline acetyltransferase but no tyrosine hydroxylase activity; and clone N-18 with neither enzyme; as well as an uncloned line derived from neuroblastoma C1300, glioma clone C6, and L-cell clone B82. In most cases 100-mm petri dishes were inoculated in quintuplicate with 10*, 105, 104, 10%, and 10? cells. Colonies were scored after approxi- mately 30 days of incubation. Duplicate experiments were per- formed. 2532 Cell Biology: Breakefield and Nirenberg Tasie 2. Tyrosine hydroxylase activity of neuroblastoma lines selected without tyrosine   pmol of *H* released from (*H]tyrosine/min   Cell lines per mg of protein N-TD6 324 N-T4 162 N-T7 101 N-TN10 74 N-T13 65 N-TN1 63 N-T1 42 N-T12 34 N-TD4 . 33 N-T6 30 N-T16, N-TD2, N-T8, N-T14, N-T15, N-T16, N-TN2, N-TN12, N-TN16, N-TD7, N-TD10 <10   An uncloned line of neuroblastoma C-1300 with negligible tyrosine hydroxylase activity (<10 pmoles of *Ht+ released from [?H]tyrosine per min/mg of protein) was grown in F-14 medium without tyrosine for 30 days (see Table 1). Well-isolated colonies were picked, grown, and assayed for tyrosine hydroxylase as described in Methods. Tyrosine hydroxylase reactions were supplemented with 0.25 mM N-(3-hydroxybenzyl)-N-methylhydrazine, an in- hibitor of aromatic amino-acid decarboxylase. Reaction prod- ucts were eluted from alumina oxide columns and character- ized by thin-layer and paper chromatography with four solvent systems (Table 3). The major reaction product was identical in chromatographic mobility to authentic dopa. Tasiu 3. Chromatographic characterization of the ‘H-labeled product of the tyrosine hydroxylase reaction   Solvent systems A B Cc D % of the *H-labeled product       Cell lines with the mobility of dopa N-TD6 95 89 95 89 N-T4 96 85 97 85 N-T7 95 79 94 91 N-T1 94 87 95 89 N-115 99 95 95 93   Tyrosine hydroxylase reactions were performed as described in Methods. Tritiated catechol products were characterized by thin- layer chromatography with the following solvent systems: solvent A, 1-butanol-concentrated acetic acid-water (12:3:5); solvent B, methylethylketone-concentrated formic acid—water (24:1:6); sowent C, ethylacetate-concentrated acetic acid—water (15:- 15:10); solvent D, 1-butanol-1 N acetic acid-absolute ethanol (35:10:10). Solvent A was used with Whatman No. | filter paper; solvents B, C, and D were used with thin-layer cellulose plates (Eastman 6064). The Rr values of authentic dopa were similar to those reported previously. One hundred percent corresponds to the following dpm recovered after chromatography: 6060, N-TD6; 7860, N-T4; 7550, N-T7; 9590, N-T1; and 17,200, N-115. The numbers in the table correspond to the percent of tritiated product recovered with the chromatographic mobility of authentic dopa. Proc. Nat. Acad. Sct. USA 71 (1974) In addition, several unidentified radioactive products were found, which represented <10% of the tritiated reaction products, In other experiments performed with Tom Lloyd, 3-iodotyrosine and antiserum prepared against purified bovine adrenal medulla tyrosine hydroxylase (14) were found to inhibit the tyrosine hydroxylase activity of N-TD6 homog- enates in a dose-dependent manner. These results, together with studies reported previously (5), strongly suggest that dopa synthesis is catalyzed by tyrosine hydroxylase. Some, but not all, of the cell lines obtained in the absence of tyrosine showed catecholamine histofluorescence induced by formaldehyde, suggesting that catecholamines are both syn- thesized and stored (David Jacobowitz, paper in preparation). A small proportion of cells from the C1300 neuroblastoma tumor grown 7 vive (15) and in vitre (16, 17) have been shown to exhibit catecholamine fluorescence. DISCUSSION The results show that neuroblastoma cell lines differentiated with respect to adrenergic neurotransmitter synthesis can be selected on the basis of their ability to grow in medium lacking tyrosine. Clones derived from various cell types, including glia and fibroblasts, were tested, but multiplication in the absence of tyrosine was found only in neuroblastoma cells with tyrosine hydroxylase activity. When uncloned popula- tions of neuroblastoma cells were selected by this procedure, approximately one out of 7 X 10‘ cells gave rise to a cell line that multiplied well without tyrosine. Approximately 50% of the cell lines thus obtained had tyrosine hydroxylase activity. Shiman, Akino, and Kaufman have shown that tyrosine hydroxylase preparations catalyze the synthesis of tyrosine and its conversion to dopa at similar rates in the presence of tetrahydrobiopterin, the in vivo cofactor (3). Studies of brain metabolism in vivo (19, 20) and in synaptosome preparations (21) suggest that phenylalanine is converted to tyrosine in the normal nervous system. The ability of tyrosine hydroxylase to catalyze tyrosine synthesis may be advantageous since adren- ergic neurons thus can generate the substrate needed for transmitter synthesis by an independent endogenous route. The observation that adrenergic neuroblastoma cells support the growth of neighboring cells in the absence of tyrosine suggests that tyrosine is released by neuroblastoma cells and has a tropic effect upon L cells which determines the relative positions of these two cell types. The interaction of certain cells in the normal nervous system may be determined in a similar manner. It should be possible to select dividing, as well as nondivid- ing, cells on the basis of their ability to multiply or survive in the absence of tyrosine. In addition, the selection procedure coupled with somatic cell hybridization affords a means of generating and selecting different neural phenotypes on the basis of their specificity in encoding neural information. Ultimately, it may be possible to obtain five classes of cells with respect to putative neurotransmitters, those that syn- thesize dopamine, norepinephrine, epinephrine, serotonin, or melatonin, since tryptophan hydroxylase also catalyzes the formation of tyrosine from phenylalanine (21). E. Held- man in our laboratory has shown that one of the cell lines, N-T16, that grows in the absence of tyrosine and does not have tyrosine hydroxylase activity, does synthesize sero- tonin (unpublished observations). Proc. Nat. Acad. Set. USA 71 (1974) We gratefully acknowledge the help of Elliot Richelson, Perola Nirenberg, John Minna, and Tom Lloyd. 1, 2. Eagle, H. (1955) J. Biol. Chem. 214, 839-852. Ikeda, M., Levitt, M. & Udenfriend, 8. (1965) Biochem. Biophys. Res. Commun. 18, 482-488. . Shiman, R., Akino, M. & Kaufman, §, (1971) J. Biol. Chem. 246, 1330-1340. Kaufman, S. (1962) in Methods in Enzymology, eds. Colowick 8. P. & Kaplan, N. D. (Academic Press, New York and London), Vol. 5, pp. 809-816. Amano, T., Richelson, E. & Nirenberg, M. (1972) Proc. Nat. Acad. Sci, USA 69, 258~263. Benda, P., Lightbody, J., Sato, G., Levine, L. & Sweet, W. (1968) Sczence 161, 370-371. Littlefield, J. W. (1966) Exp. Cell Res. 41, 190-196. Vogel, Z., Sytkowski, A. J. & Nirenberg, M. W. (1972) Proc. Nat. Acad. Sci. USA 69, 3180-3184. Blume, A., Gilbert, F., Wilson, §., Farber, J., Rosenberg, R. & Nirenberg, M. (1970) Proc. Nat. Acad. Sci. USA 67, 786- 792. . Nagatsu, T., Levitt, M. & Udenfriend, S. (1964) J. Biel. Chem, 239, 2910-2917. 11. 12, 13. 14. 15. 16. 17. 18. 19, 20. 21. Selection for Neuroblastoma Cells 2533 Schrier, B. K., Wilson, 8. H. & Nirenberg, M. (1974) in Methods in Enzymology, eds. Fleischere, S., Packer, L. & Estabrook, R. W. (Academic Press, New York and London), in press. Udenfriend, 8, & Zaltzman-Nirenberg, P. (1964) Life Sct. 3, 695-702. Richelson, E. & Nirenberg, M. (1974) in Methods in Enzy- mology, eds. Fleischere, S., Packer, L., & Estabrook, R. W. (Academic Press, New York and London), in press. Lloyd, T. & Kaufman, 8. (1973) Mol. Pharmacol. 9, 438-444, Anagnoste, B., Freedman, L. S., Goldstein, M., Broome, J. & Fuxe, K. (1972) Proc. Nat. Acad. Sci. USA 69, 1883-1886. DeLellis, R. A., Rabson, A. S. & Albert, D. (1970) J. Histo- chem. Cytochem. 18, 913-914. Hermeter, J. C., Ciesielski-Treska, J. & Mandel, P. (1972) J. Histochem. Cytochem. 20, 137-138. Bagchi, 8. P. & Zarycki, B. P. (1970) Life Sci. 9, 111-119. Bagchi, S. P. & Zarycki, E. P. (1973) Biochem. Pharmacol. 22, 1353-1368. Karobath, M. & Baldessarini, R. J. (1972) Nature New Biol. 236, 206-208, Jequier, E., Robinson, D, 8., Lovenberg, W. & Sjoerdsma, A, (1969) Biochem. Pharmacol. 18, 1071-1081.", "Nirenberg, Marshall W. ; Breakefield, Xandra O.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8cbk_dpge~eve3", "00000000-0000-0000-62BD-5CEE30C588DE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Acetylcholine Receptor-Mediated Regulation of Adenylate Cyclase in Hybrid Cells\"", "101584910X226", "101584910X222", "1976", "September 1976", "Summary of work on this project as indicated on the report: \"The activity of adenylate cyclase in neuroblastoma x glioma hybrid NG108-15 cells is regulated by the interaction of acetylcholine and its analogues with the muscarinic acetylcholine receptors of the cells.  Addition of the acetylcholine analogue carbamylcholine inhibits adenylate cyclase activity.  However, growth of cells with carbachol results in a prolonged increase in adenylate cyclase activity.  Adenylate cyclase activity increases slowly and after 18-24 hours is 1.5-3 times higher than control values.  Intracellular cAMP is also 1.5-3 -fold times higher in carbachol-grown cells than in controls.  These changes are maintained for at least three days in the presence of carbachol, but the activity returns to control values if carbachol is removed.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00016-01 LBG Period Covered: July 1, 1975 through June 30, 1976 Title of Project: Acetylcholine Receptor-Mediated Regulation of Adenylate Cyclase in Hybrid Cells Names, Laboratory and Institute Affiliations, and Titles of principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, Lab. of Biochem. Genetics, LBG NHLI Neil M. Nathanson, Guest Worker, LBG NHLI Cooperating Units (if any): Muscular Dystrophy Association Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20014 Total Man Years: 1.05 Professional: 1.05 Summary of Work:  The activity of adenylate cyclase in neuroblastoma x glioma hybrid NG108-15 cells is regulated by the interaction of acetylcholine and its analogues with the muscarinic acetylcholine receptors of the cells.  Addition of the acetylcholine analogue carbamylcholine inhibits adenylate cyclase activity.  However, growth of cells with carbachol results in a prolonged increase in adenylate cyclase activity.  Adenylate cyclase activity increases slowly and after 18-24 hours is 1.5-3 times higher than control values.  Intracellular cAMP is also 1.5-3-fold higher in carbachol-grown cells than in controls. These changes are maintained for at least three days in the presence of carbachol, but the activity returns to control values if carbachol is removed. Project Description: Objectives:  The objective of this project is to study muscarinic acetylcholine-receptor mediated regulation of adenylate cyclase activity, in an attempt to understand how neurotransmitter-receptor interactions modulate synaptic transmission. Major Findings:  The activity of adenylate cyclase in neuroblastoma x glioma hybrid NG108-15 cells is regulated by the interaction of acetylcholine and its analogues with the muscarinic acetylcholine receptors of the cells.  Addition of the acetylcholine analogue carbamylcholine inhibits adenylate cyclase activity.  However, growth of cells with carbachol-results in a prolonged increase in adenylate cyclase activity.  Adenylate cyclase activity increases slowly and after 18-24 hours is 1.5-3 times higher than control values.  Intracellular cAMP is also 1.5-3-fold higher in carbachol-grown cells than in controls.  These changes are maintained for at least three days in the presence of carbachol, but the activity returns to control values if carbachol is removed. Significance to Biomedical Research:  The results obtained suggest that neurotransmitter-receptor interactions can exert long-lived effects on macro-molecules required for synaptic transmission. Proposed Course:  Work on this project will be incorporated into other projects in the coming year.", "Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Nathanson, Neil M.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tw8w_bban_dwkr", "00000000-0000-0000-6D1E-38E1B2969597", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Acetylcholine Receptors in the Developing Nervous System\"", "101584910X227", "101584910X222", "1976", "September 1976", "Summary of work for this project as indicated on the report: \"The goal of this project is to define the properties of muscarinic and nicotinic acetylcholine receptors of chick embryo retina before and after synaptogenesis in the retina.  Thus far we have elucidated (1) the specificity and affinities of muscarinic and nicotinic acetylcholine receptors for cholinergic agonists and antagonists, (2) the number of receptors were defined as a function of developmental age of the retina, and (3) the location of nicotinic receptors within the retina was determined.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00017-01 LBG Period Covered: July 1, 1975 through June 30, 1976 Title of Project: Acetylcholine Receptors in the Developing Nervous System Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, LBG, LBG NHLI Hiroyuki Sugiyama, Research Associate, LBG NHLI Cooperating Units (if any): None Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20014 Total Man Years: 1.6 Professional: 1.3 Other: .3 Summary of Work:  The goal of this project is to define the properties of muscarinic and nicotinic acetylcholine receptors of chick embryo retina before and after synaptogenesis in the retina.  Thus far we have elucidated (1) the specificity and affinities of muscarinic and nicotinic acetylcholine receptors for cholinergic agonists and antagonists, (2) the number of receptors were defined as a function of developmental age of the retina, and (3) the location of nicotinic receptors within the retina was determined. Project Description: Objectives:  The objectives are to define the biochemical properties of acetylcholine receptors before and after synaptogenesis in the retina. Major Findings:  Neurons dissociated from chick embryo retina and maintained in vitro were found to reaggregate and form, in vitro, approximately 1 x 10^9 synapses per mg of protein.  Three types of synapses and several subtypes were identified which closely resemble those of the intact retina. Chick embryo retina was found to be a rich source of both muscarinic and nicotinic acetylcholine receptors.  Both muscarinic and nicotinic acetylcholine receptors are synthesized before synapses appear in the retina; however, during development, nicotinic acetylcholine receptors become associated predominantly with neurites in the synaptic layers of the retina.  The properties of muscarinic acetylcholine receptors were determined at different developmental ages and were compared with the properties of muscarinic inhibitory and excitatory receptors of neuroblastoma cells. Significance to Biomedical Research:  This study gives fundamental information and serves as a basis for further studies on the role of this receptor protein in synapse formation. Proposed Course:  More detailed studies of synaptogenesis and receptor properties are planned. Publications: 1.  Vogel, Zvi and Nirenberg, Marshall:  Localization of acetylcholine receptors during synaptogenesis in retina. Proc. Natl. Acad. Sci. USA, In Press. 2.  Vogel, Zvi, Daniels, Mathew P. and Nirenberg, Marshall:  Synapse and acetylcholine receptor synthesis by neurons dissociated from retina. Proc. Natl. Acad. Sci., USA, In Press. 3.  Gouras, P., Chader, G., Enrigues, N. and Gibbons, R.G.: Calcium-induced spikes in cultures pigment epithelium of chick retina.  Invest. Ophthalmol. 15: 62-64, 1976.", "Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Sugiyama, Hiroyuki", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-w4f6.zupw_dydn", "00000000-0000-0000-4BEB-B14CFADE2081", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Acetylcholine Receptors\"", "101584910X228", "101584910X222", "1976", "September 1976", "Summary of work on this project as indicated on the report: \"Our aim is to study the distribution of nicotinic acetylcholine receptors in intact and cultured tissues of the peripheral and central nervous system in relationship to the development and function of synapses.  To this purpose histochemical localization of alpha-bungarotoxin bound to the receptors is used in conjunction with light and electron microscopy.  In the past year we have studied the ultrastructural distribution of receptors on cultured skeletal muscle fibers and have initiated the following investigations: 1) location and characterization of synapses formed by neuroblastoma hybrid cells in culture 2) modification of histochemical methods in order to permit ultrastructural analysis of receptor distribution in the central nervous system and 3) analysis of the distribution of receptors in the visual system of the goldfish with relationship to optic nerve damage and regeneration.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00001-05 LBG Period Covered: July 1, 1975 through June 30, 1976 Title of Project: Acetylcholine Receptors Names, Laboratory and Institute Affiliations, and Titles of Principal Investigations and All Other Professional Personnel Engaged on the Project: PI: Mathew P. Daniels, Staff Fellow, LBG NHLI OTHER: Marshall W. Nirenberg, Chief, Lab of Biochem. Genetics, LBG NHLI P. Nelson, Chief, Behavioral Biology Branch, LBG NHLI C. Christian, Special Fellow, BB NICHD G. Maloney, NIH Postdoctoral Fellow, LBG NHLI Zvi Vogel, Assistant Professor, Weizmann Institute Cooperating Units (if any): Behavioral Biology Branch, NICHD Neurobiology Unit, Weizmann Institute of Science Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20014 Total Man Years: 2.0 Professional: 1.5 Other: 0.5 Summary of Work: Our aim is to study the distribution of nicotinic acetylcholine receptors in intact and cultured tissues of the peripheral and central nervous system in relationship to the development and function of synapses.  To this purpose histochemical localization of a-bungarotoxin bound to the receptors is used in conjunction with light and electron microscopy.  In the past year we have studied the ultrastructural distribution of receptors on cultured skeletal muscle fibers and have initiated the following investigations:  1) location and characterization of synapses formed by neuroblastoma hybrid cells in culture 2) modification of histochemical methods in order to permit ultrastructural analysis of receptor distribution in the central nervous system and 3) analysis of the distribution of receptors in the visual system of the goldfish with relationship to optic nerve damage and regeneration. Project Description: Objectives:  Investigators in this laboratory and others have utilized 125I labeled alpha-bungarotoxin (alphaBT) as a label for nicotinic acetylcholine receptors in intact and cultured skeletal muscle, and in embryonic and mature retina.  The objectives of this study were to devise a histochemical technique of greater sensitivity and resolution for localizing bound alphaBT and to apply this technique to studying the ultrastructural distribution of acetylcholine receptors in the peripheral and central nervous system during development, in culture, and in the mature state. Methods Employed:  We have employed indirect immunoperoxidase staining of cryostat sectioned, teased, or monolayered cultured materials to which alphaBT has been bound.  These materials are subsequently examined by light and electron microscopy. Major Findings:  Acetylcholine receptor-rich regions on the surface of muscle fibers grown in culture had previously been observed by light microscope autoradiography with [125I]-labeled alphaBT.  The appearance of these regions could be explained either by (1) the localized presence of complex folds in the plasma membrane or (2) a high local concentration of receptors in the plasma membrane, unrelated to membrane folding.  Using the alphaBT-iummunoperoxidase technique with light- and electronmicroscopy we have shown that hypothesis 2 is correct; the plasma membranes of these regions contain at least 7 times the concentration of receptors found in other regions, with no distinctions in cell surface topography. Significance to Biomedical Research:  Knowledge of ultrastructural distribution of acetylcholine receptor is of clear importance in any attempt to understand the role of neurotransmitters and their receptors in the function and development of the nervous system.  The alpha-bungarotoxin-immunoperoxidase technique already has shown promise for the diagnosis and analysis of mechanisms in human neuromuscular disorders. Proposed Course:  (1) We are using the alphaBT-immunoperoxidase technique to help locate and characterize the ultrastructure of synapses which have been detected electrophysiologically in cultures of neuroblastoma hybrid cells with skeletal muscle fibers.  (2) We are developing new reagents to adapt the histochemical technique to ultrastrucutral visualization of acetylcholine receptor sites in mature and developing central nervous system tissues.  (3) We plan to study the distribution of acetylcholine receptors in the visual system of the goldfish with relationship to the destruction and reformation of synapses during optic nerve degeneration and regeneration. Publications: 1.  Ringel, S. P., Bender, A. N., Festoff, B. W., Engel, W. K., Vogel, Z. and Daniels, M. P.:  Ultrastructural demonstration and analytical application of extrajunctional receptors of denervated human and rat skeletal muscle fibres.  Nature 255:  730-731, 1975. 2.  Vogel, Z. and Daniels, M. P.:  The ultrastructure of acetylcholine receptor clusters on cultured muscle fibers.  J. Cell Biol. 69:  in press. 3.  Vogel, Z. and Daniels, M. P.:  The acetylcholine receptor of intact and cultured chicken retina cells.  Proc. VI Int. Cong. Pharmacol. Vol. 1 Receptors and Cellular Pharmacology.  Pergamon Press, New York, 1976, pp. 59-66.", "Daniels, Mathew P. ; Vogel, Zvi ; Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Nelson, Phillip G. ; Maloney, G. ; Christian, Clifford", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-t2kw.nt9e.hyd5", "00000000-0000-0000-3908-D3D5A3D27B5C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Morphine Receptors as Regulators of Adenylate Cyclase\"", "101584910X229", "101584910X222", "1976", "September 1976", "Summary of work for this project as indicated on the report: \"The objectives are to elucidate the mechanisms of dependence upon opiates and of tolerance to these compounds to define the normal functions of the newly discovered endogenous opiate peptides.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00002-03 LBC Period Covered: July 1, 1975 through June 30, 1976 Title of Project: Morphine Receptors as Regulators of Adenylate Cyclase Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, Lab. of Biochemical Genetics, LBG NHLI OTHER: Arthur Lampert, Staff Fellow, LBG NHLI Werner Klee, Research Chemist, LGCB NIMH Cooperating Units (if any): Laboratory of General and Comparative Biochemistry, NIMH Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20014 Total Man Years: 1.2 Professional: 1.2 Summary of Work:  The objectives are to elucidate the mechanisms of dependence upon opiates and of tolerance to these compounds and to define the normal functions of the newly discovered endogenous opiate peptides. Project Description: Major Findings:  Clonal cell lines with morphine receptors were found and were used to study the mechanism of action of narcotics.  Morphine and other narcotics were found to affect adenylate cyclase in two ways, mediated by the opiate receptor:  (1) narcotics inhibit adenylate cyclase activity, and (2) when cells are cultured in the presence of morphine for 12 to 48 hours an increase in adenylate cyclase activity is observed which compensates for the inhibition of enzyme activity by morphine.  Cells then have normal cAMP levels and appear tolerant to morphine because the increase in adenylate cyclase activity is approximately equal to the inhibition of enzyme activity by morphine.  However, the cells then are dependent upon morphine to maintain normal cAMP levels.  Withdrawal of morphine, or displacement of the narcotic from the opiate receptor by the antagonist, naloxone, reverses the inhibition and results in the synthesis of abnormally high levels of cAMP.  Thus, dual regulation of adenylate cyclase by narcotics accounts for narcotic dependence and tolerance.  The recently discovered endogenous opiate peptides, Met-enkephalin and Leu-enkephalin, also were shown to be potent inhibitors of adenylate cyclase.  These results show that the endogenous opiate peptides and narcotics act as pleiotropic regulators of other species of receptors which are coupled to the activation of adenylate cyclase.  In this way, the opiates alter the perception of neurons to incoming messages. Significance to Biomedical Research:  The biochemical basis for narcotic dependence and tolerance has been established and the mode of action and the normal role of the endogenous opiate peptides have been clarified. Proposed Course:  Further studies on the mechanism of dual regulation of adenylate cyclase are in progress. Publications: 1.  Sharma, Shail K., Klee, Werner A. and Nirenberg, Marshall: Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance.  Proc. Natl. Acad. Sci. USA 72: 3092-3096, 1975.", "Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Lampert, Arthur ; Klee, Werner A.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xuih~3pn6_mwhz", "00000000-0000-0000-E003-B522ED1A40A9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Storage and Release of Molecules Required for Synaptic Communication\"", "101584910X230", "101584910X222", "1976", "September 1976", "Summary of work on this project as indicated on the report: \"The objectives are to devise biochemical assays for neurotransmitter release from neuroblastoma and hybrid cell lines and then to define the steps which are required for neurotransmitter storage and release and factors which regulate these reactions.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "SMITHSONIAN SCIENCE INFORMATION EXCHANGE U.S. DEPARTMENT OF PROJECT NUNBER PROJECT NUMBER (Do NOT use this space) [HEALTH, EDUCATION. AND WELFARE PoeLic Heatth. Service } INTRAMURAL RESEARCH PROJECT Z01 HL 00014-01 LBG       PERIOD COVERED July 1, 1975 through June 30, 1976   TITLE OF PROJECT (80 characters or less) Storage and Release of Molecules Required for Synaptic Communication   NAMES, LABORATORY ANO INSTITUTE AFFILRATIONS, ANO TITLES OF PRINGIPAL INVESTIGATORS AND ALL OTHER PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT PI: Marshall Nirenberg Chief, Lab. of Biochem. Genetics LBG NHLI OTHER: Richard McGee Staff Fellow LBG NHLI Saburo Ayukawa Visiting Fellow LBG NHLI ‘Clifford Christian Special Fellow BB NICHD Phillip Nelson Chief, Behavioral Biology Branch BB NICHD   COOPERATING UNITS (if any) Behavioral Biology Branch, NICHD   LAB/ BRANCH . Laboratory of Biochemical Genetics   SECTION Section on Molecular Biology   INSTITUTE AND LOCATION NHLI, NIH, Bethesda, Maryland 20014   TOTAL MANYEARS: PROFESSIONAL: OTHER: 2.2 2.2       SUMMARY OF WORK (200 words or less — underline keywords) The objectives are to devise biochemical assays for neurotransmitter release from neuroblastoma and hybrid cell lines and then to define the steps which are required for neurotransmitter storage and release and factors which regulate these reactions.     PHS-6040 (12-75)   Z01 HL 00014-01 LBG Project Description: Major Findings: The results show that the uptake of 34-choline into neuroblastoma x glioma hybrid cells, the rate of acetylcholine synthesis and the storage of acetylcholine are regulated by the conditions of cell growth. The results suggest that the evoked release 6f H-acetylcholine can be ob- tained but further work is needed to clarify the release process products. The results also showed the presence of a dopamine storage mechanism in some cell lines. Proposed Course: When the assays for transmitter release have been validated, they will be used to determine the reactions which are required for transmitter release and to determine whether the steps are regulated. Publications: 1. Breakefield, Xandra 0., Neale, Elaine A., Neale, Joseph H. and Jacobowitz, David M.: Localized catecholamine storage associated with granules in murine neuroblastoma cells. Brain Res. 92: 237-256, 1975. 2. Rotman, Avner, Daly, John W., Creveling, Cyrus R. and Breakefield, Xandra O.: Uptake and Binding of dopamine and 6—-hydroxydopamine in murine neuro- blastoma and fibroblast cells. Biochem. Pharmacol: 25: 383-388, 1976.", "Nirenberg, Marshall W. ; McGee, Richard ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Ayukawa, Saburo ; Nelson, Phillip G. ; Christian, Clifford", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fnk5.9zk4.vyc4", "00000000-0000-0000-0813-8E37C3AA0074", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Regulation of Cyclic Nucleotide Biosynthesis by Neurotransmitters and Opiates\"", "101584910X231", "101584910X222", "1976", "September 1976", "Summary of work on this project as indicated on the report: \"The role of the cyclic nucleotides adenosine 3'5' monophosphate and guanosine 3'5' monophosphate in synaptic transmission is under study using cultured cells of neutral origin.  The topics of interest during the current year have been the following: 1) The receptor-mediated inhibition of adenylate cyclase activity by alpha-adrenergic agents in neuroblastoma x glioma hybrid cells; 2) Demonstration of a compensatory induction of adenylate cyclase activity in cells treated for one or more days with alpha-adrenergic agents, as had been previously demonstrated by others for opiates, and study of the mechanism of this compensatory induction; 3) The effect of opiates and alpha-adrenergic agents on guanosine 3'5' monophosphate levels in the hybrid cells; 4) Search for a cultured-cell system which synthesizes a peptide with opiate-like properties.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00015-01 LBG Period Covered: July 1, 1975 through June 30, 1976 Title of Project: Regulation of cyclic nucleotide biosynthesis by neurotransmitters and opiates Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, Lab. of Biochem. Genetics, LBG NHLI Steven L. Sabol, Research Associate, LBG NHLI Cooperating Units (if any): None Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20014 Total Man Years: 2.0 Professional: 1.5 Other: .5 Summary of Work:  The role of the cyclic nucleotides adenosine 3'5' monophosphate and guanosine 3'5' monophosphate in synaptic transmission is under study using cultured cells of neural origin. The topics of interest during the current year have been the following:  1) The receptor-mediated inhibition of adenylate cyclase activity by alpha-adrenergic agents in  neuroblastoma x glioma hybrid cells; 2) Demonstration of a compensatory induction of adenylate cyclase activity in cells treated for one or more days with alpha-adrenergic agents, as had been previously demonstrated by others for opiates, and study of the mechanism of this compensatory induction; 3) The effect of opiates and alpha-adrenergic agents on guanosine 3'5' monophosphate levels in the hybrid cells; 4) Search for a cultured-cell system which synthesizes a peptide with opiate-like properties. Project Description: Objectives:  Previous work in this laboratory demonstrated that opiates, alpha-adrenergic agonists, and muscarinic cholinergic agonists lower cyclic AMP (adenosine 3'5' monophosphate) levels by receptor-mediated mechanisms in the neuroblastoma x glioma hybrid cell line NG108-15. Also demonstrated was the inhibition of adenylate cyclase by opiates and a compensatory induction of adenylate cyclase activity (measured in the absence of opiates) in cells grown in the presence of opiates.  One aspect of the present project attempts to demonstrate and analyze similar effects of adrenergic agonists on adenylate cyclase in cell-free systems.  Another aspect is to relate these findings to the action of the enkephalins, recently discovered endogenous opiate-like peptides. Major Findings:  (1) Adrenergic agents, like opiates, inhibit adenylate cyclase activity in NG108-15 homogenates.  The order of agonist potencies is consistent with an alpha receptor, and the effect is blocked by alpha receptor antagonists. (2) Cultivation of NG108-15 with norepinephrine for 1 to 4 days results, as in the case with opiates, in an increase in adenylate cyclase activity which compensates for the inhibition of this enzyme by norepinephrine.  Cells then appear tolerant to norepinephrine, but are dependent upon norepinephrine for maintenance of normal cAMP levels.  Withdrawal of norepinephrine or the addition of an alpha-adrenergic antagonist results in an increase in cellular cyclic AMP levels to abnormally high values. Significance to Biomedical Research:  The results show that cells exposed to alpha-receptor activators for several days become tolerant to and dependent upon these compounds and that tolerance and dependence are established by the mechanism of dual regulation of adenylate cyclase. Proposed Course:  It is intended to continue the investigation of the induction of adenylate cyclase activity by cyclase inhibitors, including the possible mediation of cyclase activators.  Also the relationship among the opiate, adrenergic and cholinergic receptors in NG108-15 should be studied.  Finally a search for endogenous \"endorphin\" synthesis in cell lines of neural origin is being initiated, with the goal of developing a tissue culture system for the study of the biosynthesis of this peptide or peptides.", "Sabol, Steven L. ; Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-te2n.qxk6_gqsb", "00000000-0000-0000-538E-C33094C28F0D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Regulation of Receptor Activity\"", "101584910X232", "101584910X222", "1976", "September 1976", "Summary of work for this project as indicated on the report: \"Evidence for a new type of PGE1 receptor coupled to cGMP accumulation was obtained.  Cell lines with PGE1 receptors coupled only to cAMP were found as well as cell lines with 2 species of PGE1 receptors, one coupled to cAMP accumulation, the other to cGMP accumulation.  The 2 species of PGE1 receptors also desensitize at different rates.  These results show that the coupling of PGE1 to increases in cAMP and cGMP levels are clonally inherited properties which can be expressed independently.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "PROJECT NUMBER (Do NOT use this space) ee pag HEALTH a ico EALTH_SERV TICE INTRAMURAL RESEARCH PROJECT SMITHSON! AN ER (Do INFORMATION EXGHANGE U.S. DEPARTMENT OF PROJECT NUMBER ZO1 HL 00003-03 LBG       PERIOD COVERED July 1, 1975 through June 30, 1976 TITLE OF PROJECT (80 characters or less)   Regulation of Receptor Activity   NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES GF PRINCIPAL INVESTIGATORS AND ALL OTHER PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT PI: Marshall Nirenberg Chief, Lab. of Biochemical oo, _ Genetics LBG NHLI Hiroshi Matsuzawa Visiting Associate LBG NHLI   COOPERATING UNITS (if any) None   LAB/BRANCH Laboratory of Biochemical Genetics SECTION Section on Molecular Biology INSTITUTE AND LOCATION NHLI, NIH, Bethesda, Maryland 20014 TOTAL MANYEARS: PROFESSIONAL: OTHER: 1.3 1 3 SUMMARY OF WORK (200 words or less — underline keywords )             Evidence for a new type of PGE, receptor coupled to cGMP accumulation was obtained. Cell lines with PGE, receptors coupled only to cAMP were found as well as cell lines with 2 species of PGE, receptors, one coupled to cAMP accumulation, the other to cGMP accumulation. Thé 2 species of PGE, receptors also desensitize at different rates. These results show that the coupting of PGE, to increases in cAMP and cGMP levels are clonally inherited properties which can be expressed independently.         PHS-6040 (12-75) Z01 HL 00003-03 LBG . Project Description: Objectives: The objective is to define receptor~mediated responses of clonal cells which can be used as model systems for synapse studies. Major Findings: Evidence for a new type of PGE, receptor coupled to cGMP accumulation was obtained. Cell lines with PGE, receptors coupled only to cAMP were found as cell lines with 2 species of PGE, receptors, one coupled to cAMP accumulation, the other to cGMP accumulation. line 2 species of PGE, receptors also desensitize at different rates. These results show that the coupling of PGE. increases in cAMP and cGMP levels are clonally inherited properties which can be expressed independently. Publications: 1. Matsuzawa, Hiroshi and Nirenberg, Marshall: Receptor-meeiated shifts in cGMP and cAMP levels in neuroblastoma cells. Proc: Natl. Acad: Sci: USA 72: 3472-3476, 1975. ~~   2. Bachrach, Uriel: Cyclic AMP-mediated induction of ornithine decarboxylase of glioma and neuroblastoma cells.’ Proc: Natl: Acad. Sci: 'USA'72: 3087-3091, 1975. a", "Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Matsuzawa, Hiroshi", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-n5a6_bzhx.2bwi", "00000000-0000-0000-D7E4-F4DC6503E01A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Studies of Action Potential and Receptor Ionophores\"", "101584910X233", "101584910X222", "1976", "September 1976", "Summary of work for this project as indicated on the report: \"The objective of this project is to develop biochemical methods for studies of action potential and receptor ionophores leading eventually to isolation of these macromolecules and characterization at both the molecular and cellular levels.  Our current efforts are directed toward developing reagents, mainly neurotoxins, which act on the action potential Na+ ionophore, preparing radioactively labelled (sic) derivatives, and using these reagents to characterize the ionophore at the cellular level and to solubilize and eventually isolate it.  The nicotinic acetylcholine receptor ionophore is also under study at the cellular level.\"", "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00004-03 LBG Period Covered: July 1, 1975 through June 30, 1976 Title of Project: Studies of Action Potential and Receptor Ionophores Names, Laboratory and Institute Affiliations, and Titles of Principal Investigations and All Other Professional Personnel Engaged on the Project: PI: W.A. Catterall, Staff Fellow, LBG NHLI R. Ray, MARC Fellow/NIGMS, LBG NHLI L. M. Huang, NIH Postdoctoral Fellow, LBG NHLI and BP NINCDS Cooperating Units (if any): Laboratory of Biophysics, NINCDS Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20014 Total Man Years: 2.7 Professional: 1.7 Other: 1 Summary of Work: The objective of this project is to develop biochemical methods for studies of action potential and receptor ionophores leading eventually to isolation of these macromolecules and characterization at both the molecular and cellular levels.  Our current efforts are directed toward developing reagents, mainly neurotoxins, which act on the action potential Na+ ionophore, preparing radioactively labeled derivatives, and using these reagents to characterize the ionophore at the cellular level and to solubilize and eventually isolate it.  The nicotinic acetylcholine receptor ionophore is also under study at the cellular level. Project Description: Objectives:  The objectives of this project are (1) to develop biochemical methods for study of action potential and receptor ionophores, (2) to use these methods to study the mechanism of action of these macromolecules at the cellular and membrane levels, and (3) to solubilize, purify, and characterize these ionophores at the molecular level. Methods Employed:  Biochemical assays which measure changes in passive Na+ influx were used to study the acetylcholine receptor ionophore and the action potential Na+ ionophore. Major Findings: Previous results led to the conclusion that (1) the neurotoxic alkaloids veratridine, batrachotoxin, and aconitine activate the action potential Na+  ionophore by interaction with a single class of sites; (2) scorpion venom activates the ionophore by interaction with a different class of sites; (3) the sites of action of these 2 classes of toxin are allosterically coupled in a highly cooperative manner; and (4) the inhibitors tetrodotoxin and saxitoxin act at a separate site directly involved in ion transport by the ionophore. The active component of scorpion venom has been purified using its ability to activate the action potential Na+ ionophore as a specific assay.  The toxin is a polypeptide having a molecular weight of 6700, an isoelectric point of 9.8, and lacking methionine and histidine. The purified toxin retains the ability to act cooperatively with each of the 3 alkaloids. It acts reversibly at a single class of sites with an apparent dissociation constant of 1 to 2 nM.  The action of the toxin is highly membrane potential dependent.  Depolarization of the cells causes a 30 fold increase in apparent dissociation constant.  These results suggest that scorpion toxin binds to a voltage sensitive component of the Na+ ionophore that acts cooperatively in regulating its ion transport activity. We have prepared an 125I-labeled derivative of scorpion toxin which retains biologic activity.  Using this derivative we have detected a small class of saturable binding sites in electrically excitable neuroblastoma cells but not in neuroblastoma cells defective in electrical activity. Binding of scorpion toxin to these sites is voltage dependent as is the effect of the toxin on ion transport activity.  Preliminary estimates of the number of sites are in the range of 3 to 6 fmole/mg cell protein or less than 1 site per mu-m2 of cell membrane.  This labeled toxin derivative appears to provide an important new tool in studies of the Na+ ionophore. Significance to Biomedical Research:  The results provide new insights into the mechanism of action and regulation of membrane macromolecules involved in information transfer and processing in the nervous system and in maintenance of normal beating in heart. Proposed Course:  Planned investigations include (1) completing the analysis of scorpion toxin binding to excitable membranes of neuroblastoma cells, nerve axons, and heart muscle; (2) preparing labeled derivatives of saxitoxin and comparing binding with scorpion toxin; (3) studying the voltage dependent aspects of scorpion toxin binding in detail and relating them to the electrophysiologic properties of the ionophore; and (4) attempting to solubilize and purify the binding sites for scorpion toxin and saxitoxin and thus isolate the action potential Na+ ionophore. Publications: 1. Catterall, W. A.:  Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin.  J. Biol. Chem. 250:  4053-4059, 1975. 2. Catterall, W. A.:  Cooperative activation of the action potential Na+ ionophore by neurotoxins.  Proc. Natl. Acad. Sci. USA 72: 1782-1786, 1975. 3. Catterall, W. A. and Ray, R.:  Interactions of neurotoxins with the action potential Na+ ionophore.  J. Supramolecular Structure, in press. 4. Catterall, W. A.:  Purification of a topic protein from scorpion venom which activates the action potential Na+ ionophore.  J. Biol. Chem., in press. 5. Catterall, W. A., Ray, R. and Morrow, Cynthia S.:  Membrane potential dependent binding of scorpion toxin to the action potential Na+ ionophore. Proc. Natl. Acad. Sci. USA, in press.", "Ray, Radharaman ; National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Huang, L. M. ; Catterall, W. A.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ywys.aeyn_m3t2", "00000000-0000-0000-4ED3-412D3440B266", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics [summary of laboratory projects]", "101584910X234", "101584910X222", "1976", "September 1976", null, "Reports, Excerpts", null, "Neuroblastoma Research, 1967-1976", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Annual Report of the Laboratory of Biochemical Genetics National Heart and Lung Institute July 1, 1975 through June 30, 1976 Fusion of clonal neuroblastoma cells with rat glioma cells yielded clonal hybrid cell lines which synthesize, store and excrete acetylcholine; properties which are not expressed by the parental cell lines.  Cells from one hybrid line were found to form synapses with cultured striated muscle cells. Synapses between hybrid cells and muscle cells closely resemble the synapses between normal motor neurons and striated muscle before they are fully developed.  Under appropriate conditions, hybrid cells establish synaptic connections with virtually every muscle cell tested; thus, synaptic connections are formed in abundance.  Marked differences were observed in the efficiency of transmission across different synapses.  Axonal activities which were found to be regulated include choline acetyltransferase, acetylcholinesterase, Na+ action potential ionophore specific activities, and the rate of choline transport into cells. Eight species of receptors have been found thus far with the hybrid cell line which forms synapses.  Receptor mediated shifts in cAMP levels, cGMP levels and membrane potentials have been identified and characterized.  Thus, the foundation has been laid for studies on the effects of receptor-mediated reactions on synaptic transmission.  In addition, more than 100 cell lines which synthesize acetylcholine have been obtained and are being studied to determine whether some cell lines are defective with respect to synapse formation.  Much remains to be done but it seems clear that the experimental approach and model systems which have been established afford extraordinary opportunities to explore synapse properties and correlate biochemical events with developmental and electrophysiological phenomena. Synaptogenesis by normal neurons also was studied. Neurons dissociated from chick embryo retina and maintained in vitro were found to reaggregate and form in vitro approximately 1 x 10^9 synapses per mg of protein.  Three types of synapses and several subtypes were identified which closely resemble those of the intact retina.  Studies with this system are described in other sections of this report. A histochemical technique for detecting and localizing nicotinic acetylcholine receptors was devised previously which depends upon the formation of a complex between peroxidase coupled to an antibody for alpha-bungarotoxin and the nicotinic acetylcholine receptor.  Using this method, clusters of nicotinic acetylcholine-receptors on cultured muscle cells were shown to contain at least 7 times the concentration of receptors found in other membrane regions.  Receptor clusters are not characteristically associated with folds in the plasma membrane. The hybrid cells which form synapses possess abundant morphine receptors.  Morphine and other narcotics are potent inhibitors of adenylate cyclase in cells which possess opiate receptors but not in cells which lack these receptors.  Exposure of cells with opiate receptors to morphine for 12 to 48 hours results in an increase in adenylate cyclase activity.  Cells have normal cAMP levels and appear tolerant to morphine because the increase in adenylate cyclase activity is approximately equal to the inhibition.  However, the cells then are dependent on morphine to maintain normal cAMP levels.  Withdrawal of morphine, or displacement of the narcotic from the opiate receptor by the antagonist, naloxone, reverses the inhibition and results in the synthesis of abnormally high levels of cAMP.  Thus, dual regulation of adenylate cyclase by narcotics accounts for the phenomena of narcotic dependence and tolerance.  The recently discovered endogenous opiate peptides, Met-enkephalin and Leu-enkephalin, also were shown to be potent inhibitors of adenylate cyclase.  These results show that the endogenous opiate peptides and narcotics act as pleiotropic regulators of other species of receptors which are coupled to the activation of adenylate cyclase.  By this mechanism opiates alter the perception of neurons to incoming messages. A cell line with muscarinic inhibitory acetylcholine receptors, and another line with muscarinic excitatory receptors were found.  The number of receptors, the receptor affinities for cholinergic ligands and other receptor properties were determined by measuring the binding of [3H]-quinuclidinyl benzilate to receptors.  The apparent dissociation constant of the ligand receptor complex is 6 x 10^(-11)M.  Activation of muscarinic acetylcholine receptors results in a transient increase in cGMP, and a profound, long-lived inhibition of adenylatc cyclase activity.  Exposure of cells to acetylcholine or carbachol for 24 hours markedly decreases the number of acetylcholine receptors and increases adenylate cyclase activity 50 to 500%. Removal of carbachol results in the return of adenylate cyclase activity and acetylcholine receptor levels to normal values after approximately 3 and 24 hours, respectively.  Similarly, exposure of cells with alpha-receptors to norepinephrine inhibits adenylate cyclase activity and elicits a delayed, compensatory increase in adenylate cyclase activity.  These results show that prolonged activation of alpha-receptors, muscarinic acetylcholine receptors, or opiate receptors results in cell tolerance to and dependence upon norepinephrine, acetylcholine, or opiates, respectively.  Withdrawal of the receptor activator elevates intracellular cAMP levels and shifts cells to a supersensitive state with respect to other species of receptors which activate adenylate cyclase. Desensitization of muscarinic receptors decreases the affinity of the receptor for agonists by a process which exhibits negative cooperativity; whereas interactions between the receptor and antagonists are not cooperative.  Both muscarinic excitatory and inhibitory acetylcholine receptors were solubilized and the properties of membrane-bound and soluble receptors were compared. Chick embryo retina was found to be a rich source of both muscarinic and nicotinic acetylcholine receptors.  Both muscarinic and nicotinic acetylcholine receptors are synthesized before synapses appear in the retina; however, during development, nicotinic acetylcholine receptors become associated predominantly with neurites in the synaptic layers of the retina. The properties of muscarinic acetylcholine receptors were determined at different developmental ages and were compared with the properties of muscarinic inhibitory and excitatory receptors of neuroblastoma cells. Evidence for a new type of PGE1 receptor coupled to cGMP accumulation was obtained.  Cell lines with PGE1 receptors coupled only to cAMP were found as well as cell lines with 2 species of PGE1 receptors, one coupled to cAMP accumulation, the other to cGMP accumulation.  The 2 species of PGE1 receptors also desensitize at different rates.   These results show that the coupling of PGE1 to increases in cAMP and cGMP levels are clonally inherited properties which can be expressed independently. Two pathways for gamma-aminobutyric acid synthesis were found in chick embryo retina.  The first pathway depends upon the conversion of putrescine to ornithine, catalyzed by ornithine decarboxylase and the subsequent conversion of ornithine to gamma-aminobutyric acid.  The second route of synthesis is dependent upon the conversion of glutamic acid to gamma-aminobutyric acid, catalyzed by glutamic acid decarboxylase.  Elevation of cAMP levels in neuroblastoma cells was shown to induce ornithine decarboxylase activity.  In the developing embryo, neurotransmitters which affect cAMP levels may regulate ornithine decarboxylase activity and thereby control the rate of GABA synthesis from ornithine. Previous results led to the conclusions that (1) veratridine, batrachotoxin, and aconitine activate the action potential Na+ ionophore by interaction with a single class of sites; (2) scorpion venom activates the ionophore by interaction with a different class of sites; (3) two species of toxin bound to separate sites are allosterically coupled and interact in a cooperative manner; and (4) tetrodotoxin and saxitoxin act at a 3rd site which is involved in ion transport. A toxin which activates the action potential Na+ ionophore has been purified from scorpion venom.  The toxin binds to a single class of sites and acts cooperatively with each of the three alkaloids.  Depolarization of cells causes a 30-fold increase in the apparent dissociation constant.  The results suggest that the scorpion toxin binds to a voltage sensitive component of the Na+ ionophore that acts cooperatively in regulating ion transport activity.  Binding studies with an 125I-labeled derivative of scorpion toxin showed that the concentration of toxin binding sites is approximately 3 to 6 fmole per mg protein. Clonal skeletal muscle myoblasts have substantial action potential Na+ ionophore activity.  A small increase in activity accompanies cell fusion.  The activity in both myoblasts and myotubes is relatively insensitive to inhibition by saxitoxin and tetrodotoxin and thus resembles denervated rat striated muscle which has been shown to be relatively insensitive to these toxins.  Chronic electrical stimulation of muscle cells in vitro does not increase tetrodotoxin sensitivity. At least 3 ionophores are involved in the action potential in adult heart: a rapidly activated axon-like Na+ ionophore responsible for the rising phase of the action potential, a slower Ca++/Na+ ionophore responsible for the plateau phase, and a K+ ionophore responsible for the repolarization phase.  Studies with specific inhibitors of the fast Na+  ionophore (tetrodotoxin) and the slow Ca++/Na+ ionophore (D-600) show that the role of these two types of ionophore in beating changes during development of the embryonic chick heart.  In early embryonic hearts, the fast Na+ ionophore is present but is not required for beating.  During development in ovo or in monolayer or aggregate culture in vitro, changes in the requirement for activity of the fast Na+ ionophore in beating are accompanied by changes in the sensitivity of the slow Na+/Ca++     ionophore to D-600.  When the slow Na+/Ca++ ionophore is able to maintain beating without participation of the fast Na+ ionophore, its sensitivity to D-600 is high whereas when the fast Na+ ionophore is required for beating, the slow Na+/Ca++  ionophore is relatively insensitive to D-600.  Transitions between these two states can be induced in 2 hours in vitro by inhibition of beating.  Thus, the activity of these ionophores is regulated during development by a process dependent on the rhythmic activity of the cells. The developmental changes in action potential ionophores of embryonic hearts occur between days 4 and 7 in ovo.  During this time, vagal innervations of the heart takes place.  Consequently we have studied the muscarinic acetylcholine receptors in embryonic hearts using both physiologic and ligand binding methods to determine whether changes in their properties are temporally correlated with changes in the action potential ionophores.  In early embryonic hearts, muscarinic agents are ineffective in inhibiting beating. Between days 5 and 7, sensitivity to inhibition by muscarinic agents increases to the adult level.  QNB binds specifically to muscarinic acetylcholine receptors in embryonic and adult heart as assessed by competition studies with muscarinic and nicotinic agents.  Receptors, as detected by QNB binding, are present in unresponsive early embryonic hearts and the number per mg heart protein does not increase dramatically during development.  Receptor desensitization is accompanied by a small change (3 fold increase) in the KD for muscarinic agonists as measured by competition with 3H QNB.  This change in KD occurs only in hearts that are responsive to muscarinic agents and thus may be associated with the physiologic action of the receptor.  Competition curves for agonists suggest the involvement of negative cooperativity in activation of the receptor.  These results suggest that muscarinic acetylcholine receptors, like action potential ionophores, are present in early embryonic hearts in modified \"precursor\" or \"inactive\" forms and undergo activation during development. Studies on the mechanism of catabolite repression in E. coli show that glucose inhibits adenylate cyclase activity reversibly in cells treated with toluene, and that phosphate is required for high adenylate cyclase activity and for glucose dependent inhibition of enzyme activity.  Other sugars also inhibit adenylate cyclase provided that transport systems for the sugars are induced.  Mutant strains of E. coli defective in phosphoenolpyruvate: sugar phosphotransferase system components were examined.  Cyclic AMP levels were normal in an HPr mutant, but were markedly depressed in a leaky Enzyme I mutant.  Adenylate cyclase activity was low in the Enzyme I mutant whereas the HPr mutant had normal enzyme activity.  The Enzyme I mutant under starvation conditions exhibits high adenylate cyclase activity and the adenylate cyclase of this mutant is unusually sensitive to variations in carbon source.  The addition of phosphoenolpyruvate leads to a substantial increase in adenylate cyclase activity in permeabilized cell preparations of the Enzyme I mutant.  These results suggest that Enzyme I is involved in the regulation of adenylate cyclase activity.  Studies are in progress to test the hypothesis that Enzyme I interacts with adenylate cyclase and that the PEP-dependent phosphorylation of Enzyme I is responsible for activation of adenylate cyclase. Levels of cGMP were compared with cAMP levels in E. coli grown under different conditions.  The results show that cGMP and cAMP concentrations are inversely coupled in E. coli and that the regulation of cGMP levels can be uncoupled from that of cAMP. Since specific species of tRNA are involved in amino acid mediated repression and end-product inhibition, the effect of amino acid deprivation upon tRNA of relaxed and stringent strains of E. coli was studied.  In relaxed control E. coli, leucine starvation results in the formation of new isoacceptor species of leucine, histidine, arginine, valine, and alanine-specific tRNA and quantitative changes in the concentration of some other isoacceptors.  Experiments with stringent strains or the use of uracil starvation or rifampicin addition provided evidence for the de novo synthesis of new species of leucine-tRNA.  The new species of leucine-tRNA is not formed by aggregation of tRNA or nuclease catalyzed hydrolysis, and it is not grossly deficient with respect to methylation.  Since there is some evidence from the recent work of others that tRNA formed under conditions of amino acid starvation is deficient in the minor bases 5,6-dihydrouridine and 4-thiouridine, we think that the biochemical explanation for the accumulation of new tRNA species under conditions of amino-acid starvation may be that the enzymes responsible for these tRNA modifications are unstable and require continued protein synthesis to maintain their levels of activity. Further information was obtained on the mechanism of arginyl-tRNA synthetase which does not catalyze an amino acid dependent ATP-PPi exchange in the absence of added tRNA.  A new purification procedure was devised which yields homogeneous enzyme in approximately 10% yield.  Pulse labeling experiments indicate that no enzyme bound arginyl-adenylate is formed in the absence of added tRNA.  Equilibrium experiments show that no arginyl-adenylate accumulates either in the presence or absence of tRNAarg.  These data further validate our earlier suggestion that the mechanism of this reaction is probably concerted. A series of additional studies carried out with the purified preparation of arginyl-tRNA synthetase from E. coli indicate that metals may have two functional roles in the catalytic mechanism.  Complete metal activation is observed when MgCl2, MnCl2, CoCl2, or FeCl2 is present at a concentration (5 mM) in excess of the total ATP concentration (2 mM).  When CaCl2 is substituted for MgC12, activity is not observed unless a small amount (0.1 mM) of MgCl2, MnCl2, CoCl2, FeCl2, or ZnCl2 is added.  On the basis of these experiments, we visualize a model in which the enzyme possess a site for free metal which, when filled, lowers the Km for the three substances (arginine, tRNAarg, and metal ATP) and increases the Vmax of the reaction.", "Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ehnd~fnpg.urke", "00000000-0000-0000-A863-BBDB39F04994", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory of Biochemical Genetics", "101584910X236", "101584910X235", "1988", "[1988?]", "This rough draft summarizing work completed in the Laboratory of Biochemical Genetics from 1987-1988 describes the latest results from neuroblastoma research and the first year of work with Drosophila homeobox genes.  The summary was authored as part of a requirement for the justification of funding.", "Drafts (documents)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Neuroblastoma and related lines of cultured cells were used as model systems for studies on synapse formation and other aspects of neuronal development.  Prolonged elevation of cyclic AMP levels of neuroblastoma cells has profound, long-lived, stimulatory effects on the ability of the cells to form synapses and transmit information to other cells, which is due, in part, to the appearance of functional voltage-sensitive calcium channels.  cDNA and genomic DNA clones were obtained that correspond to the alpha-subunit of L-type voltage-sensitive calcium channels of rat brain.  In addition, seventeen cDNA clones were obtained that correspond to species of RNA that increase in abundance when neuroblastoma-glioma hybrid cells are treated with dibutyryl cAMP.  Two of the cDNA clones were identified by nucleotide sequence analysis.  Clone pNG-10 DNA corresponds to RNA transcribed from the light strand of mitochondrial DNA that functions as an RNA primer needed for the initiation of synthesis of heavy strand mitochondrial DNA.  This species of RNA increases 40-fold in response to dibutyryl cAMP.  Clone NG-32 DNA corresponds to mRNA for ATP synthase subunit 6, which is transcribed from a heavy strand mitochondrial gene and codes for a protein that is part of the H+ channel of the ATP synthase complex.  This species of mRNA increases 8-fold in response to dibutyryl cAMP.  These results show that RNA transcripts from both light and heavy mitochondrial DNA strands increase in abundance when NG108-15 cells are treated with dibutyryl cAMP.  The possibility that cAMP regulates the replication of mitochondrial DNA or the ability of mitochondria to synthesize ATP are problems for future studies. Four novel Drosophila homeobox genes were cloned and partially sequenced.  The homeobox family of genes code for proteins that regulate the expression of genes during development, and some Drosophila homeobox genes are known to regulate pathways of differentiation.   Three of the 4 new homeobox genes, NK-1, NK-3, and NK-4, were mapped to the 93 E region of the right arm of the third chromosome.  The fourth novel homeobox gene was mapped to the 2C region of the sex chromosome.  The NK-1 gene is expressed in embryos 3 to 12 hours after fertilization.  Nucleotide sequence analysis showed that the NK-1 gene has 3 exons, and that 1 of the 2 introns detected resides within the homeobox region.  These genes provide an experimental system that can be used to define the mechanisms that regulate the expression of these homeobox genes as well as the regulatory effects of the homeobox protein products on the expression of other genes.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p2t2~sead_pein", "00000000-0000-0000-28C9-12E5995EEA43", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Memorandum from Marshall W. Nirenberg to Claude Lenfant", "101584910X237", null, "1988", "22 August 1988", "This memorandum from Marshall Nirenberg to Claude Lenfant, Director of the National Heart, Lung and Blood Institute, lists the four major accomplishments of the Laboratory of Biochemical Genetics Laboratory during the period from 1978-1988.", "Memorandums", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Date: August 22, 1988 From: Marshall Nirenberg Subject: Accomplishments of the Laboratory of Biochemical Genetics To: Dr. Claude Lenfant The major accomplishments of the Laboratory of Biochemical Genetics during the last ten years are as follows: 1. Demonstrated that treatment of neuroblastoma cells with cAMP results in increases in the abundance of some species of poly A+ RNA and the expression of voltage-sensitive ion channel proteins and other neuronal proteins that are required for synaptogenesis. 2. Elucidated the mechanism by which cellular metabolites regulate the activity of E. coli adenylate cyclase. 3. Demonstrated complex synergistic regulation of genes coding for enkephalin and neuropeptide Y by cAMP, glucocorticoids, activators of protein kinase C, and nerve growth factor. 4. Elucidated mechanisms involved in the formation, stabilization, and elimination of nicotinic acetylcholine receptor aggregates during the development of neuromuscular synapses.", "Nirenberg, Marshall W.", null, null, null, "Lenfant, Claude", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-x8zm-28jw-rnaw", "00000000-0000-0000-F37A-C9BAA3AF4DE9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X238", null, "1987", "September 1987", "This annual report includes descriptions of the main project undertaken by Nirenberg's laboratory during the late 1970s and well into the 1980s--Cell Recognition and Synapse Formation.  Major findings and publications are included.", "Reports", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "9", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT O01 HL 00009-13     PERIOD COVERED October 1, 1986 - September 30, 1987 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Cell Recognition and Synapse Formation fRINcl ey VegTiGar R (List other rere ional personnel below the Principal investigator.) (Name, title, laboratory, and institute affiliation) Mars Nirenberg, C > LBC, NHLBI Hemin Chin, Staff Fellow, LBG, NHLBI > Patricia Bray, Biologist, LBG, NHLBI Li-Shan Hsieh, Visiting Fellow, LBG, NHLBI Wu-Hong Tsai, Visiting Fellow Maria Giovanni, Staff Fellow, NLBG, NHLBI David Trisler, Guest Worker, LBG, NHLBI Haruhiro Higashida, Visiting Scientist, LBG, NHLBI       erald Aurbacn Chief, MDB, NIADDK Allen Spiegel, MDB, NIADDK’ Bruce Schrier, LDN, NICHD Lou Hirsch, Dept. of Biochem., U. of Texas, Dallas, Texas LAB/BRANCH Laboratory of Biochemical Genetics SECTION Section of Molecular Biology INSTITUTE AND LOCATION NHLBI, NIH, Bethesda, Maryland 20892               TOTAL MAN-YEARS: PROFESSIONAL: OTHER: 1l 9 2 CHECK APPROPRIATE BOX(ES) C] (a) Human subjects C] (b) Human tissues (3 (c) Neither L] (a1) Minors C] (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Mouse neuroblastoma-rat retina hybrid cell lines were established to rescue the expression of retinal genes. A monoclonal antibody that binds to cells from one hybrid line recognizes an antigen expressed by few cell types in retina and brain. cDNA corresponding to the neural antigen was cloned and sequenced. Additional monoclonal antibodies were obtained that bind to antigens that are markers of cell types in retina. Clones for 4 species of ag cDNA and 1 ag genomic DNA were obtained that correspond to the ag subunit of G, signal transduction protein. Two a;-1 cDNA clones were obtained and sequenced. In addition, DNA clones for rat and human 4, genes were obtained. Monospecific antibodies to an subunit of voltage-sensitive Ca2+ channels from rat T-tubules were shown to activate voltage-sensitive calcium channels of parathyroid cells. Both cDNA and genomic DNA clones were obtained that correspond to species of poly At RNA that increase or decrease in abundance when cells are treated with dibutyryl cAMP. TOP, a cell membrane protein that is distributed in a dorsal-ventral topographic gradient in chick retina was shown to be distributed in an inverted but matching ventral-dorsal gradient in chick embryo tectum. Proteins rich in lysine markedly stimulate the activity of a protein kinase in Xenopus oocyte membranes and NG108-15 membranes. NG108-15 cells respond to bradykinin by increased hydrolysis of phosphatidylinositol-4,5-bisphosphate and accumulation of inositol-1,4,5-trisphosphate, which releases Ca2+from intracellular stores, and diacylglycerol, which activates protein kinase C. Cytoplasmic Ca2t activates a Ca2t -dependent Kt channel leading to cell hyperpolarization. The activation of protein kinase C by bradykinin leads to an increase in the phosphorylation of 4 proteins and the inhibition of M-channels. Activation of protein kinase C greatly potentiates Ca2t dependant acetylcholine secretion from NG108-15 cells.         PHS 6040 (Rev. 1/84) GPO 014-918 ZO! HL 00009-13 LBG Project No. Z0l HL 00009-13 LBG Major Findings: N18TG-2 mouse neuroblastoma cells were fused with 18-day rat embryo retina cells, and somatic cell hybrid lines then were established to obtain clonal cell lines that continue to express genes characteristic of cells in retina. One of the hybrid cell lines generated, N18RE-103 possesses high tyrosine hydroxylase activity and synthesizes dopamine; whereas, tyrosine hydroxylase is not expressed by the parental N18TG-2 cells. N18RE-103 cells also possess voltage-sensitive Nat, Kt, and Ca2+ channels. N18RE-103 cells were used as an immunogen for the production of hybridoma cell lines that synthesize monoclonal antibodies to antigens expressed by N18RE-103 cells and retina. Thirty-three of the 374 hybridoma cell lines obtained synthesize antibodies to antigens expressed by N18RE-103 cells and rat retina cells, but not with cell lines from other tissues. Some of the antigens recognized by the monoclonal antibodies are expressed by different cell types in adult rat retina. For example, antibody 41C5 binds to antigen expressed by some, but not all, retinal ganglion neurons. Antibody 41A4 binds to antigen associated with the ganglion neuron layer. Antibody 89A5 binds to molecules restricted predominantly to the outer segments of photoreceptor cells. Antibody 38B6 binds to an abundant antigen on photoreceptor cell bodies and to less abundant antigen associated with horizontal, bipolar, and amacrine neuron soma. In contrast, antibody 35F8 recognizes antigen distributed in a punctate manner in the inner nuclear layer, the inner synaptic layer, and ganglion neuron layer of retina. A Agt1l cDNA expression library was prepared and the recombinants were screened with antibody 41C5. One positive recombinant clone, A41C5, was detected, which directs the synthesis of a 41C5-Bgalactosidase fusion protein recognized by antibody 41C5. Northern blot analysis with N18RE-103 poly At RNA revealed one species of 41C5 poly At RNA with a chain length of approximately 1 Kb. The nucleotide sequence of the A41C5 insert was determined. One open reading frame was found, but no homology was detected between the predicted amino acid sequence of 41C5 and other proteins. The distribution of 41C5 protein was shown by indirect immunofluorescence to be highly restricted to certain cells in rat retina and brain; the antigen was not detected in striated muscle, cardiac muscle, liver, or kidney. The distribution of 41C5 mRNA also was shown by in situ hybridization with a synthetic labeled oligodeoxynucleotide probe to be highly restricted in rat retina ZOI HL 00009-I3 LBG and brain. 41C5 cDNA will be used as a probe to study the mechanisms that enable only a few cell types in the nervous system to express the 41C5 gene. Hybridoma cell lines were generated that synthesize antibodies directed against cells from rat or rabbit retina that were used as immunogens. One monoclonal antibody detects an antigen that is most abundant in the outer layer of axons in the optic nerve. An antibody specific for the inner and outer synaptic layers of the retina was found as well as antibodies that bind only to Muller cells. Still other antibodies are specific for astrocytes in the retinal ganglion neuron layer. Some of the antigens that are markers of cell types were found to be proteins and were partially purified. As described previously, 4 species of Q@, cDNA clones were found that differ in nucleotide sequence in the region that corresponds to amino acid residues 71-88 in G@, protein, a subunit of @, signal transduction protein. A mechanism was proposed for generating 4 species of Q@, mRNA from a common precursor RNA transcribed from a single gene by 2 types of alternative splicing. Further work on Og mRNA revealed that half the normal amount of G, mRNA is present in fibroblasts of pseudo- hypoparathyroid patients. Levels of @, mRNAs that code for low and high molecular weight forms of Og protein were reduced with no apparent change in the ratio of the different species of @, mRNA to one another. These results suggest that the genetic lesion in pseudohypoparathyroidism type Ia either decreases the rate of synthesis of a common precursor of the 4 species of Qs mRNA or increases equally the rates of turnover of the four species of Qs, MRNA. Two @j-1 cDNA clones also were obtained from a human brain cDNA library that correspond to Qj protein subunits of Gj signal-transduction proteins. One of the cDNA inserts was sequenced completely, the other partially. The nucleotide sequence of human brain @j-1 is similar to that of bovine brain Q@j;-1, but differs significantly from Q@;-2 cDNAs from human monocytes, rat glioma cells, and mouse macrophages in nucleotide and amino acid sequences (88% and approximately 73% homology, respectively). The 3'-untranslated region of human brain a -1 also differs markedly from that of human monocyte @j-2. Five genomic DNA clones for @ genes were obtained. One clone was identified as a gene for @;, and 2 clones, 1 from a human genomic DNA library, the other from a rat genomic DNA library, were identified as M% genes. Further work is needed to identify the 2 remaining @ genomic DNA clones. The focus in current ZO! HL 00009-I3 LBG studies is to define the nucleotide sequences of the 5'-upstream regulatory regions of @ genes. A Agt1l cDNA library was constructed from poly at RNA from 14 day chick embryo retina and screened for recombinants with antibodies to B-subunits. One recombinant was detected with 9 out of 10 monospecific rabbit antibody preparations directed against the B-subunit of bovine transducin. The nucleotide sequence of the cDNA insert was determined and the predicted amino acid sequence was compared to that of the bovine B-subunit. Only a small region of homology was observed; hence, the cloned DNA is not related to the B Six of 10 amino acid residues predicted for the recombinant DNA were identical to amino acid residues near the N-terminus of the B-subunit and 2 glutamine residues of the B-subunit were replaced by glutamic acid residues. A synthetic decapeptide corresponding to the amino-terminal residues of B blocked the binding of anti B antibodies to B-subunits. These results show that the amino terminal decapeptide of the B- subunit is a major antigenic site of the native protein. L-Type voltage-sensitive calcium channels are sensitive to dihydropyridine agonists and antagonists. Activation of the L- type voltage-sensitive calcium channels of parathyroid cells opens the calcium channels and permits the entry of extracellular calcium ions into the parathyroid cells, which paradoxically, inhibits parathyroid hormone release. Calcium channel antagonists that block calcium entry into cells, stimulate secretion of the parathyroid hormone. Polyclonal, monospecific mouse antibodies to highly purified preparations of the a-subunit of voltage-sensitive calcium channels from rat T-tubules were obtained and were tested for their effects on parathyroid hormone secretion by cells. Three mouse antibody preparations blocked the secretion of parathyroid hormone from cells. Parathyroid cell protein was solublized and fractionated by SDS gel electrophoresis. The antibodies bound to only one major band of protein with an apparent Mr of 150,000. Incubation of a mouse antibody with parathyroid cells resulted in the activation of voltage-sensitive calcium channels and markedly increased Ca2+ uptake by parathyroid cells and inhibited parathyroid hormone release. These results suggest that the antibodies bind to @ subunits of voltage-sensitive calcium channels of parathyroid cells and activate the calcium channels. Previously we showed that elevation of cAMP levels of NG108-15 neuroblastoma-glioma hybrid cells or neuroblastoma cells for several days results in marked increases in the activities of voltage~sensitive Nat, Kt, and Ca2+ channels, and the rate of spontaneous secretion of acetylcholine at synapses between the ZOI HL 00009-I3 LBG hybrid cells and cultured striated muscle cells. We obtained cDNA clones for species of poly At RNA that increase in abundance when NG108-15 or NS20-Y cells are treated for 5 days with dibutyryl cAMP, as well as cDNA clones for other species of mRNA that decrease in abundance. The dibutyryl cAMP-dependent increases in poly At RNA range from 5- to 90-fold above those of control cells, depending upon the species of mRNA examined. The levels of most species of poly At RNA were not affected by treatment of cells with dibutyryl cAMP. Northern blot analysis showed that some independently isolated recombinant clones hybridize to the same species of mRNA; however, cDNA clones were obtained for approximately 15 species for dibutyryl cAMP responsive species of mRNA. A mouse genomic DNA library in Charon 30 was screened with cDNA probes for some dibutyryl cAMP responsive species of mRNA. Fragments of the cloned genomic DNA will be tested for promoter and/or enhancer activities. TOP is a cell surface protein that is distributed topographically in a 35 fold gradient from the dorsal margin of chicken retina, which contains the highest concentration of TOP, to the ventral margin of the retina, which contains little TOP. An inverted gradient of TOP was detected in the tectum of 3 to 5 day chick embryos. The highest concentration of TOP is present in ventral tectum and the lowest is in dorsal tectum. The topographic map of cell position in the avian retina is inverted in its projection to the optic tectum. Dorsal retinal ganglion neuron axons project to ventral tectum and ventral retinal ganglion neurons axons project to dorsal tectum. Gradients of TOP are present in chick embryo retina and tectum before retinal axons arrive in the tectum. After 10 days of embryonic development, the number of antigenic TOP sites in the tectum decreases markedly; whereas, TOP levels in the retina remain relatively constant. The presence of corresponding TOP gradients in retina and tectum at the time of innervation of the tectum by retinal ganglion neuron axons suggests a possible role for TOP in orienting the dorsal- ventral axis of the retinal projection onto the tectum by homophilic interactions between TOP molecules. The half-life of TOP in cultured retinal cells in the presence of cycloheximide or actinomycin D was 5 or 6 hr, respectively. Ablation of cells at the poles of the gradient in 60 hr chick embryos altered TOP expression during subsequent retinal development. Cells at the dorsal pole of the 13-day embryo retinas, 11.5 days after dorsal ablation, expressed 50% less TOP than normal and those at the ventral pole, after ventral ablation, expressed 300% more TOP than normal. Cells in other regions of the retina expressed normal levels of TOP. ZO! HL 00009-13 LBG Polypeptides rich in lysine markedly stimulate the phosphorylation of some membrane proteins catalyzed by a protein kinase in Xenopus oocyte membranes or membranes from NG108-15 neuroblastoma-glioma hybrid cells. A synthetic peptide containing the last 14 amino acid residues of human Ki-ras 2 protein (KKKKKKSKTKCVIM) ,which is rich in lysyl-residues, also stimulates protein phosphorylation. These effects were not observed with polyarginine. Polylysine peptides including the synthetic c-Ki-ras 2 peptide also stimulate the in vitro phosphorylation of membrane inositolphospholipids, resulting in the synthesis primarily of phosphatidylinositol 4-phosphate and to a lesser extent, phosphatidylinositol 4,5-bisphosphate. Hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns (4,5)P2) produces two prospective intracellular messengers: inositol-1, 4,5-trisphosphate (InsP3), which releases Ca2+ from intracellular stores; and diacylglycerol (DG), which activates protein kinase C. The formation of these two substances triggered by one external messenger, bradykinin, leads to the appearance of two different sequential membrane conductance changes in NG108-15 neuroblastoma-glioma hybrid cells. The addition of bradykinin to these cells rapidly stimulates hydrolysis of PtdIns (4,5)P9 to InsP3 and DG, raises intracellular Ca‘*+ and hyperpolarizes and then depolarizes the cell membrane. By voltage-clamp recording hyperpolarization was shown to result from the activation of a pharmacologically-identifiable species of Ca2+-dependent Kt current. This is also activated by intracellular injections of Ca2+ or InsP3 so may be attributed to the formation and action of InsP3. The subsequent depolarization results primarily from the inhibition of a different, voltage-dependent K current, the M-current that is also inhibited by DG activators. Hence a dual, time-dependent role is described for these two intracellular messengers in the control of neuronal signalling by a peptide. Iontophoretic injections of inositol 1,4,5-trisphosphate inside NG108-15 cells evoked an outward Kt current across the outer cell membrane, probably activated by the release of intracellular Ca2+. No such current was produced by equivalent intracellular injections of inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate. Instead, these compounds evoked an inward current with a reversal potential of about -20 mV, which may therefore be due to a non-specific cation conductance. This suggests that these compounds are unable to release sufficient Ca to activate the Ca¢t -dependent K+ current in these cells. The role of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) as possible mediators of the membrane current ZOI HL 00009-13 LBG responses of NG108-15 cells to bradykinin (BK) has been tested using intracellular iontophoresis of InsP3 and external application of phorbol dibutyrate (PDBu) and l-oleoyl-2-acetylglycerol (DAG). Intracellular iontophoresis of InsP3 into cells clamped at -30 to -50 mV produced (i) a transient outward current, (ii) a transient outward current followed by an inward current, or (iii) an inward current. All currents were accompanied by an increased input conductance. The transient outward current reversed at between -80 and -90 mV. The reversal potential was shifted to more positive potentials on raising extracellular [Kt], suggesting that it resulted from an increased Kt -conductance. The outward current was inhibited by apamin (0.4 uM) or d-tubocurarine (0.2-0.5 mM); these drugs also inhibit the outward current produced by BK or by intracellular Ca2t injections. The outward current was also slowly reduced in the absence of external Ca2t or in the presence of a solution containing 0.5 mM Cd2+ and 2 mM Co2- Iontophoretic injection of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, guanosine trisphosphate or inorganic phosphate did not evoke an outward current but produced only an inward current with an increased conductance, reversing at between -10 and -20 mV. Bath-application of PDBu (10 nM — 1 uM) or DAG (1 - 10 YM) produced an inward current with a fall in input conductance. The inward current was voltage-dependent and was accompanied by an inhibition of the time-dependent current relaxations associated with activation or deactivation of the voltage-dependent Kt-current. PDBu did not clearly reduce the Ca2+-current or the Ca2+t-dependent Kt-current recorded in these cells. During superfusion with PDBu, the outward current produced by intracellular iontophoresis of InsP3 was greatly enhanced. Analysis of homogenates of 32p-labelled NG108-15 cells by two-dimensional gel electrophoresis revealed more than 260 phosphoproteins. Four proteins were phosphorylated due to the addition of both BK and PDBu, including the 80,000 M, substrate for protein kinase C. The results support the view that the two membrane current responses to BK might result from accelerated membrane phosphatidylinositide hydrolysis. One product, InsP3, releases Ca2+ and activates an apamin/curare sensitive outward Kt current; this effect is imitated by intracellular InsP3 iontophoresis. The second product, DAG, activates protein kinase C to inhibit the voltage-dependent K*t-current and generate an inward current; this effect is imitated by external application of PDBu or DAG, and is associated with increased protein phosphorylation. The action of bradykinin (BK), inositol 1,4,5-trisphosphate (InsP3), and phorbol dibutyrate (PDBu) on the release of acetylcholine (ACh) was studied electrophysiologically on ZO! HL 00009-I3 LBG short-distance (<20 wm) synapses formed between cultured NG108-15 cells and rat muscle cells. Intophoretic application of BK onto the somatic surface of an NG108-15 cell produced an increase in frequency of m.e.p.p.s for 40-50 s in the paired myotube. Some m.e.p.p.s were evoked during BK~induced hyperpolarization (10-20 sec) of the hybrid cell soma. A few m.e.p.p.s also were elicited during BK-induced depolarization. Iontophoretic injection of Ca2t+ into an NG108-15 cell soma generated m.e.p.p.s for a very brief period (less than 3 sec), coincident with somatic hyperpolarization. No increase was observed during a subsequent somatic depolarization induced by a larger current of Ca Iontophoretic injection of InsP3 into the cytoplasm of an NG108-15 cell soma transiently evoked m.e.p.p.s during the InsP3-induced hyperpolarizing phase. A large InsP3 injection caused sustained generation of m.e.p.p.s for 2-4 min, associated with InsP3-evoked depolarization. Within 3-5 min after exposure of NG108-15-myotube pairs to 1 WMPDBu, the m.e.p.p. frequency increased by 2-5 times and reached a plateau after 8 min. The increase continued after washout of the drug. The PDBu-induced increase of m.e.p.p.s was still observed when the menmbrane potential of the NG108-15 cell was clamped at -30 mV. The data suggest that the BK-induced facilitation results from the action of two intracellular second messengers: an InsP3-dependent release of Ca2+ from the intracellular storage sites and protein phosphorylation by DAG-activated protein kinase C. Publications: l. Bray, P., Carter, A., Simons, C., Guo, V., Puckett, C., Kamholz, J., Spiegel, A. and Nirenberg, M.: Human cDNA Clones for Four Species of Ggsg Signal Transduction Protein. Proc, Natl, Acad, Sci,, USA 83, 8893-889 (1986). 2. Bray, P., Carter, A., Guo, V., Puckett, C., Kamholz, J., Spiegel, A., and Nirenberg, M.: Human cDNA Clones for an @ Subunit of Gy Signal-transduction Protein. Proc, Natl, Acad. Sci., USA 84, 5115-5119 (1987). 3. Carter, A., Bardin, C., Collins, R., Simons, C., Bray, P. and Spiegel, A.: Reduced Expression of Multiple Forms of Gg alpha in Pseudohypoparathyroidism Type Ia. USA, In Press. 4. Gatica, M., Allende, C. C., Antonelli, M. and Allende, J.E.: Polylysine-containing Peptides, Including the Carboxyl-terminal Segment of the Human c-Ki-ras 2 Protein, Affect the Activity of Some Key Membrane Enzymes. Proc, Natl. Acad. Sci., USA 84, 10. ll. 12. 13. ZOI HL 00009-I3 LBG 324-328 (1987). Trisler, D. and Collins, F.: Corresponding Spatial Gradients of TOP Molecules in the Developing Retina and Optic Tectum. Science, In Press. Trisler, D.: Synapse Formation in Retina is Influenced by Molecules that Identify Cell Position. Current Topics in Devel, Biol. 21, 277-308 (1987). Trisler, D.: Molecular Markers of Cell Position in Avian Retina are Involved in Synapse Formation. American Zoologist 2i, 189-206 (1987). Higashida, H. and Brown, D.A. Two Polyphosphatidylinositide Metabolites Control Two Kt Currents in a Neuronal Cell. Nature 323, 333-335 (1986). Higashida, H. and Brown, D.A.: Membrane Current Responses to Intracellular Injections of Inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in NG108-15 Hybrid Cells. FEBS Letters 208, 283-286 (1986). Brown, D.A. and Higashida, H.: A Note on Voltage- and Ca-activated K-currents in Mouse Neuroblastoma x Rat Glioma Hybrid Cells. J. Physiol., In Press. Brown, D.A. and Higashida, H.: Membrane Current Responses of NG108-15 Mouse Neuroblastoma x Rat Glioma Hybrid Cells to Bradykinin. J. Physiol., In Press. Brown, D.A. and Higashida, H.: Inositol 1,4,5-trisphosphate and Diacylglycerol Mimic Bradykinin effects on Mouse Neuroblastoma x Rat Glioma Hybrid Cells. J. Physiol., In Press. Higashida, H.: Acetylcholine Release Induced by Bradykinin, Inositol 1,4,5-Trisphosphate and Phorbol Dibutyrate in Mouse Neuroblastoma x Rat Glioma Hybrid Cells. J. Physiol., In Press.", "Nirenberg, Marshall W. ; Tsai, Wu-Hong ; Giovanni, Maria ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Higashida, Haruhiro ; Bray, Patricia ; Trisler, G. David ; Hsieh, Li-Shan ; Chin, Hemin", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-33um-2xmj-awfh", "00000000-0000-0000-04CE-DD4AF40017A5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on NK-2 homeobox gene", "101584910X239", "101584910X317", "1993", "[1993?]", "In this handwritten note for the annual report from the Laboratory of Biochemical Genetics (1992-1993), Nirenberg explains that studying homeobox genes in Drosophila allows the researcher to test hypotheses about the relationship between genes and nervous system development experimentally.  Furthermore, understanding the role of the homeobox genes in Drosophila may help in understanding the development of the mammalian nervous system more clearly in the future.", "Notes", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-y2gw~wy2i-4ttx", "00000000-0000-0000-4DAC-804D13E63EB8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X240", null, "1992", "September 1992", "In this annual report from the Laboratory of Biochemical Genetics, work on the Cell Recognition and Synapse Formation project is explained and major findings with mouse and Drosophila homeobox research are provided.", "Reports", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Department of Health and Human Services - Public Health Service Notice of Intramural Research Project Project Number: Z01 HL 00009-18 LBG Period Covered: October 1, 1991 - September 30, 1992 Title of Project: Cell Recognition and Synapse Formation Principal Investigator (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation): Marshall Nirenberg, Chief, LBG, NHLBI Sadamitsu Asoh, Vis. Assoc. LBG, NHLBI Wha-Seon Kwon, Predoc. Stud. LBG, NHLBI Yoshinobu Hara, Vis. Assoc., LBG, NHLBI Michael Mitas, NRC Res. Assoc. LBG, NHLBI Jacque Ferrante, Staff Fell. LBG, NHLBI Dong-Ping Tan, Vis. Assoc. LBG, NHLBI Hasi-Ping Li, Vis. Assoc. LBG, NHLBI Zheng-Mei Liu, Spec. Vol. LBG, NHLBI Lan-Hsiang Wang, Staff Fell. LBG, NHLBI Adil Nazarali, Vis. Assoc., LBG, NHLBI Alessandra Rovescalli, Vis. Fell. LBG, NHLBI Bruce Bethke, IRTA Fell., LBG, NHLBI Eduardo Marban, Spec. Vol., LBG, NHLBI Timothy Kamp, Spec. Vol., LBG, NHLBI Cooperating Units: Christine Kozak, LMM, NIAID Scott Young, LCB, NIMH Adam Hartman, LCB, NIMH Yongsok Kim, LMC, NHLBI James Ferretti, LBC, NHLBI Desiree Tsao, LBC, NHLBI Ward Oldenwald, LNC, NINDS Hemin Chin, LMB NINDS Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, MD Total Man Years: 14 Professional: 13 Other: 1 Summary of Work: Mouse Hox 1.11 homeobox DNA was cloned and sequenced. Hox 1.11 poly A+ RNA is expressed in 12 to 14 day-old mouse embryos in the hind brain up to, but not including, the pons. Hox 1.11 poly A+ RNA also was expressed in the spinal cord, the VIIth and VIIIth cranial ganglia, spinal ganglia, larynx, lungs, vertebrae, sternum, and intestine. A mouse homeobox gene, mNK-1, was cloned and approximately 5.2 kb of DNA was sequenced. Comparison of the amino acid sequences of the mouse and Drosophila NK-1 revealed 95% homology. Hox 4.1 cDNA and genomic DNA were cloned and sequenced; comparison of Hox 4.1 and Hox 2.7 proteins revealed 59% homology. Hox 4.9 also was cloned and partially sequenced.  Four Pou-domain genes expressed in embryonic and adult mouse brain were cloned and sequenced: Brain-1, Brain-2, Brain-4, and Scip.  Similar amino acid sequences were found in various regions of the proteins. No introns were detected in the coding regions of the 4 Pou-domain genes which suggests that the genes originated by reverse transcription of an ancestral species of Pou-domain mRNA followed by insertion of cDNA molecules into germ cell DNA.  The Dropsphila homeobox gene, NK-2, is expressed initially by virtually all cells in the ventral neurogenic anlage as the neuogenic anlage appears.  The nucleotide sequence of the DNA binding site for the NK-2 homeodomain was determined.  Putative genes that are regulated by NK-2 protein were cloned and were sequenced partially. Drosophila genomic DNA corresponding to 20 genes identified in enhancer trap experiments, which for the most part are expressed specifically in the nervous system, were cloned.  A nuclear protein was found that binds to a novel trinucleotide repeat sequence in the 5'-upstream region of a rat brain voltage-sensitive calcium channel a1 subunit that activates an enhancerless chloramphenicol acetyltransferase reporter gene.  cDNA clones were obtained that encode 3 kinds of DNA binding proteins, each specific for a different novel mouse enhancer sequence were cloned. Cell Recognition and Synapse Formation Project Description Major Findings Mouse Homeobox Genes.  Homeobox and Pou-domain proteins bind to DNA and regulate gene expression.  Hox-1.11 cDNA and genomic DNA were cloned and 5,856 bp were sequenced.  The Hox-1.11 gene contains 2 exons separated by a small intron. The Hox-1.11 protein, 372 amino acid residues long, contains a conserved pentapeptide, a homeodomain, and an acidic region.   The amino acid sequence of the Hox-1.11 homeodomain is identical to that of Hox-2.8, which suggests that both homeobox proteins may bind to the same or similar nucleotide sequences in DNA.   In collaboration with C. Kozak the Hox 1.11 gene was mapped to mouse chromosome 6, which contains the Hox-1 cluster of homeobox genes.  One species of Hox-1.11 poly A+ RNA (1.7 kb) was found that is most abundant in 12-day-old embryos and progressively decreases thereafter.  The most anterior expression of Hox-1.11 poly A+ RNA in 12 to 14-day-old mouse embryos is the hind brain up to, but not including, the pons.  Hox-1.11 poly A+ RNA also is expressed in the spinal cord, the VIIth and VIIIth cranial ganglia, spinal ganglia, larynx, lungs, vertebrae, sternum, and intestine. In collaboration with Y. Kim genomic DNA and cDNA for a novel mouse homeobox gene, mNK-1, were cloned and approximately 5.2 kb was sequenced.  mNK-1 is the mouse equivalent of the Drosophila homeobox gene, NK-1, which we discovered previously.  Fifty seven of the 60 amino acid residues of mNK-1 homeodomain are identical to the amino acid residues of the Drosophila NK-1 homeodomain (95% homology), which suggests that mouse and Drosophila homeodomain proteins bind to similar nucleotide sequences in DNA.   Both mouse and Drosophila NK-1 proteins contain an acidic domain.  mNK-1 poly A+ RNA was detected in 12 to 18-day-old mouse embryos. Hox 4.1 cDNA and genomic DNA were cloned and sequenced.   The deduced amino acid sequence of Hox 4.1 protein consists of 417 amino acid residues.  Hox 4.1 protein contains a conserved pentapeptide and a homeodomain that is similar to that of Hox 2.7 (97% homology).  Comparison of Hox 4.1 and Hox 2.7 proteins revealed 59% homology, which suggests that these genes evolved from a common ancestor.  Another homeobox gene, Hox 4.9, also  was cloned and partially sequenced. Pou-Domanin Genes.  The Pou-domain is a conserved amino acid sequence approximately 150 amino acid residues long that contains a Pou-specific domain and a homeodomain; both domains are required for high-affinity binding to DNA.  Genomic DNA was cloned for four Pou-domain genes that are expressed in embryonic and adult mouse brain; Brain-l (10.3 kb sequenced), Brain-2 (4.6 kb sequenced; Brain-2 cDNA also was cloned and sequenced), Brain-4, a novel Pou-domain gene, (3.2 kb sequenced), and Scip (9.9 kb sequenced).  The four proteins have similar Pou-domain amino acid sequences; sequences in other regions of the proteins also are similar, but to a lesser extent.  Although the proteins contain between 361 to 495 amino acid residue, no introns were detected in the coding regions of the four Pou-domain genes.  These results suggest that the 4 Pou-domain genes arose by duplication of an ancestral Pou-domain gene, which originated by reverse transcription of a molecule of Pou-domain mRNA followed by insertion of the cDNA into germ cell DNA. Drosophila Homebox Genes.  Previously, we cloned a novel Drosophila homeobox gene, NK-2, which initially is expressed by virtually all cells in the ventral neurogenic anlage early in embryonic development when the ventral neurogenic anlage first appears.  The results suggest that expression of the NK-2 gene results in cell commitment to the neuroblast pathway of differentiation, which is required for the development of a large portion of the CNS.  During further development, only 20% of the cells continue to differentiate as neuroblasts; whereas 80% switch to the epidermoblast pathway of development and give rise to the ventrolateral epidermis of the embryo.  Concomittantly, NK-2 mRNA levels decrease in some, but not all, neuroblasts resulting in a pattern consisting of clusters of neuroblasts with high levels of NK-2 mRNA surrounded by cells destined to become epidermoblasts with lower levels of NK-2 mRNA.  During further development, expression of the NK-2 gene is extinguished in epidermoblasts and isolated precursors of neuroblasts with high levels of NK-2 mRNA can be seen surrounded by epidermoblasts with little or no NK-2 mRNA.  It is likely that the positions in the embryo of the clusters of neuroblast precursors with high NK-2 mRNA are selected by a set of transactivating proteins.  Then 1 neuroblast precursor per cluster is selected by lateral inhibition mediated by cell-cell interactions.  Ten genes have been identified by others that are required for the neuroblast selection process. By defining the mechanisms that regulate NK-2 gene expression in future studies, we hope to understand how the number of neuroblasts and their relative positions are selected in a large part of the CNS. A peptide that contains the NK-2 homeodomain was synthesized in E. coli and purified to apparent homogeneity. Most of the peptide was given to D.  Tsao and J. Ferretti to determine the conformation of the NK-2 homeodomain in solution by nuclear magnetic resonance spectroscopy.  The NK-2 homeodomain peptide also was covalently coupled to Sepharose and synthetic double-stranded oligodeoxynucleotides that contain random sequences in the middle were passed through the column under conditions that promote NK-2 homeodomain DNA binding.   Oligodeoxynucleotides with sequences that bind to the NK-2 homeodomain were purified by repetitive affinity-column chromatography, cloned, and sequenced, and a consensus binding sequence for the NK-2 homeodomain was defined.  Putative NK-2 homeodomain binding sites were identified in the 5'-upstream region of the NK-2 gene, which suggests that NK-2 protein may regulate the expression of the NK-2 gene.   Other putative genes regulated by NK-2 protein were purified by passing Drosophila a genomic DNA fragments through the NK-2 homeodomain affinity-column.  DNA retained by the column was eluted, cloned, and 42 clones of genomic DNA were sequenced partially.  Multiple binding sites for the NK-2 homeodomain were found in all sequences examined.   Further work is needed to determine whether the cloned DNA fragments correspond to genes that are regulated by the NK-2 homeodomain protein in vivo. P-Element Transposition.  Transgenic lines of Drosophila were generated previously by transposition of a P-element that contains the B-galactosidase gene from one site in the genome to another.  Some P-element insertions into genes expressed only in the nervous system were identified as homozygous lethal mutations.  P-element insertions into some genes were found that result in gross morphological defects in the developing nervous system.   The locations of about 60 genes with P-element insertions were mapped.  P-elements with a few kb of adjacent Drosophila genomic DNA were cloned from 20 of the most interesting transgenic fly lines and the cloned Drosophila DNA genomic DNA fragments were used as probes to screen a Drosophila genomic DNA library for larger DNA fragments.  Thus far, large genomic DNA clones were obtained that correspond to P-element insertion sites for 5 transgenic fly lines.  Additional studies are in progress to characterize the genes that have been cloned that are expressed specifically in the nervous system. Regulation of a Gene for a Voltage-Sensitive Calcium Channel A-1 Subunit.  The efficiency of transynaptic communication between NG108-15 cells and cultured striated muscle cells previously was shown to be regulated by intracellular levels of cAMP or by retinoic acid, which in turn regulate the level of mRNA for a voltage-sensitive calcium channel a-l subunit required for stimulus-secretion coupling.  The 5' -upstream region of the calcium channel gene was cloned and sequenced and a novel trinucleotide repeat sequence was found that is a powerful activator of an enhancerless chloramphenicol acetyltransferase reporter gene.  Synthetic oligodeoxynucleotides inserted upstream of the reporter gene in + or - orientations or inserted downstream of the reporter gene activate reporter gene expression. In collaboration with T. Kamp and E. Marban, a nuclear protein was found that binds to the novel enhancer sequence. Enhancer Selection:  Previously we devised a selection method for mouse genomic DNA clones that contain enhancer sequences based on the demonstration that the synthesis of polyoma virus DNA in mouse cells requires viral enhancer sequencers that also are required for the synthesis of mRNA from polyoma genes.  An E. coli - mammalian cell shuttle vector was constructed that contains a library of mouse genomic DNA fragments ligated to the vector to replace the deleted polyoma virus enhancer region.  In addition, the B-lactamase gene and E. coli origin of replication from pBR322 were inserted in the polyoma coat protein gene.  Thus, only plasmids with mouse genomic DNA inserts that contain enhancers that activate plasmid DNA synthesis replicate in mouse cells and are selectively amplified.  In collaboration with W. Odenwald, the enhancer selection method was used to clone genes that may be regulated by mouse homeobox protein, Hox-1.3.  Mouse fibroblasts were cotransfected with a mouse genomic DNA library inserted in the polyoma shuttle vector and Hox-1.3 cDNA under the control of constitutive enhancer sequences so that Hox 1.3 protein is synthesized.  Two of the DNA clones that were obtained after several rounds of selection were sequenced.  One clone contained 22 and the other 44 putative Hox-1.3 binding sites.  Insertion of the cloned DNA upstream of a chloramphenicol acetyltransferase reporter gene was found to inhibit the expression of the reporter gene in cells that were cotransfected with the reporter gene construct and Hox-1.3 cDNA.  Inhibition of reporter gene expression required the homeobox region of Hox-1.3 cDNA, which suggests that the inhibition of gene expression is dependent on Hox 1.3 homeodomain binding to DNA. Previously, 3 novel mouse enhancer DNA sequences were found by the enhancer selection method.  A cDNA expression library was screened for DNA binding proteins that bind with specificity to a 32P-labeled oligodeoxynucleotide enhancer sequence.  cDNA clones were obtained that encode proteins that bind to each enhancer sequence.", "Nirenberg, Marshall W. ; Bethke, Bruce ; Tan, Dong-Ping ; Wang, Lan-Hsiang ; Rovescalli, Alessandra ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Hara, Yoshinobu ; Mitas, Michael ; Nazarali, Adil J. (Adil Jafferali), 1954-2017 ; Li, Hsi-Ping ; Ferrante, Jacqueline ; Liu, Zheng-Mei ; Marban, Eduardo ; Kamp, Timothy ; Asoh, Sadamitsu ; Kwon, Wha-Seon", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8sqz.isxs-c6vb", "00000000-0000-0000-5FCA-BD3C96871CB1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Recognition of DNA by the NK-2 Homeodomain", "101584910X241", null, "1997", "28 July 1997", "This abstract for a paper to be delivered at Cold Spring Harbor explains the role of NK-2 homeobox genes in early development.  NK-2 is expressed initially by nuclei that are precursors of neuroectodermal cells, which in turn give rise to other major parts of the nervous system.", "Abstracts (summaries)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Neurobiology of Drosophila Lan-Hsiang Wang Recognition of DNA by the NK-2 Homeodomain L.-H. Wang*, D.H.H. Tsao**, J. Ferretti**, R. Chmelik*, and M. Nirenberg*; Laboratory of Biochemical Genetics* and Laboratory of Biophysical Chemistry**, NIH, Bethesda, Maryland. The NK-2 homeobox gene (Kim,Y., and Nirenberg, M. (1989) Proc. Natl. Acad. Sci. USA, 86,77l6-7720) is expressed initially by nuclei in late stage 4 or early stage 5 Drosophila embryos that comprise the ventral half of the ventral neurogenic anlage and by some nuclei in the  procephalic region. These nuclei are precursors of neuroectodermal cells that give rise to subsets of neuroblasts, ganglion mother cells, and neurons in the subesophageal ganglion, ventral nerve cord, stomatogastric nervous system, and some cephalic ganglia. NK-2 also is expressed in the anterior and posterior midgut primordia. (Nakayama, K., Nakayama, N. Kim, Y., Webber, K., Lad, R., and Nirenberg, M., in preparation).  The NK-2 homeodomain and flanking regions (77 amino acid residues, termed NK-2H) was synthesized in E. coli and purified. Nucleotide sequences of DNA binding sites for NK-2H were identified by purifying oligonucleotides with central random sequences by repetitive NK-2H-Sepharose affinity column chromatography. Purified oligonucleotides were amplified by PCR and cloned. The consensus nucleotide sequence of the NK-2H DNA binding site, obtained by sequencing 77 clones, is T(T/C)AAGTG(G/C). The KD of NK-2H for the consensus sequence is 2 x 10^(-l0) M. Twenty putative high-affinity and 13 lower affinity NK-2 binding sites were found in 2.2 kb of the 5'-flanking sequence of the NK-2 gene, which suggests that NK-2 protein may be required to maintain NK-2 gene expression. Many NK-2 binding sites for DNA were found with overlapping or adjacent putative sites for other proteins, such as E(spl)m8, dorsal, snail, or other homeodomain proteins, which suggests that NK-2 protein may compete with some proteins that regulate gene expression for occupancy of NK-2 binding sites in DNA Circular dichroism measurements and 1D NMR spectra showed that the tm for denaturation of NK-2H is approximately 25 degrees C at pH 4.4 and that denaturation is fully reversible. NK-2H has considerably less alpha-helical content and a less stable conformation than does the Antp homeodomain (Otting, G. et al. (1989) EMBO J. 7, 4305-4309). NK-2H uniformly enriched with 15N was examined by 2D and 3D NMR. The results show that NK-2H has a novel homeodomain conformation.", "Nirenberg, Marshall W. ; Wang, Lan-Hsiang,Tsao, D. H.H. ; Ferretti, J. ; Chmelik, Rebecca", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-my8t~pyk2~zisk", "00000000-0000-0000-4638-718498CD0F8F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "NKx-1, a Mouse Homeobox Gene Expressed in Part of the Nervous System and Mesoderm", "101584910X242", null, "1993", "3 May 1993", "This abstract for the Society for Neurobiology details work on finding homologs for Drosophila homeobox genes using the polymerase chain reaction (PCR) technique.  PCR is used to amplify the number of copies of a specific region of DNA in order to produce enough DNA to be adequately tested.  Major findings from early work with mouse homeobox are included.. NOTE: The keywords in the black box at bottom of page are:  \"1. Transcription 2. Embryo 3. DNA 4. Hindbrain.\"  The second black box contains Marshall Nirenberg's signature, printed name and office phone number.", "Abstracts (summaries)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "First (Presenting) Author: Alessandra Cecilia Rovescalli Lab. Biochemical Genetics, NHLBI NIH, 9000 Rockville Pike Bldg. 36, room 1C06 Bethesda, MD 20892 Fax: (301) 402-0270 Office: (301) 496-3551 Home: (301) 718-6582 Presentation Preference: Poster Themes and Topics: 1st theme title: Devel. & Regeneration, etc. Theme letter: A 1st topic title: Molecular & Pharmacol. Correlates of Development Topic number: 17 2nd theme title: Develop. & Regen., etc. Theme letter: A 2nd topic title: Pattern Formation, etc. Topic number: 16 NKx-I, A Mouse Homeobox Gene Expressed In Part Of The Nervous System And Mesoderm. A. C. Rovescalli*, Y. Kim**, S. Kim**, J. Ferrante*, and M. Nirenberg*. Laboratory of Biochemical Genetics* and Laboratory of Molecular Cardiology**, National Heart, Lung and Blood Institute, NIH, Bethesda, MD 20692 The Drosophila NK-1 homeobox gene (S59 is a synonym) is expressed during embryonic development in a subset of neurons in the CNS and a subset of founder mus cells (Kim, Y. and Nirenberg, M., Proc. Natl. Acad. Sci. 86, 7716 (1989), and Dohmann, C., Azpiazu, N., and Frasch, M., Genes and Develop. 4, 2098 (1990)). Oligodeoxynucleotides corresponding to sequences in the NK-1 homeobox were used as primers with honeybee, salmon, Xenopus, mouse, or rat genomic DNA to amplify homologs of the NK-l homeobox by PCR.  Amplified DNA fragments were cloned and sequenced and all were shown to encode part of an NK-l-like homeodomain. The rat homeobox fragment was used as a probe to screen a mouse genomic DNA library at low stringency. A 15 kb genomic DNA clone was obtained, and 8 kb was sequenced. The ded amino acid sequence of the mouse homeodomain differs from the Drosophila NK-1 homeodomain by only 3 of 60 amino acid residues; therefore, the mouse gene was named NKx-1. Both NKx-1 and NK-1 proteins contain an acidic region before the homeodomain. Southern analysis showed that the mouse genome contains only one NKx-1 gene. NKx-1 poly A+ RNA was detected by PCR and RNase protection in 10-18 day mouse embryos; the abundance of NKx-1 poly A+ RNA is highest in 10 day embryos; then progressively decreases. Northern analysis of poly A+ RNA from adults revealed 1 major band of NKx-1 poly A+ RNA in brain RNA and trace bands in RNA from testes and spleen. Sections of 14 day mouse embryos were subjected to in situ hybridization and autoradiography. NKx-1 RNA was found in discrete regions of the mesencephalon and myelencephalon; NKx-1 RNA also was found in spinal cord, vertebrae, and ribs. These results show that the mouse NKx-1 gene encodes a homeodomain that is closely related that of Drosophila NK-1, and is expressed during embryonic development in part of the nervous system, in some mesodermal tissues, and in adult brain.", "Kim, Yongsok ; Nirenberg, Marshall W. ; Rovescalli, Alessandra ; Kim, S. ; Ferrante, Jacqueline", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dt9h-bpa7~digh", "00000000-0000-0000-F4CF-1ADCAB2E342A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Purification of a Membrane Protein Distributed in a Topographic Gradient in Chicken Retina", "101584910X244", null, "1986", "July 1986", "This article is part of a series involving chick embryo retina.  Findings here suggest that protein molecules known as TOP are involved in the dividing of cells that gives rise to retinal cells.  The working hypothesis of the article is that the TOP gene or genes influence the location of the accumulation of TOP.  Furthermore, results suggest that after division the cell progeny inherit the parental ability to express the TOP gene--significant because it implies cellular memory.", "Articles", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "4", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Proc. Natl. Acad. Sci. USA Vol. 83, pp. 4730-4733, July 1986 Biochemistry Purification of a membrane protein distributed in a topographic gradient in chicken retina (neuronal differentiation /embryonic development /monoclonal antibodies /cell surface antigen) JosEPH R. MosKAL*, DAviD TRISLER, MICHAEL D. SCHNEIDER’, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Marshall Nirenberg, March 21, 1986 ABSTRACT Antigenic molecules termed TOP, which are distributed in a dorsal > ventral concentration gradient in chicken retina, are expressed early in development (by 48 hr after fertilization) in the optic cup of chicken embryos and continue to be expressed in retina thereafter. **S-labeled- TOP-antibody complexes were purified by protein A-Seph- arose column chromatography and subjected to NaDodSO,/ polyacrylamide gel electrophoresis and autoradiography. TOP also was purified from dorsal retina by anti-TOP IgG-Affi- Gel 10 affinity column chromatography. In both cases, one major band of protein at M, ~ 47,000 was obtained. A protein of M, ~ 47,000 also was purified from chicken embryo brain. Cultured cells dissociated from 8-day chicken embryo retinas accumulated the amount of TOP expected of cells in the intact retina, depending on the position of the cells in the retina. TOP accumulations by cells dissociated from dorsal or ventral retina, mixed in different proportions, and cocultured were additive. These results show that TOP is a protein, that the gradient of TOP is established early in development, and that perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusable molecules or on maintenance of interactions between cells.   Previously, a monoclonal antibody was obtained that recog- nizes a cell surface antigen, TOP, that is distributed in a topographic dorsal > ventral concentration gradient in chick- en retina (1). The gradient is established during embryonic development and also is expressed at all developmental stages thereafter, including adult retina. The antigen was detected on most or all cell types in retina and also was found in the cerebral cortex and thalamus, but little or no antigen was detected elsewhere in the nervous system or in other tissues. The antigen defines a dorsal—ventral axis of the retina and can be used to identify cell position on this axis. The concentration of TOP detected near the dorsal margin of chicken retina was at least 35-fold higher than that found near the margin of ventral retina, and the amount of TOP detected changed smoothly and continuously between the dorsal and ventral poles of the gradient (1). In this report we describe the purification and character- ization of TOP and other results that pertain to the problem of how a topographic gradient of protein is generated and perpetuated by retina cells. Preliminary accounts of some of the results have been presented (2, 3). MATERIALS AND METHODS Retina Cell Culture. Chicken embryo retinas were disso- ciated by incubation with trypsin and either collagenase or DNase I. Cells were plated on poly(L-lysine)-coated tissue culture dishes (8 x 10’ cells per 100-mm dish). Culture   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement”’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4730 medium was Eagle’s minimal essential medium supplement- ed with 10% fetal bovine serum. Cultures were maintained in an atmosphere of 5% CO,/95% air at 100% humidity at 37°C. TOP Radioimmunoassay. The assay has been described in detail (1). In brief, retina cell membranes were incubated either with anti-TOP monoclonal antibody or with antibody synthesized by parental P3X63 Ag8 cells. The membranes then were washed and incubated with affinity-purified }>I- labeled rabbit F(ab’), directed against mouse IgG. Purification of >°S-Labeled TOP by Protein A-Sepharose Column Chromatography. *°S-labeled TOP was obtained by culturing 8-day chicken embryo dorsal retina cells (8 x 10’) with 1 mCi (1 Ci = 37 GBq) of L-[°S]methionine per 6 ml of medium with serum (adjusted to a final concentration of 3.33 4M [7°S]methionine) per 100-mm Petri dish for 40 hr. [Better conditions for obtaining S-labeled retina protein, which were found in experiments not shown here, are as follows: 8-day chicken embryo dorsal retina cells (6 x 10’) were incubated in 10 ml of Eagle’s minimal essential medium containing 10 uM [S]methionine (10 wCi) supplemented with 10% fetal bovine serum per 100-mm Petri dish. Cells were washed, harvested, and incubated at 4°C for 1 hr with 1 ml of solubilization buffer [Dulbecco’s phosphate-buffered saline (without Ca? and Mg?*) supplemented with 1% Triton X-100, 0.2% NaDodSO,, 25 mM Hepes (adjusted to pH 7.4), 0.005% pancreatic DNase I, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1% ethanol]. The suspension was centrifuged for 60 min at 100,000 x g and the supernatant fraction was frozen. S-labeled protein (2 x 10° cpm) in the supernatant fraction was incubated with ascites fluid containing either anti-TOP antibody or antibody synthesized by P3X63 Ag8 cells. The solutions were adjusted to 200 mM sodium phos- phate (pH 8.0) and were applied to protein A-Sepharose columns (1.5-ml bed volume) that had been washed with 200 mM sodium phosphate, pH 8.0/1% Triton X-100/0.2% Na- DodSOQ,. Antibody-antigen complexes were eluted from each column with 100 mM citrate/phosphate, pH 5.9/1% Triton X-100, 0.2% NaDodSO,/600 mM NaCl (4—6). Eluted protein was precipitated with 15% (wt/vol) trichloroacetic acid and subjected to NaDodSO,/7% polyacrylamide gel electrophoresis. Purification of TOP by Antibody-Affi-Gel 10 Affinity Col- umn Chromatography. Anti-TOP IgG and control IgG were obtained by culture of hybridoma and of parental P3X63 Ag8 cells as described (1). The media were collected and the antibodies were purified by protein A-Sepharose affinity column chromatography (4).. Antibodies were precipitated with ammonium sulfate (70% saturated), dialyzed against 50 mM sodium borate, pH 8.5/150 mM NaCl, and applied to protein A-Sepharose columns. The columns were washed, and antibodies were eluted with 0.1 M citrate/phosphate, pH   *Present address: Laboratory of Cell Biology, Building 36, Room 3A-17, National Institute of Mental Health, Bethesda, MD 20892. tPresent address: Section of Cardiology, Baylor College of Medi- cine, 6535 Fannin, MSF905, Houston, TX 77030. Biochemistry: Moskal e¢ al.   TRYPSIN DAY 0 TRYPSIN DAYS 0,4 Ww T | to 125] jabeled F(ab’), specifically bound, pmol/mg of protein       OD Mv DM V Fic. 1. Expression of TOP antigen by retina cells from dorsal, middle, or ventral 8-day chicken embryo retina that had been dissociated with 0.05% trypsin and 0.003% collagenase and cultured for 10 days. On the 4th day of culture, where indicated, cells were dissociated again with trypsin and collagenase as described above, washed, and transferred to new 100-mm dishes and incubated for an additional 6 days. Radioimmunoassay of TOP was performed as described in Materials and Methods. D, M, and V refer to dorsal, middle, and ventral retina, respectively; A and P refer to anterior and posterior retina, respectively. 3.5/1 M NaCl, dialyzed against phosphate-buffered saline, and coupled to Affi-Gel 10 (Bio-Rad). Each antibody-Affi-Gel 10 column was adjusted so that 1 mg of IgG was covalently coupled to 1 ml of Affi-Gel 10. One hundred freshly dissected dorsal halves of 14-day chicken embryo retinas or 20 brains minus the cerebella were homogenized with 20 strokes in a Dounce homogenizer and incubated at 4°C for 60 min in solubilization buffer supplemented with 5 mM EGTA (5 mg of protein per ml). The suspensions were centrifuged for 30 min at 30,000 x g, and equal portions of the supernatant fractions containing solubilized protein were applied to the antibody-Affi-Gel 10 columns at flow rates of ~5 ml/hr. Each effluent was cycled through the appropriate column two additional times. Each column was washed with 100 ml of solubilization buffer and then with 100 ml! of solubilization buffer with 0.1%, rather than 1%, Triton X-100 and without NaDodSO,. Protein was eluted with 200 mM acetic acid, pH 3.0/0.1% Triton X-100/150 mM NaCl (7). Eluates were neutralized with sodium carbonate, and dithiothreitol and NaDodSOQy, were added to portions (1.5 ml) of the eluate (total eluate collected, 4 ml) at final concentrations of 5 mM and 2%, respectively. The samples were diluted 1:10 with Laemmilli’s tank buffer (8) and were subjected to NaDodSO,/ polyacrylamide gel electrophoresis as described by Neville (9). RESULTS TOP is expressed early in embryonic development in the optic cup, approximately 48 hr after fertilization. As deter- mined by TOP radioimmunoassay, tissue from the optic cup, head, and trunk of 48-hr embryos specifically bound 3.78, 0.88, and 0.59 pmol of }*°I-labeled F(ab’). per mg of protein, Proc. Natl. Acad. Sci. USA 83 (1986) 4731              T T T   8 days in ovo + 6 days in vitro   125] labeled F(ab’) specifically bound, pmol/mg of protein % dorsal cells 25 50 75, i j i 0 100 75, 50 25 0 % ventral cells   Fic.2. TOP antigen expression in monolayer cultures containing varying proportions of cells dissociated with 0.05% trypsin in the presence of 0.005% pancreatic DNase I from dorsal or ventral 8-day chicken embryo retina. Each 100-mm Petri dish contained 8 x 10’ retina cells and 15 ml of medium. After 6 days of culture, TOP was quantitated by radioimmunoassay. The open circle and open square on the left ordinate refer to pmol of '*I-labeled F(ab’), specifically bound per mg of protein in freshly dissected 8-day embryonic ventral retina and 14-day embryonic ventral retina, respectively. The open circle and open square on the right ordinate refer to pmol of 125]-labeled F(ab’), specifically bound per mg of protein in freshly dissected 8-day chicken embryo dorsal retina and 14-day embryo dorsal retina, respectively. D, V, A, and P refer to dorsal, ventral, anterior, and posterior, respectively. respectively. The number of TOP molecules detected in the optic cup approached the levels detected in the adult retina. Low levels of TOP were detected in optic vesicles, 33 hr after fertilization (data not shown). We next asked whether dissociated chicken embryo retina cells from dorsal, middle, or ventral retina that were cultured in vitro express the amount of TOP that would be expected of cells in those regions of the intact retina in ovo (Fig. 1). Little or no TOP was detected immediately after 8-day chicken embryo retina cells were dissociated to single cells with trypsin, because trypsin destroys the antigenicity of TOP (1). However, after 10 days of culture, TOP accumu- lations by cultured cells from dorsal, middle, or ventral retina were similar to the amounts of TOP detected in the appro- priate regions of the intact retina. Similar results were obtained when retina cells were trypsinized, cultured for 4 days, trypsinized again, cultured for an additional 6 days, and then assayed for TOP. These results show that TOP is synthesized by cultured retina cells, and that the amount of TOP detected is dependent on the original position of the cells in the intact retina. Trypsinized celis from 8-day chicken embryo dorsal retina and ventral retina were mixed in different proportions, and cells then were cultured for 6 days to determine whether interactions between cells from different topographic regions of retina regulate the expression of TOP. The results (Fig. 2) show that TOP accumulations by cocultured dorsal and ventral retina cells were approximately additive. Thus, in- 4732 Biochemistry: Moskal et al. Proc. Natl. Acad. Sci. USA 83 (1986) 1.0   2S S S S Ss) be - a T T T pmol/mg of protein S an 1251_Jabeled F(ab’), bound,     0 4 8 12 16 2 O 4 8 12 16 Time, hr teractions between cocultured dorsal and ventral retina cells had little or no effect on TOP accumulation. Cycloheximide (2 pg/ml) or actinomycin D (1 pg/ml) almost completely inhibited the synthesis of TOP by cultured cells dissociated from dorsal 8-day chicken embryo retina with trypsin (Fig. 3). These results suggest that the expres- sion of TOP by cultured retina cells is dependent on de novo protein synthesis, rather than on posttranslational modifica- tion of precursor protein molecules. Incubation of 8-day chicken embryo retina cells with 50 ng of tunicamycin per ml of medium for 24 hr resulted in a decrease in the amount of TOP detected, to 32% of the value found with cells cultured without tunicamycin (Table 1). This suggests that TOP may be a glycoprotein synthesized via a dolichol-oligosaccharide intermediate. Incubation of dorsal 8-day chicken embryo retina cells with wheat germ agglutinin decreased antibody binding to retina cell membranes, whereas concanavalin A or Ulex europaeus I lectins increased antibody binding to retina cell membranes 204% and 130%, respectively (Table 1). In contrast, incuba- tion of retina cells with bovine brain gangliosides had little or no effect on the binding of anti-TOP antibody to retina cell membranes (1). These results show that the amount of antibody specifically bound to TOP can be increased or decreased by the binding of a lectin to an appropriate receptor. Table 1. Characterization of TOP   125]-Jabeled F(ab’). specifically bound to retina Treatment membranes, % control   Experiment I* None (control) 100 (467 cpm per well) Wheat germ agglutinin 58 Concanavalin A 204 Ulex europaeus ‘ectin 130 Experiment 2+ None (control) 100 (1390 cpm per well) Tunicamycin 50 ng/ml 32 100 ng/ml 19   125]-labeled F(ab’), concentration was 440 nM. *Each 50-yl reaction mixture (containing 10° dissociated 8-day chicken embryo retinal cells) was incubated for 15 min with or without the indicated lectin (50 wg) before quantitation of TOP by radioimmunoassay (1). tCells were exposed to tunicamycin for 24 hr before TOP was assayed. At these concentrations, tunicamycin inhibited [*H]man- nose incorporation into trichloroacetic acid-precipitable protein but did not significantly reduce incorporation of [4C]leucine. Leupeptin (50 4M) had little or no effect on the amount of TOP detected in the absence or presence of tunicamycin.                      Fic. 3. Inhibition of TOP synthesis in cultured retina cells by cycloheximide (A) or actinomycin D 49.8 (8). Cells were dissociated from dorsal halves of 8-day chicken embryo retina with 0.05% trypsin (which destroys the antigenicity of TOP) and 0.005% pancreatic DNase I in phosphate-buffered saline without Ca?* or Mg?* and were washed with to.4 medium containing 1 mM phenylmethylsulfonyl fluoride. Each 100-mm Petri dish coated with poly(L-lysine) was inoculated with 8 x 10’ cells. 0.2 Cells were cultured in the absence (@) or presence (4) of cycloheximide (2 ug/ml of medium) or actinomycin D (1 ug/ml of medium). Cells were harvested at the indicated times and frozen until assayed for TOP as described in Materials and Methods.     24 In addition, each well of 96-well multiwell plates was coated with 0.1-100 ng of a glycosaminoglycan, either heparin, chondroitin sulfate, or hyaluronic acid. Wells were washed, incubated with antibody to TOP, washed, and then incubated with !I-labeled F(ab’), (440 nM) directed against mouse immunoglobulin, produced in rabbits. No specific binding of anti-TOP antibody to the glycosaminoglycans tested was detected. [In this experiment, in the absence of glycosaminoglycans, 1390 cpm of )I-labeled F(ab’), was bound per well.] 35§-labeled TOP was obtained by culturing dissociated 8-day chicken embryo retina cells with [2>S]methionine for 40 hr. Cells were washed, and 25S-labeled protein was solubil- ized with 1% Triton X-100/0.2% NaDodSO, and incubated either with antibody to TOP or with antibody synthesized by parental P3X63 Ag8 cells. >°S-labeled protein-IgG complexes and free IgG were retained by protein A-Sepharose at pH 8.0. The columns were washed and the labeled protein-IgG complexes and free IgG were eluted at pH 5.9. The eluted 3§-labeled protein then was subjected to nonreducing NaDodSO,/polyacrylamide gel electrophoresis. Under these conditions anti-TOP IgG-TOP complexes dissociate, whereas protein subunits of IgG do not dissociate from one another. 35§-labeled proteins were visualized by staining with Coomassie blue and by autoradiography (Fig. 4). Only one major band of protein was detected by Coomassie blue staining, at M, ~ 150,000, which corresponds to IgG directed against TOP or IgG synthesized by P3X63 Ag8 cells. Autoradiography of the same gel revealed only one band of Coomassie blue Anti-TOP P3X63 Autoradiography Anti-TOP P3X63     200 — 116.5 — 68 — B—)   Fic. 4. Purification of 3°S-labeled TOP by protein A-Sepharose column chromatography. Cell extracts were incubated with ascites containing either anti-TOP or antibody synthesized by P3X63 Ag8 cells. Antigen-antibody complexes were isolated by chromatogra- phy on protein A-Sepharose, precipitated with 15% trichloroacetic acid, and analyzed by NaDodSO,/PAGE followed by staining with Coomassie blue and then by autoradiography. Positions of molecular weight standards (M, x 1073) are at left. Biochemistry: Moskal et al. 35§-labeled protein dissociated from anti-TOP IgG-antigen complexes, at M, ~ 47,000. No band of S-labeled protein was detected with P3X63 Ag8 IgG. Anti-TOP IgG or IgG synthesized by parental P3X63 Ag8 cells was purified from medium harvested from hybridoma or P3X63 Ag8 cells by protein A-Sepharose affinity column chromatography. Each antibody preparation then was cou- pled to Affi-Gel 10 (agarose with a 10-atom-spacer arm terminating in a reactive N-hydroxysuccinimide ester). Solubilized protein samples from 14-day chicken embryo retina or brain were applied to an anti-TOP IgG-Affi-Gel 10 column and a P3X63 Ag8 IgG-Affi-Gel 10 column. Columns were washed, antigens were eluted and then were subjected to NaDodSO,/polyacrylamide gel electrophoresis under re- ducing conditions, and proteins were visualized by silver staining. Only one band of protein (M, ~ 47,000) from retina or brain was detected in eluates from the anti-TOP IgG- Affi- Gel 10 column (Fig. 5). Little or no protein was detected in the eluates from the P3X63 Ag8 IgG-Affi-Gel 10 column. Protein eluted from the anti-TOP IgG-Affi-Gel 10 column also was subjected to isoelectric focusing. Only one band of protein from retina or brain was detected, with a pI ~ 4.1 (data not shown). DISCUSSION TOP, a plasma membrane protein that is distributed in a large dorsal > ventral gradient in chicken retina (1), was solubil- ized and purified extensively. The apparent M, of TOP is =47,000 and the apparent pI is 4.1. A protein with the same 1 2 4 94 — 67— 43— 30— Fo 20—— |   14— Fic. 5. Purification of TOP by antibody-Affi-Gel 10 column chromatography. After NaDodSO,/PAGE, proteins were visualized by silver staining (10). Lane 1: retina protein eluted from an anti-TOP IgG-Affi-Gel 10 column. Lane 2: retina protein eluted from a P3X63 Ag8 IgG- Affi-Gel 10 column. Lane 3: Cerebrum and thalamus protein eluted from an anti-TOP IgG-Affi-Gel 10 column. Lane 4: Cerebrum and thalamus protein eluted from a P3X63 Ag8 IgG-Affi-Gel 10 column. Positions of molecular weight standards (Mf, x 1073) are at left. Proc. Natl. Acad. Sci. USA 83 (1986) 4733 M, and pI also was purified from chicken embryo brain. We estimate that TOP is a moderately abundant membrane glycoprotein in 14-day chicken embryo dorsal retina, com- prising perhaps 0.2% of the plasma membrane protein but only about 0.01% of total retina protein. Cells dissociated from 8-day chicken embryo retina with trypsin, which destroys TOP antigenicity, synthesize and accumulate the amount of TOP during subsequent culture that would be expected of cells in the intact retina, dependent upon the original position of the cells in the retina. Thus, populations of single cells that were dissociated from retina and cultured continue to synthesize and accumulate the amounts of TOP that would constitute a gradient in the intact retina. Coculture of cells dissociated from dorsal or ventral retina and mixed in different proportions did not affect TOP accumulation by the cells; hence, no regulation of TOP expression was detected that could be attributed to interac- tions between cells from dorsal and ventral retina. TOP is expressed early in development, approximately 33 hr after fertilization, by cells in the optic vesicles of chicken embryos before the retina is formed. At approximately 48 hr after fertilization, as the optic cup is formed, the number of TOP molecules detected per milligram of total protein is similar to the levels that are found in adult retina. These results suggest that TOP is expressed by dividing cells that give rise to retinal cells and that the gradient of TOP is established as retinal cells are generated. The results also suggest that the gradient of TOP is established early in development by transient events that regulate either the expression or the accumulation of TOP in each cell. The gradient, once formed, is relatively stable. Perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusable molecules or on the maintenance of interactions between cells. We assume that cell progeny inherit the parental ability to express the TOP gene or genes. The possibility that multiple rounds of cell division are required to establish a gradient of TOP expression (for example, by differential DNA methyla- tion) requires further study. Our working hypothesis is that the gradient in cellular accumulation of TOP reflects a gradient in the rate of expression of the TOP gene or genes, but other possibilities, such as regulation of turnover of TOP mRNA or protein, are not ruled out. We gratefully acknowledge Dr. Maria Giovanni for helpful dis- cussions and for assistance in the glycosaminoglycan experiments. 1. Trisler,G. D., Schneider, M. D. & Nirenberg, M. (1981) Proc. Natl. Acad. Sci. USA 78, 2145-2149. 2. Trisler, G. D., Schneider, M. D., Moskal, J. R. & Nirenberg, M. (1981) Cold Spring Harbor Rep. Neurosci. 2, 231-245. 3. Moskal, J. R., Trisler, G. D. & Nirenberg, M. N. (1982) Soc. Neurosci. Symp. 8, 693 (abstr.). 4. Dimitriadis, G. J. (1979) Anal. Biochem. 98, 445-451. 5. Ey, P. L., Prowse, S. J. & Jenkin, C. R. (1978) Immunochem- istry 15, 429-436, 6. MacSween, J. M. & Eastwood, S. L. (1981) Methods Enzy- mol. 73, 459-471. 7. Staehelin, T., Hobbs, D. S., Kung, H.-f., Lai, C.-Y. & Pestka, S. (1981) J. Biol. Chem. 256, 9750-9754. 8. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 9. Neville, D. M., Jr. (1971) J. Biol. Chem. 246, 6328-6334. 10. Merril, C. R., Goldman, D. & Van Keuren, M. L. (1982) Electrophoresis 3, 17-23.", "Moskal, Joseph R. ; Nirenberg, Marshall W. ; Schneider, Michael D. ; Trisler, G. David", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-d3kb.ic6i.fz6a", "00000000-0000-0000-AD2C-80BCDA79E36B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Yongsok Kim to Marshall W. Nirenberg", "101584910X245", null, "1986", "16 December 1986", "Kim thanks Nirenberg for arranging a postdoctoral fellowship for him at NIH.  He notes that he considers Nirenberg's work on the genetic code as one of the three milestones (along with Mendelism and DNA structure) in the history of the life sciences.  Kim would subsequently be the key figure in the discovery of the four new homeobox genes.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Yongsok Kim.", "Copyright may apply", null, null, "December 16, 1986. Dear Dr. Nirenberg Thank you very much for the arrangement of NIH postdoctoral fellowship, which I heard from my major professor, Dr. Rho, whom you sent the letter with the lucky news for me.  Professor Rho told me that he was going to write a Thank-you letter and I am accepting arrangement with a great honor.  This is why I write the accepting letter late.  Of course, I accept your fellowship arrangement with a great happiness.  In the meantime, I almost filled up the Application for Federal Employment (form 171) and I will send you with my C.V./ reprints within a short period of time. After I heard from my professor that you were visiting Korea, I was getting excited and impatiently waited to see you, because I admired, since I was an undergraduate student, your works on genetic code, which I think, is one of the three milestones (Mendelism, DNA structure, genetic code) in the history of life sciences.  I attended your seminar twice.  I was deeply impressed with your studies on neurobiology.  I had a feeling in a broad sense that you are trying to relate your genetic code work and memory phenomena, which is, I think, a tangible approach. I have no words with which to thank you for your giving me the opportunity that I can study in your laboratory.  I am anxious to join your laboratory to take part in your research plan.  However, I have to finish up my dissertation writing in August, 1987.  So I will be at your laboratory on early September of 1987.  My research experiences are on the area of restriction enzymes, DNA sequencing, gene cloning and expressions.  I promise you that I will do my best in your laboratory. With all my best regards I remain, Yours Faithfully, Yong Sok Kim Department of Zoology Seoul National University Seoul 151, Korea", "Sŏul Taehakkyo ; Kim, Yongsok", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5r5m.yiv3_25bn", "00000000-0000-0000-2C75-E24C0CABACEF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Michael Hanley to Marshall W. Nirenberg", "101584910X246", null, "1986", "9 December 1986", "Hanley updates Nirenberg on the progress of research, using cell lines obtained from Nirenberg earlier, in the Molecular Neurobiology Unit at the University of Cambridge Medical School.  Hanley also requests additional clonal cell lines for future study.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "9 December, 1986 Dear Marshall: To keep you up to date on our progress with the cell lines, we have now started sub-cloning by limiting dilution each of the populations you sent to us.  It seems likely that most, if not all, of the NCBS and NCFL series are oligoclonal.  The resulting sub-cloned lines are being sent to Anna Borsodi for her programme as well as being evaluated here. Nothing very interesting has yet emerged. On another matter, we should still be very grateful if the clones for the G-proteins were to be made available to us.  We should be delighted to receive anything we could, but the clones for Gs and Gi were muted, and we should be particularly interested if a Go probe were also possible. Thank you for your help. Best wishes, and Merry Christmas, yours sincerely, Michael Hanley", "Medical Research Council (Great Britain) ; Hanley, Michael", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4izv~ubmg_ujuq", "00000000-0000-0000-82DD-6EF305007909", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from R. Naresh Singh to Marshall W. Nirenberg", "101584910X247", null, "1990", "12 February 1990", "Singh invites Nirenberg to an international conference for leading scientists in the field of neurobiology on \"Principles of Design and Function of the Nervous System\" to be held in Goa, India.  Nirenberg is invited to be a member of the International Advisory Council for the Conference.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of R. Naresh Singh.", "Copyright may apply", null, null, "February 12, 1990 Dear Dr. Nirenberg, We, the organizers of ICONDSS, 1988, are planning to hold a second International Conference at Goa, about one and half years from now, between September 22-28, 1991. The theme of this International conference will be 'Principles of Design and Function in the Nervous System'.  The conference is being organized to bring together leading scientists in the field of neurobiology to present and discuss their work in relation to the design and function of the nervous system including the sensory systems.  Interdisciplinary studies on nervous systems drawing upon the techniques of anatomy, biochemistry, developmental biology, genetics,   immunology, molecular biology, physiology, tissue culture and behavioral biology would be encouraged. This conference aims at inspiring the younger scientists to visualize a wider horizon in the field of neurobiology.  Scientists working with specialized biological systems will be motivated to compare their results with those obtained in other systems in an effort to deduce common principles.   Approximately 120 participants from international and national institutions are expected to attend the conference, the proceedings of which will be published. We would be delighted to have you as a Member of the International Scientific Advisory Council for the Conference and would like your opinion regarding the list of the participants, the scientific programme and the other organizational aspects of the conference. Since we are initiating the necessary steps now, we would be happy to hear from you as early as possible about your concurrence to be a Member of the International Scientific Advisory Council of the Conference. With regards, Yours sincerely R. Naresh Singh Convenor", "Tata Institute of Fundamental Research ; Singh, R. Naresh", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rejw-if7h-jc4w", "00000000-0000-0000-80FA-E106D0E0669E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Joel Yager, Gordon Strauss to Marshall W. Nirenberg", "101584910X248", null, "1980", "21 November 1980", "In this letter the authors identify Nirenberg as a \"leader in the field of Neurobiology (Neurochemistry, Neurophysiology, etc) and Behavior.\"  They request advice for directions in the field and include a questionnaire designed to identify the \"cutting edges\" in psychiatry and the behavioral sciences.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Joel Yager.", "Copyright may apply", null, null, "November 21, 1980 Dear Dr.  Nirenberg: We are trying to identify the \"cutting edges\" in psychiatry and the behavioral sciences.  We are surveying the leaders in the field and the faculty of the Department of Psychiatry and Biobehavioral Sciences at UCLA named you as a leader in the field of Neurobiology (Neurochemistry, Neurophysiology, etc.) and Behavior.   We would like you to answer four broad questions, both in your identified area of special expertise and in psychiatry as a whole, including basic and clinical sciences.  Having your answers will be especially important if our study is to be both complete and representative. In answering the enclosed questionnaires don't hesitate to include individuals, events or articles which are off the beaten path.  Also, while unbridled modesty is not called for here, please do not list more than two of your own publications.  Finally, treat the numbers on the questionnaires as upper limits, not as minimums. We realize that answering our questions will take time and thought, but we believe you'll find the process stimulating.  If we receive a substantial return we intend to publish the results.  We hope this information will alert others to up-and-coming issues, increase awareness of how colleagues perceive the significance of new developments, and aid in curriculum development for mental health educational programs. We have enclosed a pre-addressed envelope and would appreciate receiving your completed questionnaires as soon as possible.   Thank you for your assistance. Sincerely, Joel Yager, M.D. Associate Professor Gordon Strauss, M.D. Assistant Professor", "Yager, Joel ; University of California, Los Angeles ; Strauss, Gordon", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-thr6_vib4_7wdy", "00000000-0000-0000-493C-90BB1CB91550", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Distinguished Lecture Series in Basic and Medical Neuroscience", "101584910X249", null, "1983", "[1983-1984]", "This program is for the Distinguished Lecture Series in Basic and Medical Neurosciences--1983-84, presented by the Department of Psychiatry and the Neuroscience Association at the University of California, Irvine.  Nirenberg is listed for his lecture on \"Synapse Formation by Neuroblastoma Cells.\"", "Programs (documents)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Reproduced with permission of University of California, Irvine.", "Copyright may apply", null, null, "Distinguished Lecture Series in Basic and Medical Neurosciences--1983-84 Presented by Department of Psychiatry and Neuroscience Association University of California, Irvine. Schedule of Speakers and Topics October 5, 1983 Neurotransmitter Transformation During the Development of the Sympathetic Nervous System Story C. Landis, Ph.D., Department of Neurobiology, Harvard University Medical School, Boston, Massachusetts November 2, 1983 Brain Transplants in Animals and Man Lars Olson, M.D., Professor of Histology, Karolinska Institute, Stockholm, Sweden December 7, 1983 Schizophrenia: Biological and Genetic Components Seymour S. Kety, M.D., Associate Director, National Institute for Mental Health (Intramural Program), Bethesda, Maryland January 4, 1984 Biochemical Basis of Alzheimer's Disease Dennis J. Selkoe, M.D., Department of Neurology, Harvard Medical School--Mailman Research Center, McLean Hospital, Belmont, Massachusetts The lectures will be held the first Wednesday of the month (second Wednesday in March) from 7:00 p.m. to 8:30 p.m., on the University of California, Irvine campus in the Science Lecture Hall. February 1, 1984 Synapse Formation by Neuroblastoma Cells Marshall W. Nirenberg, Ph.D. (Nobel Laureate) Chief, Laboratory of Biochemical Genetics, National Heart, Lung & Blood Institute, Bethesda, Maryland March 14, 1984 Structure and Function of the Acetylcholine Receptor Protein Jean-Pierre Changeux, M.D., Ph.D. Pasteur Institute, Paris, France April 4, 1984 High Specificity Antibodies in Biology and Medicine Richard A. Lerner, M.D. Department of Cellular and Developmental Immunology Scripps Clinic and Research Foundation La Jolla, California May 2, 1984 Looking and Seeing: The Visual Functions of the Parietal Lobe Vernon B. Mountcastle, M.D. Professor of Neuroscience, Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland (Grass Foundation Lecture) Additional Information: For additional information concerning this program, please call (213) 595-3811. The Department of Psychiatry reserves the right to cancel a program at any time caused by circumstances not under its control. Persons registered in a cancelled program will be notified by telephone, using the telephone number listed on the application form. Registration fees will be refunded for those programs cancelled. Please note that there may be changes in faculty or minor changes in the program content caused by circumstances beyond the Department of Psychiatry control. Accreditation: As an organization accredited for continuing medical education, the University of California, Irvine, College of Medicine, designates each lecture as meeting the criteria for 1 1/2 credit hours in Category 1 of the Physician's Recognition Award of the American Medical Association and the California Medical Association Certificate. Fee: A registration fee of $150.00 prior to October 1, 1983, for physicians wishing to receive Category 1 credit. After October 1, 1983, the fee for physicians wishing to receive Category 1 credit will be $250.00 for the entire series. No fee will be charged for allied health professionals, students, residents, or the lay public. Please make check payable to the CENTER FOR HEALTH EDUCATION, and mail with application to: Assistant Director, Center for Health Education, 2801 Atlantic Avenue (P.O. Box 1428), Long Beach, CA 90801 [MAP=University of California, Irvine\"] DISTINGUISHED LECTURE SERIES In Basic and Medical Neuroscience October 5, 1983 to May 2, 1984 This lecture series provides a survey of the newest and most exciting topics in the basic and medical neurosciences by the leaders of the field. This research has an impact on the diagnosis, treatment and causes of mental disease, neurological illness and on each person's view of himself. All lecturers are internationally known for their research achievements and for their ability to present their story in an exciting and clear manner to scientists, physicians and the public at large. This series is ideal for persons involved in the health care system and those who enjoy learning about basic and medical neurosciences and now wish to hear about it from the scientists who made the discoveries. Several topics are immediately clinically relevant, both for the diagnosis and treatment of disease. Others involve new findings in the basic neurosciences which are certain to have wide application in the future.", "University of California, Irvine", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-edum-mqsr~7wqd", "00000000-0000-0000-D3F2-C3527DF5A04F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics [summary of laboratory projects]", "101584910X251", "101584910X250", "1979", "September 1979", "This annual report includes a summary of research undertaken in the Laboratory of Biochemical Genetics and specific descriptions of two projects listing Nirenberg as the principal investigator: 1) Receptor Mediated Regulation of Adenylate Cyclase; and 2) Cell Recognition and Synapse Formation.  Major findings, significance to biomedical research, and associated publications are detailed.", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "6", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Annual Report of the Laboratory of Biochemical Genetics National Heart, Lung, and Blood Institute October 1, 1978 through September 30, 1979 Biochemistry of Synaptogenesis. Clonal lines of hybrid cells derived by fusion of neuroblastoma cells with other cell types were shown previously to form synapses with striated muscle cells with high frequency. The formation of synapses between clonal cells of neural origin, such as NBr10A or NG108-15 hybrid cells, and rat striated muscle cells was found to be regulated. Exposure of hybrid cells for 3-7 days to PGE1, which results in activation of adenylate cyclase, or exposure to various cyclic nucleotide phosphodiesterase inhibitors markedly increases the number of synapses formed. The effects of putative neurotransmitters or hormones on intracellular cyclic AMP or cyclic GMP levels, voltage-sensitive Ca2+ channel activity, and acetylcholine secretion were determined. Receptor-mediated increases in intracellular cyclic AMP or cyclic GMP levels had no immediate effect on K+-dependent 45Ca2+ uptake by cells or on acetylcholine secretion from cells. However, prolonged exposure of hybrid cells to PGE1 results both in an increase in cellular cyclic AMP and the gradual acquisition by cells of functional voltage-sensitive Ca2+ channels. Concomitantly cells acquire the ability to secrete acetylcholine in response to a depolarizing stimulus and can then form functional synapses with muscle cells. These results show that the acquisition of voltage-sensitive Ca2+ channels is regulated and that this reaction in turn controls the formation of synapses. D600 inhibits 45Ca2+ uptake dependent on 80 mM K+ (IC50 = 2 x 10-7 M), but has little or no effect on 45Ca2+ uptake in the presence of 5mM K+. 45Ca2+ uptake also is inhibited by 10 mM La3+, Co2+, Ni2+, Mn2+, Sr2+, or Ba2+, but not by 10 uM tetrodotoxon, 20 mM tetraethylammonium, or 1 mM 3,4-diaminopyridine. Other cell lines were found that synthesize acetylcholine but do not form synapses with striated muscle cells. Various types of synapse defects were detected; including defects in voltage-sensitive 45Ca2+ channels, vesicles, and an additional unidentified reaction that is required for acetylcholine secretion. To identify molecules required for synaptogenesis or communication across the synapse, hybrid cell lines which synthesize mono-specific antibodies were obtained by fusion of clonal myeloma cells with spleen cells immunized against cells from the nervous system. Some of the hybridoma cell lines that were obtained synthesize monospecific antibodies of high titre directed against membrane antigens found on some cells from the nervous system that were not detected with cells from other tissues. One of these cell lines, A2B5, synthesizes antibody directed against an antigen that was shown by indirect immunofluorescence to be associated with plasma membranes of most, or all, neuron cell bodies in chick retina; however, the antigen was not detected on axons or dendrites of neurons, on retina Muller cells, or pigment cells, or on cells from non-neural tissues. Antigen A2B5 activity is relatively stable at 100 degrees C, is insensitive to trypsin, exhibits the solubility properties of a ganglioside, and is destroyed by neuraminidase. Antibody A2B5 cytotoxicity against retina cells is inhibited by a tetrasialo GQ ganglioside fraction from bovine brain (estimated half-maximal inhibition, 0.2 uM), or N-acetylneuraminic acid (half-maximal inhibition, 5,000 uM), but not by other purified gangliosides tested. These results suggest that the antigen is a GQ ganglioside in plasma membranes of retina neuron cell bodies but not membranes of axons or dendrites. A solid-phase 125 I-Protein A radioassay for anti-cell surface antibodies was devised which employs target cell monolayers cultured on fenestrated polyvinyl chloride 96-well plates (\"transfer plates\"). The calibrated aperture in the bottom of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to enter the wells when transfer plates are lowered in receptacles containing washing buffer or test sera. To assay for antibodies directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with corresponding 96-well geometry, thereby simultaneously sampling 96 wells. This assay allows rapid screening of hundreds of hybrid cell colonies for production of antibodies with desired specificity. Methyltransferases can be inhibited by S-adenosyl homocysteine or by analogs which either increase S-adenosyl homocysteine levels or inhibit methyltransferases directly such as 3-deazaadenosine(DZA), adenosine-2',3'-diazido-5'-carboxamide (744-99), 5'-deoxy-5'-isobutylthioadenosine(SIBA), and 5'-deoxy-5'-isobutylthio-3-deazaadenosine (DZ-SIBA). In collaboration with P. Chiang and G. Cantoni the effects of these and other compounds on synapses between dissociated chick embryo retina neurons and cultured rat striated muscle cells were investigated. The frequency of spontaneous synaptic responses of muscle cells was markedly reduced by these compounds; half-maximal inhibition was obtained with 1.5 x 10-6 M DZ-SIBA, 1.5 x 10-5 M DZA, 3 x 10-5 M SIBA, or 1 x 10-4 M 744-99. Homocysteine thiolactone, 5-deoxy-adenosine, or tubercidin, which do not in- crease levels of S-adenosine homocysteine or inhibit methyltransferase activity, do not affect the frequency of spontaneous synaptic responses of muscle cells. These results suggest that a transmethylation reaction may be required for acetylcholine secretion or vesicle cycling in synaptic terminals of neurons. Regulation of Adenylate Cyclase of Cell Lines From The Nervous System. The inhibition of adenylate cyclase by morphine and the gradual increase in adenylate cyclase activity that results when NG108-15 cells are incubated for 12 or more hours in the presence of morphine was previously proposed as a model for the analgesic action of opiates and for the phenomena of opiate dependence and tolerance. We now find that linoleic acid or serum lipids are required for the morphine-dependent increase in adenylate cyclase activity, but not for inhibition of the enzyme. Similar results were obtained with norepinephrine which activates a-receptors of NG108-15 cells, In this model system, therefore, the inhibition of NG108-15 adenylate cyclase by morphine or norepinephrine can be dissociated from the acquisition of dependence upon opiates or norepinephrine. Ten uM morphine or norepinephrine completely inhibit the activation of adenylate cyclase by Ca2+ ions, but inhibit basal or PGE1-activated adenylate cyclase by no more than 55 percent in NG108-15 homogenates. The extent of inhibition of adenylate cyclase by morphine or norepinephrine thus is a function of the Ca2+ ion concentration and the proportion of adenylate cyclase molecules that are activated by Ca2+ ions. Activation of serotonin receptors of NG108-15 or NCB-20 hybrid cells by serotonin results in cell depolarization, action potentials, and secretion of acetylcholine into the medium. These responses desensitize in less than 15 sec and are not inhibited or mimicked by LSD. Serotonin also stimulates adenylate cyclase activity of NCB-20 hybrid cells, but the effect of serotonin does not desensitize. Eadie-Scatchard analysis suggests a bimolecular interaction and reveals no evidence of receptor heterogeneity. The Hill interaction coefficient is 1.0, indicating independent, noncooperative reactions. LSD activates adenylate cyclase (Kact = 12 nM) and also inhibits the activation of the enzyme by serotonin (Ki = 10 nM). In addition, mianserin and cyproheptadine inhibit serotonin activation of adenylate cyclase K1 = 43 nM and 95 nM, respectvely) and LSD activation of adenylate cyclase (Ki = 100 nM and 64 nM, respectively). These results show that serotonin and LSD interact during activation of adenylate cyclase. Binding sites for [3H]LSD were detected in NCB-20 homogenates; the Kdapp was 36 nM, the Hill coefficient was 1.0, and the receptor concentration was 385 fmol/mg of protein. [3H]LSD was displaced by serotonin (Ki = 110-180 nM). These results agree well with those found to be mediated by a serotonin receptor responsive to LSD that mediates activation of adenylate cyclase. Two binding sites for [3H]serotonin were detected in NCB-20 homogenates [Kdapp = 200 nM and 3750 nM] and serotonin-LSD interactions also were detected. We conclude that NCB-20 hybrid cells possess two species of serotonin receptors, one coupled to activation of adenylate cyclase, the other to cell depolarization and acetylcholine release; that activation of adenylate cyclase does not affect the rate of acetylcholine release, and, conversely, that serotonin-dependent cell depolarization does not affect intracellular levels of cAMP or cGMP in the hybrid cells tested. Muscarinic Acetylcholine Receptors. [3H]-Quinuclidinyl-benzilate (QNB) was used to study muscarinic acetylcholine receptors in NG108-15 membrane preparations. The apparent dissociation constant of [3H]-QNB is 1 x 10-10 M; the average NG108-15 cell possesses 30,000 specific sites for [3H]-QNB. Activation of the receptors with acetylcholine or carbachol results in cell depolarization, a small increase in cellular cGMP, and inhibition of adenylate cyclase. Cell depolarization and rise in cGMB levels desensitize in 30 sec; whereas, the inhibition of adenylate cyclase does not desensitize. Scatchard analysis revealed only one homogeneous class of [3H]-QNB binding sites; however, biphasic rates of [3H]-QNB association with and dissociation from receptors were found. Evidence was obtained for the formation of a dissociable [[3H]-QNB-Receptor] complex which then is converted to a form which dissociates only slowly. Hill coefficients of approximately 1.0 were found for receptor antagonists and approximately 0.5 for receptor activators. A sequential series of reactions were proposed to account for these observations and for the various states of the muscarinic acetylcholine receptor that were detected. Nicotinic Acetylcholine Receptors. An aBT-horseradish peroxidase conjugate was used to study the distribution of nicotinic acetylcholine receptors in developing chick retina. Incubation of the retina in vitro with the conjugate allowed quantitative comparison of developmental stages. aBT-binding synapses were found at the early stages of synapse formation and comprised between 5 and 11% of the inner plexiform layer synapse population during in ovo development. The acetylcholine receptor aggregation factor from neuroblastoma x glioma hybrid cells was partially purified by ion exchange chromatography, gel filtration, and  preparative isoelectric focusing.  Factors with similar activity were detected in embryonic brain and cultures of sympathetic ganglion neurons and spinal cord neurons, but not in liver, adult brain, or embryonic glial cell cultures. Detergent treatment under appropriate conditions removed most lipid and soluble protein from cultures skeletal muscle cells, but left the cytoskeleton and bound components intact.  This extraction was used to distinguish tightly bound and loosely bound populations of acetylcholine receptors, which may be correlated with the degree of receptor aggregation. Endorphin Synthesis and Secretion.  AtT-20 mouse pituitary tumor cells were shown to synthesize and secrete B-endorphin (B-lipotropin61-91).  The cells contain at least 1 nmole B-endorphin equivalents of opioid peptides per mg cell protein.  Analysis of cell extracts by gel filtration and high pressure liquid chromatography indicate that the activity is due to B-endorphin, a-endorphin (B-lipotropin61-76), and y-endorphin (B-lipotropin61-77), in the approximate proportions 70%, 24%, and 6%, respectively. Subcellular fractionation indicated that most of the activity is located in the granular fraction. Electron microscopy revealed the presence of osmiophilic granules resembling the secretory granules of corticotrophs of the anterior pituitary.  These granules were positive for B-endorphin/B-lipotropin immunoreactivity, when assayed.  Thus, AtT-20 tumor cells possess a mechanism similar to that of normal endocrine cells for packaging peptides destined for secretion. In the absence of serum, basal secretion of B-lipotropin/B-endorphin immunoreactivity is 20-30 pmoles per mg protein per hr and secretion is linear for at least 12 hr.  Fifty to 70% of the immunoreactivity secreted is due to B-linotropin-like peptides and the rest to B-endorphin-like peptides.  Thus much B-lipotropin is secreted with further processing. Secretion is stimulated 5-8 fold by brief exposure of cells to elevated K+ ion concentration; this stimulation is dependent on Ca++ ions.  Glucocorticoids, such as dexamethasone, reduce the secretion of B-lipotropin/B-endorphin within 2 hr; for example, secretion is reduced by 33% or 67% after 2 or 8 hrs, respectively.  During this time the intracellular content remains the same, however, intracellular content diminishes after 24 hr of treatment. The half-maximally effective dexamethasone concentration is 2 nM.  The effect of dexamethasone on secretion is abolished by cycloheximide or actinomycin D which inhibit protein synthesis and RNA synthesis, respectively.  This suggests that glucocorticoids act at the transcriptional level to induce the synthesis of protein(s) which inhibit the secretion of ACTH and B-endorphin. Other workers have shown recently that corticotropin/S-lipotropin mRNA is gradually reduced by glucocorticoid treatment for 1-4 days.  The present results with AtT-20 cells suggest that glucocorticoids have an earlier different effect on secretion than the slower reduction in mRNA for the prohormone. Cyclic Nucleotides In E. Coli.  Our previous studies led to the development of a model for the regulation of adenylate cyclase involving the phosphoenolpyruvate:sugar phosphotransferase system (PTS).  The proposal has been made that Enzyme I of the PTS interacts in a regulatory sense with the catalytic unit of adenylate cyclase: Phosphoenolpyruvate + Enzyme I--Enzyme I-P  sugar sugar phosphate pyruvate stimulates Adenylate Cyclase The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of 'adenylate cyclase.  The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase.  Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase.  Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase.  While PEP activates the enzyme, either glucose or pyruvate inhibit it.  The unique relationships of PEP and Enzyme I to adenylate cyclase activity provide further support for the model outlined above. The interaction of adenylate cyclase with sugars that are transported by systems other than the PTS also were explored.  Sugars such as lactose are transported without modification by a mechanism involving proton cotransport; this mechanism requires a proton motive force across the cell membrane.  We have been able to show that uptake of sugars through the lactose transport system results in inhibition of adenylate cyclase activity if the proton symport mechanism is also active.  The protonophore carbonyl cyanide m-chlorophenylhydrazone also inhibits adenylate cyclase activity.  These data suggest that the steady-state electrochemical proton gradient regulates the activity of adenylate cyclase.  We propose that sugar-dependent inhibition of adenylate cyclase activity may occur by either of two mechanisms.  Sugars transported by the PTS inhibit adenylate cyclase activity by dephosphorylation of a regulatory protein, while sugars transported by the proton motive force system inhibit adenylate cyclase activity as a result of collapse of the proton electrochemical gradient. Metabolism of Thyrotropin Releasing Hormone.  Previously we described an enzyme (pyroglutamate aminopeptidase) in brain extracts that converts TRH to histidyl-prolineamide which spontaneously cyclizes to histidyl-proline ketopiperazine. We also presented evidence of the presence in hypothalamic extracts of an enzyme (TRH deamidase) that converts TRH to pryoglutamyl-histidyl proline. Further studies have led to the isolation from brain of an imidopeptidase for histidyl-prolineamide not previously described.  The enzyme was found in extracts of porcine brain acetone powder and purified by conventional column chromatography on DEAE cellulose resulting in the separation of the enzyme from other enzymes that metabolize TRH.  The best substrates for the imidopeptidase contain an a-amino group on histidine and a blocked carboxyl group on proline, as is found in histidyl-prolineamide.  Other polypeptide hormones were shown to inhibit imidopeptidase activity.  Inhibition of the enzyme by adrenocorticotropic hormone. (l-24) is noncompetitive.  These studies have led us to propose that pituitary hormones may stimulate the production of histidyl-proline diketopiperazine by inhibiting alternate routes of TRH metabolism. The Biological Activity of Histidyl-Proline Diketopiperazine. Previously we showed that injection of radioactive TRH into rat brain led to the formation of radioactive histidyl-proline diketopiperazine, establishing this compound as a naturally occurring brain peptide.  While TRH could antagonize the effects of ethanol in inducing sleep in rats, the dipeptide diketopiperazine was substantially more active than TRH.  We therefore suggested that the activity of TRH in antagonizing ethanol narcosis may require its conversion to histidyl-proline diketopiperazine. We have continued to explore the biological activities of histidyl-proline diketopiperazine and find that it plays a role in thermoregulation and in the regulation of brain cyclic nucleotide levels. Intraventricular administration of histidyl-proline diketopiperazine to rats produces a dose-dependent hypothermia at 4 degrees or 24 degrees, but not at 31 degrees. At 4 degrees administration of TRH elicits a dose-dependent hypothermia up to 0.1 umole/Kg which is not evoked at higher doses.  At 24 degrees, TRH administration results in no change in temperature, whereas it induces hypothermia at 31 degrees. At 4 degrees, TRH antagonizes and TRH antiserum potentiates the hypothermic effects of histidyl-proline diketopiperazine, suggesting opposing effects of TEUJ and histidyl-proline diketopiperazine on thermoregulation. Intraperitoneal administration of thyrotropin releasing hormone (50 umole/Kg) produced an approximately 2-fold increase in rat brain cGMP concentration with- in 15 min.  Histidyl-proline diketopiperazine produced a similar effect, but the response was faster and shorter-lasting.  Intraperitoneal administration of ethanol (1.5 g/Kg) decreased brain cGMP concentration approximately 50% within 10-15 min; thyrotropin releasing hormone or histidyl-proline diketopiperazine, injected 5 min after ethanol, antagonized the ethanol-induced increase in cGMP.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bfk6.xwbx.4cw3", "00000000-0000-0000-D716-B225F98AB2AA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Receptor Mediated Regulation of Adenylate Cyclase\"", "101584910X252", "101584910X250", "1979", "September 1979", "Summary of work for this project as indicated on the report: \"Receptor-mediated activation and inhibition of adenylate cyclase of neuroblastoma x glioma hybrid cells and other cell lines were studied.\"", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00002-61 LBG Period Covered: October 1, 1978 - September 30, 1979 Title of Project: Receptor Mediated Regulation of Adenylate Cyclase. Names, Laboratory and Institute affiliations, and titles of principal investigations and all other professional personnel engaged on the project: PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI OTHERS: Douglas Wilkening, PRAT Fellow, LBG, NHLBI John MacDermot, Visiting Fellow, LBG, NHLBI Saburo Ayukawa, Visiting Fellow, LBG, NHLBI Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLI, NIH, Bethesda, Maryland 20205 Total Man Years: 3.5 Professional: 3.0 Other: 0.5 Summary of Work: Receptor-mediated activation and inhibition of adenylate cyclase of neuroblastoma x glioma hybrid cells and other cell lines were studied. Project Description: Major Findings: The inhibition of adenylate cyclase by morphine and the gradual increase in adenylate cyclase activity that results when NG108-15 cells are incubated for 12 or more hours in the presence of morphine was previously proposed as a model for the analgesic action of opiates and for the phenomena of opiate dependence and tolerance.  We now find that linoleic acid or serum lipids are required for the morphine-dependent increase in adenylate cyclase activity, but not for inhibition of the enzyme. Similar results were obtained with norepinephrine which activates alpha-receptors of NG108-15 cells.  In this model system, therefore, the inhibition of NG108-15 adenylate cyclase by morphine or norepinephrine can be dissociated from the acquisition of dependence upon opiates or norepinephrine. Ten mu-M morphine of norepinephrine completely inhibit the activation of adenylate cyclase by Ca2+ ions, but inhibit basal or PGE1-activated adenylate cyclase by no more than 55 percent in NG108-15 homogenates. The extent of inhibition of adenylate cyclase by morphine or norepinephrine thus is a function of the Ca2+ ion concentration and the proportion of adenylate cyclase molecules that are activated by Ca2+ ions. Activation of serotonin receptors of NG108-15 or NCB-20 hybrid cells by serotonin results in cell depolarization, action potentials, and secretion of acetylcholine into the medium.  These responses desensitize in less than 15 sec and are not inhibited or mimicked by LSD.  Serotonin also stimulates adenylate cyclase activity of NCB-20 hybrid cells, but this effect of serotonin does not desensitize.  Eadie-Scatchard analysis suggests a bimolecular interaction and reveals no evidence of receptor heterogeneity.  The Hill interaction coefficient is 1.0, indicating independent, noncooperative reactions.  LSD activates adenylate cyclase (Kact = 12 nM) and also inhibits the activation of the enzyme by serotonin (Ki - 10 nM).  In addition, mianserin and cyproheptadine inhibit serotonin activation of adenylate cyclase (Ki = 43 nM and 95 nM, respectively) and LSD activation of adenylate cyclaae (Ki - 100 nM and 64 nM, respectively).  These results show that serotonin and LSD interact during activation of adenylate cyclase. Binding sites for [3H]LSD were detected in NCB-20 homogenates; the KDapp was 36 nM, the Hill coefficient was 1.0, and the receptor concentration was  385 fmol/mg of protein.  [3H]LSD was displaced by serotonin (Ki = 110-180 nM).  These results agree well with those found to be mediated by a serotonin receptor responsive to LSD that mediates activation of adenylate cyclase.  Two binding sites for [3H]serotonin were detected in NCB-20 homogenates [KDapp = 200 nM and 3750 nM] and serotonin-LSD interactions also were detected. We conclude that NCB-20 hybrid cells possess two species of serotonin receptors, one coupled to activation of adenylate cyclase, the other to cell depolarization and acetylcholine release; that activation of adenylate cyclase does not affect the rate of acetylcholine release, and, conversely, that serotonin-dependent cell depolarization does not affect intracellular levels of cAMP or cGMP in the hybrid cells tested. Significance to Biomedical Research: The results suggest that fatty acids may be required for cellular acquisition of opiate dependence and tolerance and that the analgesic action of morphine may be uncoupled from the acquisition of morphine dependence and tolerance. Publications: 1.  McDermot, J., Higashida, H., Wilson, S. P., Matsuzawa, H., Minna, J. and Nirenberg, M. Adenylate Cyclase and Acetylcholine Release Regulated By Separate Serotonin Receptors Of Somatic Cell Hybrids, Proc. Natl. Acad. Sci. USA 76, 1135-1139 (1979). 2.  Wilkening, D., and Nirenberg, M.  A Lipid Requirement For Acquisition Of Opiate Or Epinephrine Dependence By Neuroblastoma x Glioma Hybrid Cells, J. Neurochem., In Press. 3.  Wilkening, D., Sabol, S. L., and Nirenberg, M. Control of Opiate Receptor-Adenylate Cyclase Interactions By Calcium Ions and Guanosine-5'-Triphosphate, Brain Res., In Press. 4.  Sabol, S. L., and Nirenberg, M.  Regulation of Adenylate Cyclase Of Neuroblastoma x Glioma Hybrid Cells By alpha-Receptors, I. Inhibition Of Adenylate Cyclase Mediated By alpha-Receptors, J. Biol. Chem. 254, 1913-1920 (1979). 5.  Sabol, S. L., and Nirenberg, M.  Regulation Of Adenylate Cyclase Of Neuroblastoma x Glioma Hybrid Cells By alpha-Adrenergic Receptors.  II. Long-lived Increase Of Adenylate Cyclase Activity Mediated By alpha-Receptors.  J. Biol. Chem. 254, 1921-1926 (1979).", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; MacDermot, John ; Ayukawa, Saburo ; Wilkening, Douglas", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jtvc-2yhh~3zj4", "00000000-0000-0000-71A3-637AF602C101", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X253", "101584910X250", "1979", "September 1979", "Summary of work for this project as indicated on the report: \"Regulatory reactions were identified that turn synapses on or off.\"", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "4", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00009-05 LBG Period Covered: October 1, 1978 - September 30, 1979 Title of Project: Call Recognition and Synapse Formation. Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI OTHER: Andrej Rotter, Visiting Fellow, LBG NHLBI Radharaman Ray, Staff Fellow, LBG NHLBI Michael Adler, Staff Fellow, LBG NHLBI George Eisenbarth, Research Associate, LBG NHLBI Frank Walsh, Guest Worker, LBG NHLBI Jeffrey Thompson, Staff Fellow, LBG NHLBI Cooperating Units (if any): G. Cantoni and P. Chiang, Laboratory of General and Comparative Biochem., NIMH Lab/Branch: Laboratory of Biochemical Genetics Section: Section of Molecular Biology Institute and Location: NIH, NHLBI, Bethesda, Maryland 20205 Total Man Years: 7.5 Professional: 6 Other: 1.5 Summary of Work: Regulatory reactions were identified that turn synapses on or off. Project Description: Major Findings:  The formation of synapses between clonal cells of neural origin, such as NBr10A or NG108-15 hybrid cells, and rat striated muscle cells was found to be regulated.  Exposure of hybrid cells for 3-7 days to PGE1, which results in activation of adenylate cyclase, or exposure to various cyclic nucleotide phosphodiesterase inhibitors, markedly increases the number of synapses formed. The effects of putative neurotransmitters or hormones on intra-cellular cyclic AMP or cyclic GMP levels, voltage-sensitive Ca2+ channel activity, and acetylcholine secretion were determined.  Receptor-mediated increases in intracellular cyclic AMP or cyclic GMP levels had no immediate effect on K+-dependent 45Ca2+ uptake by cells or on acetylcholine secretion from cells.  However, prolonged exposure of hybrid cells to PGE1 results both in an increase in cellular cyclic AMP and the gradual acquisition by cells of functional voltage-sensitive Ca2+ channels.  Concomitantly cells acquire the ability to secrete acetylcholine in response to a depolarizing stimulus and can then form functional synapses with muscle cells. D600 inhibits 45Ca2+ uptake dependent on 80 mM K+ (IC50 = 2 X 10^(-7) M), but has little or no effect on 45Ca2+ uptake in the presence of 5 mM K+.  45Ca2+ uptake also is inhibited to 10 mM La3+, Co2+, Ni2+, Mn2+, Sr2+, or Ba2+, but not by 10 mu-M tetrodotoxin, 20 mM tetraethylammonium, or 1 mM 3,4-diaminopyridine Other cell lines were found that synthesize acetylcholine but do not form synapses with striated muscle cells.  Various types of synapse defects were detected; including defects in voltage-sensitive 45Ca2+ channels, vesicles, and an additional unidentified reaction that is required for acetylcholine secretion.  These results show that cell lines with or without defects in synapse formation can be generated and that voltage-sensitive Ca2+ channel activity can be regulated by a receptor-mediated reaction which is coupled to activation of adenylate cyclase, or by inhibition of adenlyate clyclase, or by inhibition of cyclic nucleotide phosphodies-terase.  Voltage-sensitive Ca2+ channel activity increases slowly over a period of days and this reaction is required for stimulus-dependent secretion of transmitter and the formation of functional synapses. To identify molecules required for synaptogenesis or communication across the synapse, hybrid cell lines which synthesize mono-specific antibodies were obtained by fusion of clonal myeloma cells with spleen cells immunized against cells from the nervous system.  Some of the hybridoma cell lines that were obtained synthesize mono-specific antibodies of high titre directed against membrane antigens found on some cells from the nervous system that were not detected with cells from other tissues.  One of these cell lines, A2B5, synthesizes antibody directed against an antigen that was shown by indirect immunofluorescence to be associated with plasma membranes of most, or all, neuron cell bodies in chick retina; however, the antigen was not detected on axons or dendrites of neurons, on retina Muller cells, or pigment cells, or on cells from non-neuraltissues. Antigen A2B5 activity is relatively stable at l00C, is insensitive to trypsin, exhibits the solubility properties of a ganglioside, and is destroyed by neuraminidase.  Antibody A2B5 cytotoxicity against retina cells is inhibited by a tetrasialo GQ ganglioside fraction from bovine brain (estimated half-maximal inhibition, 0.2 uM), or N-acetylneuraminic acid (half-maximal inhibition, 5,000 uM), but not by other purified gangliosides tested. These results suggest that the antigen is a GQ ganglioside in plasma membranes of retina neuron cell bodies but not membranes of axons or dendrites. A solid-phase 125I-Protein A radioassay for anti-cell surface antibodies was devised which employs target cell monolayers cultured on fenestrated poly-vinyl chloride, 96-well plates (\"transfer plates\").  The calibrated aperture in the bottom of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to enter the wells when transfer plates are lowered in receptacles containing washing buffer or test sera.  To assay for antibodies directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with corresponding 96-well geometry, thereby simultaneously sampling 96 wells.  This assay allows rapid screening of hundreds of hybrid cell colonies for production of antibodies with desired specificity. Methyltransferases can be inhibited by S-adenosyl homocysteine or by analogs which either increase S-adenosyl homocysteine levels or inhibit methyl-transferases directly such as 3-deazaadenosine (DZA), adenosine-2',3'-deazido- 5'-carboxamide (744-99), 5'-deoxy-5'-isobutylthioadenosine (SIBA), and 5'-deoxy- 5'-isobutylthio-3-deazaadenosine (DZ-SIBA).  In collaboration with P. Chiang and G. Cantoni the effects of these and other compounds on synapses between dissociated chick embryo retina neurons and cultured rat striated muscle cells were investigated to determine whether inhibition of transmethylation affects synapse formation, acetylcholine release, or muscle responses to acetylcholine mediated by nicotinic acetylcholfne receptors.  The frequency of spontaneous synaptic responses of muscle cells was markedly reduced by these compounds; half-maximal inhibition was obtained with 1.5 x 10^-6 M DZ-SIBA, 1.5 x 10^(-5) M DZA, 3 x 10^(-5) M SIBA, or 1 x 10^(-4) M 744-99.  DZ-SIBA reduced the frequency of muscle synaptic responses by 50 percent in 3.5 minutes via a reaction which exhibits first-order kinetics.  Homocysteine thiolactone, 5-deoxy-adenosine, or tubercidin, which do not increase levels of S-adenosine homoeysteine or inhibit methyltransferase activity, do not affect the frequency of spontaneous synaptac responses of muscle cells.  However, homocysteine thiolactone potentiates the inhibition of muscle synaptic responses by DZA by 6-fold.  These results suggest that a transmethylation reaction may be required for acetylcholine secretion or vesicle cycling in synaptic terminals of neurons. Significance to Biomedical Research:  Cultured cell systems have been established and used as model systems for biochemical and electrophysiological studies on synapses.  A reaction was found that regulates synapse plasticity. Proposed Course:  Current studies focus on determining the reactions which are required for synapse formation and termination and factors regulating these reactions. Publications: 1.  Nirenberg, M., Wilson, S., Higashida, H., Thompson, J., Eisenbarth, G ., Walsh, F., Rotter, A., Kenimer, J., and Sabol, S.  Synapse Plasticity.  In Pontificiae Academiae Scientarvm Scripta Varia, In Press. 2.  Eisenbarth, G. S., Walsh, F. S., and Nirenberg, M. Monoclonal Anti-body To A Plasma Membrane Antigen of Neurons, Proc. Natl. Acad. Sci., In Press. 3.  Schneider, M. D., and Eisenbarth, G. S.  Transfer Plate Radioassay Using Cell Monolayers To Detect Anti-Cell Surface Antibodies Synthesized By Lymphocyte Hybridomas, 2. Immunol. Methods, In Press. 4.  Eisenbarth, G. S., Ruffalo, R. R., Walsh, F. S., and Nirenberg, M. Lactose Sensitive Lectin Of Chick Retina And Spinal Cord, Biochem. and Biophys. Res. Comm. 83, 1246-1252 (1978). 5.  McGee, R., Smith, C., Christian, C., Mata, M., Nelson, P., and Nirenberg, M.  A New Method For Measurement Of The Uptake And Release Of Materials From Cultured Cells. Anal. Biochem, In Press. 6.  DeMello, F. G., The Ontogeny Of Dopamine-dependent Increase Of Adenosine 3', 5'-cyclic Monophosphate In The Chick Retina, J. Neurochem.  2, 1049-1053 (1978).", "Thompson, Jeffrey M. ; Eisenbarth, George S. ; Nirenberg, Marshall W. ; Ray, Radharaman ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Rotter, Andrej ; Adler, Michael ; Walsh, Frank (Frank S.), 1953-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p9nj~unau~kuxy", "00000000-0000-0000-A3C0-E144EF6BBF49", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Muscarinic Acetylcholine Receptors of Cultured Cell Lines\"", "101584910X254", "101584910X250", "1979", "September 1979", "Studies on muscarinic acetylcholine receptors focus both on ligand-binding and on defining the physical properties of muscarinic receptors.", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 000012-05 LBG Period Covered: October 1, 1978 - September 30, 1979 Title of Project: Muscarinic Acetylcholine Receptors of Cultured Cell Lines Names, Laboratory and Institute Affiliations, and Titles of Principal Investigations and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI Other:  William L. Kline, Guest Worker, LBG NHLBI Orest Hurko, Staff Associate, LBG NHLBI Cooperating Units (if any): None Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, MD 20205 Total Man Years: 0.25 Professional: 0.25 Other: 0 Summary of Work:  Studies on: muscarinic acetylcholine receptors focus both on ligand-binding and on defining the physical properties of muscarinic receptors. Project Description: Major Findings: [3H]-Quinuclidinyl-benzilate (QNB) was used to study muscarinic acetylcholine receptors in NG108-15 membrane preparations.  The apparent dissociation constant of [3H]-QNB is 1 x 10^(-10) M; the average NG108-15 cell possesses 30,000 specific sites for [3H]-QNB.  Activation of the receptors with acetylcholine or carbachol results in cell depolarization, a small increase in cellular cGMP, and inhibition of adenylate cyclase.  Cell depolarization and rise in cGMP levels desensitize in 30 sec; whereas, the inhibition of adenylate cyclase does not desensitize.  Scatchard analysis revealed only one homogeneous class of [3H]-QNB binding sites; however biphasic rates of [3H]-QNB association with and dissociation from receptors were found.  Evidence was obtained for the formation of a dissociable [[3H]-QNB Receptor] complex which then is converted to a form which dissociates only slowly.  Hill coefficients of approximately 1.0 were found for receptor antagonists and approximately 0.5 for receptor activators.  A sequential series of reactions were proposed to account for these observations and for the various states of the muscarinic acetylcholine receptor that were detected. Publications: 1. Burgermeister, W., Kline, W.L., Nirenberg, M., and Witkop, B., Mol. Pharm. 14, 751-767 (1978). 2. Hurko, O.  Specific [3H]-Quinuclidinyl Benzilate binding activity in digitonin-solubilized preparations for bovine brain.  Arch. Biochem. and Biophys. 190, 434-445 (1978).", "Hurko, Orest ; Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Kline, William L.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-apah.bmd5-2ixu", "00000000-0000-0000-268A-87313BA5E179", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Acetylcholine Receptors\"", "101584910X255", "101584910X250", "1979", "September 1979", "Summary of work on this project as indicated on the report: \"Our aim is to study the distribution of nicotinic acetylcholine receptors in intact and cultured tissues of the peripheral and central nervous system in relationship to the development and function of synapses.  To this purpose histochemical localization of alpha-bungarotoxin bound to the receptors is used in conjunction with light and electron microscopy.  In the past year we have continued our study of the formation of cholinergic synapses in developing chick embryos, using an alpha-bugartoxin(sic)-horseradish peroxidase conjugate; we have extended our studies on the control of nicotinic acetylcholine receptor aggregation on cultured skeletal muscle cells by macromolecular factors secreted by the neuroblastoma-glioma hybrid cells and embryonic neurons; and we have initiated work on the structural interaction between the cytoskeleton and nicotinic acetylcholine receptors in cultured skeletal muscle cells.", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00017-04 LBG Period Covered: October 1, 1978 - September 30, 1979 Title of Project: Acetylcholine Receptors Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Mathew P. Daniels, Research Biologist, LBG NHLBI OTHERS: P. Nelson, Chief, Laboratory of Developmental Neurobiology, LDN NICHD C. Christian, Senior Staff Fellow, LDN NICHD Z. Vogel, Assistant Professor, Weizmann Institute Marshall Nirenberg, Chief, LBG, LBG NHLBI Hans Bauer, Visiting Scientist, LDN NICHD Joav Prives, Guest Worker, LDN NICHD Anne Schaffner, Guest Worker, LBG NHLBI Cooperating Units (if any): Laboratory of Developmental Neurobiology, NICHD Neurobiology Units, Weizmann Institute of Science Lab/Branch: Laboratory of Biochemical Genetics Section:  Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, MD 20205 Total Man Years: 6.5 Professional: 4.5 Other: 2.0 Summary of Work: Our aim is to study the distribution of nicotinic acetylcholine receptors in intact and cultured tissues of the peripheral and central nervous system in relationship to the development and function of synapses.  To this purpose histochemical localization of alpha-bungarotoxin bound to the receptors is used in conjunction with light and electron microscopy.  In the past year we have continued our study of the formation of cholinergic synapses in developing chick retina, using an alpha-bungartoxin-horseradish peroxidase conjugate; we have extended our studies on the control of nicotinic acetylcholine receptor aggregation on cultured skeletal muscle cells by macromolecular factors secreted by neuroblastoma-glioma hybrid cells and embryonic neurons; and we have initiated work on the structural interaction between the cytoskeleton and nicotinic acetylcholine receptors in cultured skeletal muscle cells. Project Description: Methods Employed:  We have used fluorescence staining of monolayer cultured muscle cells with rhodamine-labeled alpha-bungarotoxin (alphaBT) and peroxidase staining of tissues incubated in vitro with peroxidase-labeled alphaBT. These materials are subsequently examined by light or electron microscopy to visualize and quantitate nicotinic acetylcholine receptor sites (AChR). Ion exchange chromatography, ultrafiltration, and isoelectric focusing have been used to characterize and purify the AChR aggregation factor. Primary cultures of dissociated embryonic neurons and serial cultures of clonal cell lines have been grown as sources of AChR aggregating factor. 125 I-alphaBT binding, detergent treatment and light and electron microscopy have been used to study AChR-cytoskeleton interactions. Major Findings:  An alphaBT-horseradish peroxidase conjugate was used to study the distribution of AChR (alphaBT binding sites) in developing chick retina. Incubation of the retina in vitro with the conjugate allowed quantitative comparison of developmental stages.  alphaBT-binding synapses were found at the early stages of synapse formation and comprised between 5 and 11% of the inner plexiform layer synapse population during in ovo development. The AChR aggregation factor from neuroblastoma x glioma hybrid cells was partially purified by ion exchange chromatography, gel filtration, and preparative isoelectric focusing.  Factors with similar activity were detected in embryonic brain and cultures of sympathetic ganglion neurons and spinal cord neurons, but not in liver, adult brain or embryonic glial cell cultures. Detergent treatment under appropriate conditions removed most lipid and soluble protein from cultured skeletal muscle cells, but left the cytoskeleton and bound components intact.  This extraction was used to distinguish tightly bound and loosely bound populations of AChR, which may be correlated with the degree of receptor aggregation. Significance to Biomedical Research:  Knowledge of the ultrastructural distribution of acetylcholine receptors is of clear importance in any attempt to understand the role of neurotransmitters and their receptors in the function and development of the nervous system. The results obtained with developing chick retina represent the beginning of an understanding of the role of neurotransmitter receptors in the formation and maturation of chemical synapses, as seen on the ultrastructural level. The cultured muscle studies may lead to a better understanding of the mechanism whereby neurons control the distribution of receptors on muscle cells and on other neurons. Proposed Course of Research: (1) We have developed a monolayer culture system for physiological and histochemical observation of rabbit retina neurons, which we hope to exploit to learn more about the relationships between alphaBT binding sites and AChR in central neurons. (2) We will continue the biochemical characterization of the AChR aggregation factor, adding immunochemical techniques to the array. We will also continue to probe the cellular specificity of factor formation and target receptor specificity of the factor. (3) We will pursue the study of AChR-cytoskeletal interactions with biochemical and morphological techniques. Publications: 1)  Christian, C.N., Daniels, M.P., Sugiyama, H., Vogel, Z., Jacques, L., and Nelson, G.:  A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells.  Proc. Natl. Acad. Sci. USA 75:  4011-4015 (1978) 2)  Vogel, Z., Towbin, M., and Daniels, M.P.:  alpha-Bungarotoxin-horseradish peroxidase conjugate:  Preparation, properties and utilization for the histochemical detection of receptors to acetylcholine.  J. Histochem. Cytochem. 27:  846-851, 1979.", "Daniels, Mathew P. ; Vogel, Zvi ; Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nelson, Phillip G. ; Bauer, Hans ; Prives, Joav ; Schaffner, Anne ; Christian, Clifford", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e5z2.zryk_uz4k", "00000000-0000-0000-AF5B-AF04AC7929BC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on sequencing and Henikoff method", "101584910X256", null, "1988", "21 March 1988", "These pages from Nirenberg's laboratory notebook elaborate an idea for sequencing genetic information utilizing a method developed by Steven and Jorja Henikoff for finding protein similarities using nucleotide sequence databases.", "Laboratory notes", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "= Sdediey broths 2 ae (ye Keb SA Iose aes ba fon y", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bkb7-qaq5.pt8r", "00000000-0000-0000-E08F-245ACAFC6357", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on strains of E-coli to use to make cDNA library", "101584910X257", null, "1987", "7 September 1987", "This page has a \"To Do\" list which includes making a complementary DNA (cDNA) library for E-coli. Complementary DNA libraries, or collections of DNA nucleotide sequences, eventually allowed for comparing the genetic composition of human cells to those of bovine and rats.", "Laboratory notes", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "\\\\) Dr ee mS PEE ie pe CE EAL a) ded E< A oy 2163 cht? 2) Vee wien im) ESLER TIS EC Uy i9 alg ste, NEB / nares aa pele NS RE caver", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-m449.m5gq-c4ut", "00000000-0000-0000-2C48-263526E43FC5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Robert E. Akins to Marshall W. Nirenberg", "101584910X258", null, "1995", "11 September 1995", "Akins requests cell lines for a research project in the Nemours Children's Clinic at the Alfred I. Dupont Institute on the differentiation and maintenance of muscular tissues and neuromuscular interactions.  Akins provides a summary of research findings and details the plans for using Nirenberg's cell line.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Robert E. Akins.", "Copyright may apply", null, null, "September 11, 1995 Professor Nirenberg, I am writing to request some NG108-15 cells for a research project that is commencing in our lab. We are interested in the role of heme metabolism in the differentiation and maintenance of muscular tissues especially if it has a role in neuro/muscular interactions. As you know, heme catabolism has been implicated in neuronal communication (Verma, et al., 1993). The enzymatic activity of heme oxygenase (with the release of CO, biliverdin IX, and iron) may serve as a retrograde signaling mechanism among neurons, and heme catabolism has been implicated in long term potentiation (Zhuo, et al., 1993; Stevens and Wang, 1993), the modulation of cGMP levels in neurons (Vincent et al., 1994), neurofibrillary pathology in Alzheimer's patients (Smith et al., 1994), as well as carotid body chemoreception (Prabhakar, et al., 1995). In addition, the direct addition of hemin to cultured neuronal cells (Neuro 2a, neuroblastoma cells) has been shown to induce rapid neurite outgrowth (Ishii and Maniatis, 1978). We wish to investigate the possibility that hemin affects NG108-15 differentiation through the activity of heme oxygenase; further, we wish to determine whether the products of heme catabolism are involved in neuronal differentiation. We plan to compare cellular proliferative activity with measures of cellular differentiation (expression of both choline acetyl transferase and monosialyl-ganglioside, GM2). The effects of hemin and heme oxygenase inhibitors on the proliferation and differentiation of NG108-15 cells will indicate the involvement of heme catabolism in neurite outgrowth in cholinergic neurons. In addition, we will examine the role of heme catabolism in the establishment and maintenance of neuromuscular junctions using co-cultures of NG108-15 cells with cardiac or skeletal muscle cells. If possible, please send a vial of NG108-15 cells to me at the address given below. Also, please indicate the formulation of the medium which is currently used to propagate the cells as well as what you would consider to be an appropriate \"splitting\" ratio for propagation of the cells. I look forward to hearing from you. Sincerely, Robert E. Akins PhD Assistant Research Scientist Department of Medical Cell Biology", "Akins, Robert E.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cfay-8q2m_wr8u", "00000000-0000-0000-AA70-8750D60088EE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Regulation of the NK-2 Homeobox Gene in the Developing Nervous System", "101584910X243", null, "1991", "[ca. 1991]", "This abstract for a paper to be delivered at Cold Spring Harbor explains the conditions under which the NK-2 homeobox gene is expressed differently in different organisms.  Results suggest that the NK-2 gene receives and integrates information and then generates cells that are precursors of neuroblasts.", "Abstracts (summaries)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Neurobiology of Drosophila Dervla Mellerick Regulation of the NK-2 Homeobox Gene in the Developing Nervous System. Mellerick, D., Nakayama, K., Nakayama, N., Kim, Y., Webber, K., Lad, R., and Nirenberg, M.; Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland. Nuclei in the ventral half of the Drosophila ventrolateral neurogenic anlage and in the procephalic region initially express NK-2 in late stage 4/early stage 5 embryos.  These nuclei give rise to subsets of NK-2 positive neuroectodermal cells, neuroblasts, ganglion mother cells (GMC), and neurons in the subesophageal ganglion, ventral nerve cord, stomatagastric nervous system and some cephalic ganglia.  NK-2 mRNA also is expressed in the anterior and posterior midgut primordia.  Later in development, NK-2 is detected in the PNS. Initially, NK-2 is expressed in a fairly uniform horizontal stripe, about 7 nuclei in width, on each side of the embryo that extends from 0 to 90% EL.  During gastrulation, the horizontal stripe of NK-2 positive cells is subdivided into 12 vertical stripes due to decreases in NK-2 mRNA in some cells.  As development proceeds NK-2 expression decreases in additional cells resulting in the formation of 2 clusters of NK-2 positive neuroectodermal cells per hemisegment adjacent to the mesectoderm in stage 9 or 10 embryos.  Predominantly medial neruoblasts segregate from these clusters and continue to express NK-2 in GMC and neuronal progeny. Genes that affect NK-2 expression were identified by in situ hybridization in various mutant backgrounds.  In snail mutants, the developmental fate of mesodermal prescursor cells was changed to cells that expressed the NK-2 gene, while in twist/snail double mutants, cells that develop as mesoderm or mesectoderm in wild type embryos expressed NK-2, as well as ventral neuroectodermal cells.  In single-minded mutants, which lack mesectodermal cells, NK-2 expressing neuroectodermal cells, neuroblasts, and their progeny were detected at the ventral midline.  A similar pattern of NK-2 expression was detected in E(splD)m8 mutants.  These results suggest that NK-2 is activated in the ventral 45% of the embryo, presumably by dorsal, but is not expressed in mesoderm due to repression by snail, or in mesectoderm due to repression by single-minded and m8 protein. snail is expressed in neuroblasts, which should repress activation of the NK-2 gene by dorsal.  The 5'-flanking region of the NK-2 gene contains many binding sites for NK-2 protein, which suggests that NK-2 protein may by required to maintain NK-2 gene expression (Wang et al, these abstracts).  Putative sites for dorsal, snail, and m8 overlap, or are adjacent to, many NK-2 protein binding sites.  These results suggest that the NK-2 gene receives and integrates information from the ventral-dorsal and anterior-posterior gradients of gene regulators to generate an alternating pattern of clusters of neuroectodermal cells that are precursors of different types of neuroblasts.", "Nakayama, Noriko ; Nakayama, Kohzo ; Kim, Yongsok ; Nirenberg, Marshall W. ; Webber, Keith O. ; Mellerick-Dressler, Dervla ; Lad, Rajnikant", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xve4_u6va_yjsa", "00000000-0000-0000-8264-5B56BAFAE7D9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Martine F. Roussel to Marshall W. Nirenberg", "101584910X260", null, "1996", "2 April 1996", "Roussel, form the Department of Tumor Cell Biology at St. Jude Children's Research Hospital, asks Nirenberg for some of his mouse neuroblastoma cell line for the study of neuronal differentiation.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Martine F. Roussel.", "Copyright may apply", null, null, "April 2, 1996 Dear Dr. Nirenberg: Dr. James Morgan suggested that I contact you to obtain the mouse neuroblastoma cell line NIE115. We are interested in testing the role of INK4s (inhibitors of G1 cyclin-dependent kinases) in neuronal differentiation. If you are unable to provide us with this cell line, would you let us know who has it so that we can contact them? Our Federal Express # is 0380-0214-7/0246. Please feel to contact me at 901/495-3597 or 3481, FAX at 901/495-2381 or e-mail at martine.roussel@stjude.org should you need additional information. Thank you in advance for your help. Sincerely, Martine F. Roussel, Ph.D. Member Department of Tumor Cell Biology", "Roussel, Martine F. ; St. Jude Children's Research Hospital", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-29wq-tqhm~vsxm", "00000000-0000-0000-9D85-56C62C29B2F9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Draft of \"Annual Report of the Laboratory of Biochemical Genetics\" [summary of laboratory projects]", "101584910X262", "101584910X250", "1977", "21 March 1977", "These drafts and project reports are from a highly prolific period in the Laboratory of Biochemical Genetics.  Major research projects discussed include: 1) regulation of adenylate cyclase; 2) acetylcholine receptors and nervous system development; 3) the development of chick embryo retina; and 4) morphine receptors.  The major findings, significance for biomedical research, and publications are included for each.", "Reports, Excerpts, Drafts (documents)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "6", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "3/21/77 Two topics will be discussed: (A) the phenomena of cell dependence upon narcotics and tolerance to these compounds, and (B) the problem of how neurons form synaptic connections with specificity.  The mouse neruoblastoma x rat glioma hybrid cell line was used as a model system for studies on both topics.  This cell line is one of many somatic cell hybrid and neuroblastoma cell lines which were established during the past decade and characterized with respect to neural properties in the search for experimentally advantageous model systems for problems in neurobiology.  The results which gradually emerged from these studies show that genes for enzymes of neurotransmitter synthesis, receptors, and action potential ionophores which are characteristically expressed by non-dividing neurons, also can be expressed by clonal lines of cells, that the parental programs of gene expressed on are inherited and thus can be perpetuated in a fairly stable fashion, and that somatic cell hybrids can be generated which either have acquired new neural properties not expressed by the parental cell lines or which have lost neural properties. The new programs of gene expression which have been generated also are inherited and thus can be perpetuated easily. Fusion of clonal neuroblastoma cells with normal embryonic neuroblasts or neurons is a particularly promising approach for, in effect, genes expressed by developing neurons can be rescued and may continue to function in clonal hybrid cell lines.  For example, fusion of clonal mouse neuroblastoma cells with mouse embryo sympathetic ganglion neurons yielded a hybrid cell line which had acquired a set of neural properties which were not found with the neuroblastoma parental cells, such as tyrosine hydroxylase activity, small dense core vesicles, and excitatory muscarinic acetylcholine receptors.  Similarly, fusion of clonal mouse neuroblastoma cells with clonal rat glioma cells yielded hybrid cell lines such as NG108-15, which have choline acetyltransferase activity, clear vesicles 500 A in diameter and excitatory muscarinic acetylcholine-receptors; properties not found with the parental cell lines. Most neural properties of clonal cells which have been examined thus far seem to be regulated by environmental factors. Populations of cells can be shifted in synthrony from a poorly differentiated, neuroblsst-like state to a well-differentiated, neuron-like state by either selecting for non-dividing cells, decreasing the rate of cell division, or increasing intracellular levels of cAMP. Neuroblastoma x glioma NG108-15 hybrid cells also synthesize stereospecific high affinity morphine receptors. The average cell has 3 x 10^5 opiate receptors; whereas, the average parental neuroblastoma cell has approximately 0.6 x 10^5 opiate receptors.  Opiate receptors were not detected with glioma parental cells. Morphine and other opiates reduce cAMP levels of intact neuroblastoma x glioma hybrid cells and inhibit basal and PGE1-stimulated adenylate cyclase activity in homogenates.  The effects of opiates are reversed completely by the narcotic antagonist, naloxone, which competes with narcotics for sites on the opiate receptor but lacks the pharmacologic activities of opiates.  The relative affinities of narcotics for the opiate receptor agree well with their effectiveness as inhibitors of adenylate cyclase and with their pharmacologic potency.  Morphine sensitive and insensitive cell lines were found and the degree of sensitivity to morphine was shown to be related to the abundance of opiate receptors. A new class of peptides, the endorphins or enkephalins, discovered in 1975 by Hughes and Kosterlitz and associates, possess properties of morphine and other opiates and are present in mammalian pituitary and in certain neurons.  A pituitary prohormone, Beta-lipotropin, is thought to be a precursor of both the endorphins and Beta-malanotropin.  Endorphins are derived from the carboxy-terminal portion of Beta-lipotropin (residues 61-91) and Beta-melanotropin from residues 41-58. The smallest fragment of Beta-lipotropin which retains opiate activity is methionine-enkephalin which corresponds to Beta-lipotropin (61-65).  Met-enkephalin is the most potent peptide inhibitor of adenylate cyclase known, for the concentration required for half-maximal inhibition of adenylate cyclase is 11 nM. We wondered whether exposure of cells to an opiate for hours or days would result in cell dependence or tolerance to the opiate.  The results showed that growth of NG108-15 cells in the presence of morphine or met-enkephalin for 12 to 48 hours results in an inhibition of adenylate cyclase and in a slow, compensatory increase in adenylate, cyclase activity which is delayed in onset but then can be expressed in the absence of the opiate and is relatively long-lived.  Cells then have normal cAMP levels and appear tolerant to the opiate because the increase in adenylate cyclase activity is approximately equal to the inhibition of enzyme activity by the opiate.  However, the cells are dependent upon the opiate to maintain normal cAMP levels.  If morphine is withdrawn or displaced from the opiate receptor by the opiate antagonist, naloxone, the enzyme is no longer inhibited and abnormally high adenylate cyclase activity is revealed.  Dual regulation of adenylate cyclase by opiates thus accounts for the phenomena of narcotic dependence and tolerance.  Thus opiate peptides and narcotics can acts as pleiotropic regulators of the cells response to neurotransmitters and hormones which are coupled to the activation of adenylate cyclase.  Opiates thus alter the preception of neurons to incoming messages which are destined for adenylate cyclase. NG108-15 hybrid cells also possess a-adrenergic receptors and excitatory muscarinic acetylcholine recpetors which also are coupled to the inhibition of adenylate cyclase.  Cells were cultured in the presence of norepinephrine or carbamylcholine for 10-48 hours, then the effects of withdrawal of the receptor ligand either by replacing the medium or by the addition of a receptor antagonist was tested.  Withdrawal of norepinephrine or carbamylcholine resulted in 9-and 5-fold increases in cAMP levels of intact cells respectively.  Adenylate cyclase activity also increases but to a lesser extent.  Studies on the specificity of receptor antagonists showed that the inhibitions of adenylate cyclase by norepinephrine, acetylcholine or opiates are mediated by different species of receptors.  These results show that dual regulation of adenylate cyclase is a general phenomenon and that cells become dependent upon norepinephrine and acetylcholine as well as opiates.  The cells develop an apparent tolerance but in fact remain sensitive to the compound, NG108-15 hybrid cells synthesize, store, and excrete acetylcholine and generate action potentials in response to electrical stimulation.  When the hybrid cells were cocultured with striated muscle cells which have abundant nicotinic acetylcholine receptors, they were found to form synapses with muscle cells in abundance. Studies on the embryonic development of the mammalian neuromuscular synapse reveal that at an early developmental stage when nicotinic acetylcholine receptors are distributed over the entire surface of the muscle cell, a single muscle cell usually is innervated by multiple neurons.  At this stage transmission across the synapse is relatively inefficient since some, but not all, neuron action potenitals evoke muscle responses.  As the synapse matures the number of synaptic vesicles which release acetylcholine per motor neuron action potential increases 100-300 fold with a concomitant increase in the efficiency of transynaptic communication, and the concentration of nicotinic acetylcholine receptors decreases at all sites on the muscle cell other than the site of one synapse.  Thus, the muscle cell is converted from a permissive state with respect to synapse formation to a nonpermissive state, and each muscle cell then is innervated by only one motor neuron. The synaptic connections which form between NG108-15 hybrid cells and cultured striated muscle cells closely resemble the synapses which form between normal motor neurons and muscle cells at an early developmental stage.  Although some NG108-15 action potentials evoke muscle action potentials, most muscle responses are between the threshold for initiation of action potentials. Little is know at the molecular level about the process of synapse formation and the basis for the specificity of synaptic connections between cells.  Two kinds of hypothesis have been suggested to account for synapse specificity:  (1) that cells prior to synapse formation complimentary molecules are present on the surface of neurons and their synaptic partners which must interact correctly prior to synapse formation; i.e., a molecular cell recognition code for synapse formation, or (3) synaptic circuits assemble with little specificity and then those which function appropriately are maintained by a process of selection whereas the others disappear. This clonal hybrid cell line provides an opportunity to test the specificity of synapse formation.  If the formation of synaptic connections between neurons and muscle cells were coded by specific cell recognition molecules, then discrete classes of muscle cells with different specificities for synpase formation might be expected.  However the results revealed that NG108-15 cells form synapses with clonal muscle cells and with myotubes from different embryonic muscles, and from different organisms such as the chick, mouse and rat.  Most of the muscle cells tested were innervated by hybrid cells.  The results strongly suggest that most striated muscle cells have the same specificity for synpase formation.  No evidence was found for discrete classes of muscle cells with different specificities for synapse formation. In essence the results suggest that the formation of the neuromuscular synapse is not dependent upon a cell recognition code.  The results also indicate that mechanisms of synapse formation are conserved during evolution and thus may be largely universal. The evidence suggest that much of the specificity of the normal neuromuscular synapse is acquired after synpases form by a process of selection which reduces the number of synapses and which is dependent upon effective transmission across the synapse, rather than by a process of matching complimentary molecules on neurons and muscle cells which code for different synaptic connections.  It seems likely that the amount of acetylcholine excretion from the neuron and the distribution of nicotinic acetylcholine receptors on the muscle cells are regulated and that both are involved in the synapse selection process.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j4cr_dcxe_ti4x", "00000000-0000-0000-DB1A-A14B586A0162", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Draft of \"Annual Report of the Laboratory of Biochemical Genetics, October 1, 1975 - September 30, 1976\"", "101584910X263", "101584910X222", "1976", "14 June 1976", "These drafts and project reports are from a highly prolific period in the Laboratory of Biochemical Genetics.  Major research projects discussed include: 1) regulation of adenylate cyclase; 2) acetylcholine receptors and nervous system development; 3) the development of chick embryo retina; and 4) morphine receptors.  The major findings, significance to biomedical research, and publications are included for each.. It appears as though page five is misnumbered.", "Reports, Excerpts, Drafts (documents)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "11", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Fusion of clonal neuroblastoma cells with rat glioma cells yielded clonal hybrid cell lines which synthesize, store and excrete acetylcholine; properties which are not expressed by the parental cell lines.  Cells from one hybrid line were found to form synapses with cultured striated muscle cells.  Synapses between normal hybrid cells and muscle cells closely resemble the synapses between motor neurons and striated muscle before they are fully developed.  Under appropriate conditions, hybrid cells establish synaptic connections with virtually every muscle cell tested; thus, synaptic connections are formed in abundance.  Marked differences were observed in the efficiency of transmission across different synapses.  Axonal activities which were found to be regulated include choline acetyltransferase, acetylcholinesterasa, Na+ action potential ionophore specific activities, and the rate of choline transport into cells.  In muscle ce11s, the distribution of nicotinic acetylcholine receptors is regulated. Eight species of receptors have been found thus far with the hybrid cell line which forms synapses.  Receptor mediated shifts in cAMP levels, cGMP levels and membrane potentials have been identified and characterized.  Thus the foundation has been laid for studies on the effects of receptor mediated reactions on synaptic transmission.  In addition, more than 100 cell lines which synthesize acetylcholine have been obtained.  Preliminary results suggest that some cell lines may be defective with respect to synapse formation. Although these studies are in their infancy, it seems clear that the experimental approach and model systems which have been established afford extraordinary opportunities to explore synapse properties and to correlate biochemical events with developmental and electrophysiological phenomena. Synaptogenesis by normal neurons also was.  Neurons dissociated from chick embryo retina and maintained in vitro reaggregate and were also shown to form in vitro approximately 1 x 10^9 synapses per mg of protein.  Three types of synapses and several subtypes were identified which closely resemble those of the intact retina.  Studies with this system are described in other sections of this report. A preliminary histochemical technique for detecting and localizing nicotinic acetylcholine receptors was devised which depends upon the formation of a complex between peroxidase coupled to an antibody for alpha-bungarotoxin and the nicotinic acetylcholine receptor.  Using this method, clusters of nicotinic acetylcholine receptors on cultured muscle cells were shown to contain at least 7 times the concentration of receptors found in other membrane regions.  Receptor clusters were not characteristically associated with folds in the plasma membrane. The hybrid cells which form synapses possess abundant morphine receptors. Morphine and other narcotics were shown to be potent inhibitors of adenylate cyclase in cells which possess opiate receptors and to be without effect in cells which lack these receptors.  Exposure of cells with opiate receptors to morphine for 12 to 48 hours results in an increase in adenylate cyclase activity which compensates for the inhibition of enzyme activity by morphine.  Cells have normal cAMP levels and appear tolerant to morphine because the increase in adenyalte cyclase activity is approximately equal to the inhibition of enzyme activity by morphine.  However, the cells are dependent upon morphine to maintain normal cAMP levels.  Withdrawal of morphine, or displacement of the narcotic from the opiate receptor by the antagonist, naloxone, reverses the inhibition and results in the synthesis of abnormally high levels of cAMP.  Thus, dual regulation of adenylate cyclase by narcotics accounts for the phenomena of narcotic dependence and tolerance.  The recently discovered endogenous opiate peptides, Met-enkephalin and Leu-enkephalin, also were shown to be potent inhibitors of adenylate cyclase.  The endogenous opiate peptides and narcotics act as pleiotropic regulators of other species of receptors which are coupled to the activation of adenylate cyclase.  Thus opiates alter the perception of cells to messages. The apparent dissociation constant of one liquid receptor complex is 6 x 10^(-11) M. A cell line with muscarinic inhibitory acetylcholine receptors, and another with muscarinic excitatory receptors were found.  The number of receptors, the affinities of receptors for cholinergic ligands and other properties of the receptors were determined by measuring the [3H]-quinuclidinyl benzilate binding of receptors to the muscarinic acetylcholine receptors present, results in a transient increase in cGMP, and a profound long lived inhibition of adenylate cyclase activity.*  Exposure of cells to acetylcholine or carbachol for 24 hours markedly decreases the number of acetylcholine receptors and increases adenylate cyclase activity 50 to 500%.  Removal of carbachol results in the return of adenylate cyclase activity and acetylcholine receptors levels to normal values after approximately 3 and 24 hours, respectively.  Similarly, exposure of cells with a-receptors to norepinephrine inhibits adenylate cyclase activity and elicits a delayed, compensatory increase in adenylate cyclase activity.  These results show that cells with a-receptors, muscarinic acetylcholine receptors, or opiate receptors can become tolerant to and dependent upon norepinephrine, acetylcholine, and opiates, respectively, and that withdrawal of the receptor ligand elevates intracellular cAMP levels and shifts cells to a supersensitive state with respect to other species of receptors which activate adenylate cyclase. Desensitization of muscarinic receptors was found to decrease the affinity of the receptor for ligands which activate the receptor.  Although the interaction of muscarinic acetylcholine receptors with agonists exhibits an apparent negative cooperativity; the interactions between the receptor and antagonist is not a cooperative process.  Both muscarinic excitatory and muscarinic inhibitory acetylcholine receptors were solubilized and the properties of membrane found and soluble receptors were compared. *over Chick embryo retina was found to be a rich source of both muscarinic and nicotinic acetylcholine receptors.  Muscarinic acetylcholine receptors and nicotinic acetylcholine receptors were shown to be associated predominantly with the synaptic layers of the retina.  Both muscariiric and nicotinic acetylcholine receptors are synthesized before synapses-appear in the retina; however, during the development of the retina, nicotinic acetylcholine receptors become associated predominantly with neurites in the synaptic layers of the retina.  The properties of muscarinic acetylcholine receptors were determined at different developmental ages and were compared with the properties of muscarinic inhibitory and excitatory receptors of neuroblastoma cells. Evidence for-a new type of PGE1 receptor coupled to cGMP accumulation was obtained.  Cell lines with PGEl receptors coupled to cAMP were found as well as cell lines with 2 species of PGE1 receptors, one coupled to cAMP accumulation, the other to cGMP accumulation.  PGE1 desensitizes both species of receptors but at different rates.  PGF2alpha receptors coupled to cGMP accumulation can be selectively inactivated without inactivation of PGE1 receptors and vice versa.  These results show that the coupling of PGE1 to cAMP and cGMP are clonally inherited, independently expressed, properties. Two pathways for gamma-aminobutyric acid synthesis were found in chick embryo retina.  The first pathway depends upon the conversion of putrescine to omithine, catalyzed by omithine decarboxylase and the subsequent conversion of omithine to gamma-aminobutyric acid.  The second route of synthesis is dependent upon the conversion of glutamic acid to gamma-aminobutyric acid, catalyzed by glutamic acid decarboxylase.  The omithine decarboxylase pathway accounts for 20% of the gamma-aminobutyric acid synthesized in retina on the 6th embryonic day but only 1% on the 18th embryonic day. Elevation of cAMP levels in neuroblastoma cells results in an induction of ornithine decarboxylase activity.  Thus neurotransmitters which affect cAMP levels may regulate ornithine decarboxylase activity and thus may control the rate of GABA synthesis from ornithine. Previous results led to the conclusion that veratridine, batrachotoxin, and aconitine activate the action potential Na+ ionophore by interaction with a single class of sites; scorpion venom activates the ionophore by interaction with a different class of sites; two species of toxin bound to separate sites are allosterically coupled and interact in a cooperative manner; and tetrodotoxin and saxitoxin act at a 3rd site which is involved in ion transport. A toxin which activates the action potential Na+ ionophore has been purified from scorpion venom.  The toxin binds to a single class of sites and acts cooperatively with each of the three alkaloids.  Depolarization of cells causes a 30-fold increase in the apparent dissociation constant.  The results app suggest that the scorpion toxin binds to a voltage sensitive component of the Na+ ionophore that acts cooperatively in regulating ion transport activity.  Binding studies with an 125I-labeled derivative of scorpion toxin showed that the concentration of toxin binding sites is approximately 3 to 6 fmole per mg protein. Clonal skeletal muscle myoblasts have substantial action potential Na+ ionophore activity.  A small increase in activity accompanies cell fusion. The activity in both myoblasts and myotubes is relatively insensitive to inhibition by saxitoxin and tetrodotoxin and thus resembles denervated rat striated muscle which has been shown to be relatively insensitive to these toxins.  Chronic electrical stimulation of muscle cells in vitro does not increase tetrodotoxin sensitivity. At least 3 ionophores are involved in the action potential in adult heart: a rapidly activated axon-like Na+ ionophore responsible for the rising phase of the action potential, a slower Ca++/Na+ ionophore responsible for the plateau phase, and a K+ ionophore responsible for the repolarization phase.  Studies with specific inhibitors of the fast Na ionophore (tetrodotoxin) and the slow Ca++/Na+ ionophore (D-600) show that the role of these two types of ionophore in beating changes during development of the embryonic chick heart.  In early embryonic hearts, the fast Na+ ionophore is present but is not required for beating.  During development in ovo or in monolayer or aggregate culture in vitro, changes in the requirement for activity of the fast Na+ ionophore in beating are accompanied by changes in the sensitivity of the slow Na+/Ca++ ionophore to D-600.  When the slow Na+/Ca++ ionophore is able to maintain beating without participation of the fast Na+ ionophore, its sensitivity to D-600 is high whereas when the fast Na+ ionophore is required for beating, the slow Na+/Ca++ ionophore is relatively insensitive to D-600.  Transitions between these two states can be induced in 2 hours in vitro by inhibition of beating.  Thus, the activity of these ionphores is regulated during development by a process dependent on the rhythmic activity of the cells. The developmental changes in action potential ionophores of embryonic hearts occur between days 4 and 7 in ovo.  During this time, vagal innervation of the heart takes place.  Consequently we have studied the muscarinic acetylcholine receptors in embryonic hearts using both physiologic and ligand binding methods to determine whether changes in their properties are temporally correlated with changes in the action potential ionophores.  In early embryonic hearts, muscarinic agents are ineffective in inhibiting beating.  Between days 5 and 7, sensitivity to inhibition by muscarinic acetylcholine receptors in embryonic and adult heart as assessed by competition studies with muscarinic and nicotinic agents.  Receptors, as detected by QNB binding, are present in unresponsive early embryonic hearts and the number per mg heart protein does not increase dramatically during development.  Receptor desensitization is accompanied by a small change (3 fold increase) in KD for muscarinic agonists as measured by competition with 2H QNS.  This change in KD occurs only in hearts that are responsive to mucarinic agents and thus may be associated with the physiological action of the receptor.  Competition curves for agonists suggest the involvement of negative cooperativity in activation of the receptor.  These results suggest that muscarinic acetylcholine receptors, like action potential ionophores, are present in early embryonic hearts in modified \"precursor\" or \"inactive\" forms and undergo activation during development. Studies on mechanism of catabolite repression in E. coli show that glucose inhibits adenylate cyclase activity reversibly in cells treated in toluene, and that phosphate is required for high adenylate cyclase activity for glucose dependent inhibition of enzyme activity.  Other sugars also inhibit adenylate cyclase provided that transport systems for the sugars are induced.  Mutant strains of E. coli defective in the phosphoenolpyruvate:sugar phosphotransferase system components were examined.  Cyclic AMP levels were normal in a HPr mutant, but were markedly depressed in a leaky Enzyme I mutant. Adenylate cyclase activity was low in the Enzyme I mutant whereas the HPr mutant had normal enzyme activity.  The Enzyme I mutant under starvation conditions exhibits high adenylate cyclase activity and the adenylate cyclase of this mutant is unusually sensitive to variations in carbon source.  The addition of phosphoenolpynavate leads to a substantial increase in adenylate cyclase activity in permeabilized cell preparations of the Enzyme I mutant.  These results suggest that Enzyme I is involved in the regulation of adenylate cyclase activity.  Studies are in progress to test the possibility that Enzyme I interacts with adenylate cyclase and that the PEP-dependent phosphorylation of Enzyme I is responsible for activation of adenylate cyclase. Levels of cGMP were compared with cAMP levels when E. coli was grown under different conditions.  The results showed: 1) cells starved for a carbon source for a short time have high levels of cAMP and low levels of cGMP. Addition of a carbon source leads to a decrease in cAMP and an increase in cGMP, a bi-directional change.  Washed cells starved for a carbon source for long periods have low cAMP levels which do not respond to the addition of glucose; however, the addition of glucose results in a transient increase in cGMP levels.  The results reveal the presence in E. coli of reactions which inversely couple cGMP and cAMP concentrations and show that the regulation of cGMP levels can be uncoupled from that of cAMP. Since specific species of tRNA are involved in amino acid mediated repression and end-product inhibition, the effect of amino acid deprivation upon tRNA of relaxed and stringent strains of E. coli was studied.  In relaxed control E. coli leucine starvation results in the formation of new isoacceptor species of leucine, histidine, arginine, valine, and alanine-specific tRNA and quantitative changes in the concentration of some other isoacceptors.  Experiments with stringent strains were the use of uracil starvation or rifampicin addition provided evidence for the de novo synthesis of new species of isoacceptor tRNA.  The new species of leucine RNA are not aggregation artifacts, or nuclease-damaged forms of anormal leucine isoacceptor, or grossly deficient with respect to methylation.  Since there is some evidence from the recent work of others that tRNA formed under conditions of amino acid starvation is deficient in the minor bases 5,6-dihydrouridine and 4-thiouridine, we think that the biochemical explanation for the accumulation of new tRNA species under conditions of amino-acid starvation may be that the enzymes responsible for these tRNA modifications are unstable and require continued protein synthesis to maintain their levels of activity. Further information was obtained on the mechanism of arginyl-tRNA synthetase which does not catalyze an amino acid dependent ATP-PPi exchange in the absence of added tRNA.  A new purification procedure was devised which yields homogeneous enzyme in approximately 10% yield.  Pulse labeling experiments indicate that no enzyme bound arginyl-adenylate is formed in the absence of added tRNA.  Equilibrium experiments show that no arginyl-adenylate accumulates either in the presence or absence of tRNAarg.  These data further validate our earlier suggestion that the mechanism of this reaction is probably concerted. A series of additional studies carried out with the pure preparation of arginyl-tRNA synthetase from E. coli indicate that metals may have two functional roles in the catalytic mechanism.  Complete metal activation is observed when MgCl2, MnCl2, CoCl2, or FeCl2 is present at a concentration (5 mM) in excess of the total ATP concentration (2 mM). When CaC12 is substituted for MgC12, activity is not observed unless a small amount (0.1. mM) of MgCl2, MnCl2, CoCl2, FeC12 or ZnCl2 is added.  On the basis of these experiments, we visualize a model in which the enzyme possesses a site for free metal which, when filled, lowers the KM for all three substrates (arginine, tRNAarg, and metal-ATP) and increases the Vmax of the reaction.", "Nirenberg, Marshall W. ; National Heart and Lung Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jjsb-w452-bris", "00000000-0000-0000-7F0F-9BB471FD93A1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on viruses affecting the nervous system", "101584910X259", null, "1987", "15 August 1987", "These laboratory notes list some of the relationships between retroviruses and neurological disease in humans and other animals.  Both RNA and DNA viruses are named.  An idea for comparing the relationship between protein binding and viral enhancers with different tissues or different types of cell lines is included.", "Laboratory notes", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "enn ig et", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-uin5_ev5x_hbxv", "00000000-0000-0000-FBBC-79D5D9B584A4", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Regulation of Adenylate Cyclase by Alpha-Adrenergic Receptors\"", "101584910X264", "101584910X261", "1977", "September 1977", "Summary of work on this project as indicated in the report: \"The role of the cyclic nucleotides adenosine 3':5' monophosphate (cyclic AMP) and guanosine 3':5' monophosphate (cyclic GMP) in synaptic transmission is under study using cultured cells of neural origin.  The topics of interest during the current year have been the following: 1) The alpha-receptor-mediated inhibition of adenylate cyclase activity by norepinephrine in neuroblastoma x glioma hybrid cells; and 2) characterization of a compensatory increase in adeylate cyclase activity in cells treated for 10 hours or longer with norepinephrine and study of the mechanism of this increase, which results in cell tolerance to and dependence on norepinephrine with respect to cyclic AMP synthesis.\"", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00015-02 LBG Period Covered: July 1, 1976 through September 30, 1977 Title of Project: Regulation of adenylate cyclase by alpha-adrenergic receptors Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI OTHER: Steven L. Sabol, Research Associate, LBG NHLBI Saburo Ayukawa, Visiting Associate, LBG NHLBI Lab/Branch: Laboratory of Biochemical Genetics Section:  Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20014 Summary of Work: The role of the cyclic nucleotides adenosine 3':5' monophosphate (cyclic AMP) and guanosine 3':5' monophosphate (cyclic GMP) in synaptic transmission is under study using cultured cells of neural origin.  The topics of interest during the current year have been the following: 1) The alpha-receptor-mediated inhibition of adenylate cyclase activity by norepinephrine in neuroblastoma x glioma hybrid cells; and 2) characterization of a compensatory increase of adenylate cyclase activity in cells treated for 10 hours or longer with norepinephrine and study of the mechanism of this increase, which results in cell tolerance to and dependence upon norepinephrine with respect to cyclic AMP synthesis. Project Description: Objectives:  Alpha-receptor activators such as norepinephrine rapidly lower cAMP levels of NG108-15 cells by inhibiting adenylate cyclase activity.  Furthermore, prolonged exposure of cells to alpha receptor agonists results in an increase in adenylate cyclase activity which compensates for the inhibition.  Similar rapid inhibitions and compensatory increases elicited by opiate and muscarinic cholinergic receptor agonists have been recently observed by others.  During the past year, attempts have been made to characterize these phenomena further and to elucidate the regulatory mechanisms. Major Findings:  NG108-15 hybrid cells possess alpha-adrengeric receptors which in concert with receptor activators inhibit adenylate cyclase.  Cells were cultured in the presence of norepinephrine for 0-48 hours, then the effects of withdrawal of norepinephrine either by replacing the medium or by the addition of a receptor antagonist was tested.  Withdrawal of norepinephrine resulted in a 9-fold increase in cAMP levels of intact cells.  Adenylate cyclase activity also increased but to a lesser extent.  Studies on the specificity of receptor antagonists showed that both the inhibition of adenylate cyclase by norepinephrine and the subsequent increase in adenylate cyclase activity are mediated by alpha-receptors.  These and other results show that dual regulation of adenylate cyclase is a general phenomenon and that cells can become dependent upon norepinephrine, acetylcholine, or opiates.  The cells develop an apparent tolerance to these compounds but in fact remain sensitive to the compound used. NG108-15 alpha-receptors were characterized by studying the specific binding of [3H]-dihydroergocryptine and other ligands to the receptors. The specificity of the binding sites for ligands resembles that of alpha-receptors.  The binding of the ligand to the membrane preparation is a saturable process.  The average NG108-15 cell possesses 60,000 alpha-receptors. Significance to Biomedical Research: 1. The fact that dual regulation of NG108-15 adenylate cyclase has been observed now with three classes of inhibitors each mediated by a different species of receptor, suggests that dual regulation may be a general phenomenon. 2. Norepinephrine released at adrenergic synapses may regulate cAMP levels in post-synaptic or pre-synaptic cells by the mechanism discussed here.  Such regulation may modulate the cell's responsiveness to ligands for other species of receptors which activate adenylate cyclase and thus may affect information transfer in the nervous system. Proposed Course:  The potencies of alpha-receptor activators and antagonists with respect to inhibition of adenylate cyclase will be compared with the effects of ligand binding to NG108-15 alpha-receptors.  The mechanism of coupling inhibition of adenylate cyclase with a subsequent compensatory increase in enzyme activity will be studied further. Effects of alpha-adrenergic activators and antagonists on [3H] dihydroergocryptine binding will be determined to define the specificity of the alpha-receptor and the kinetics of binding.  The regulation of receptor concentration will be studied. Publications: 1.  Archer, Ellen G., Breakefield, Xandra O. and Sharata, Mary N.:  Transport of tyrosine, phenylalanine, trytophan and glycine in neuroblastoma clones.  J. Neurochem. 28: 127-135, 1977.", "Sabol, Steven L. ; Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Ayukawa, Saburo", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-y5ed-a2x4~6gdf", "00000000-0000-0000-043F-7673C3F9259C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Acetylcholine Receptors in the Developing Nervous System\"", "101584910X265", "101584910X261", "1977", "September 1977", "Summary of work for this project as indicated in the report: \"The goal of this project is to define the properties of muscarinic and nicotinic acetylcholine receptors and acetylcholinesterase during development of chick embryo retina.  Thus far we have elucidated (1) the specificity and affinities of muscarinic and nicotinic acetylcholine receptors for receptor activation and antagonists, (2) the number of receptors were defined as a function of developmental age of the retina, and (3) the location of nicotinic and muscarinic receptors within the retina was determined.\"", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00017-02 LBG Period Covered: July 1, 1976 through September 30, 1977 Title of Project: Acetylcholine Receptors in the Developing Nervous System Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Hiroyuki Sugiyama, Visiting Associate, LBG NHLBI Marshall Nirenberg, Chief, LBG, LBG NHLBI Lab/Branch: Laboratory of Biochemical Genetics Section:  Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20014 Total Man Years: 1.6 Professional: 1.3 Other: 0.3 Summary of Work: The goal of this project is to define the properties of muscarinic and nicotinic acetylcholine receptors and acetylcholinesterase during development of chick embryo retina.  Thus far we have elucidated (1) the specificity and affinities of muscarinic and nicotinic acetylcholine receptors for receptor activation and antagonists, (2) the number of receptors were defined as a function of developmental age of the retina, and (3) the location of nicotinic and muscarinic receptors within the retina was determined. Project Description: Objectives:  The objectives are to define the biochemical properties of acetylcholine receptors before and after synaptogenesis in the retina. Major Findings:  Neurons dissociated from chick embryo retina and maintained in vitro were found to reaggregate and form, in vitro, approximately 1 x 10^9 synapses per mg of protein.  Three types of synapses and several subtypes were identified which closely resemble those of the intact retina. Chick embryo retina was found to be a rich source of both muscarinic and nicotinic acetylcholine receptors.  Both muscarinic and nicotinic acetylcholine receptors are synthesized before synapses appear in the retina; however, during development, nicotinic acetylcholine receptors become associated predominantly with neurites in the synaptic layers of the retina.  Muscarinic acetylcholine receptors also were found to localize in the inner synaptic layer of the retina, but the receptor distribution differs from that of nicotinic acetylcholine receptors.  The properties of muscarinic acetylcholine receptors were determined at different developmental ages and were compared with the properties of muscarinic inhibitory and excitatory receptors of neuroblastoma and hybrid cells. Significance to Biomedical Research:  Information was obtained which serves as a basis for further studies on the role of acetylcholine receptors in synapse formation. Proposed Course: Further studies on receptor properties are planned.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Sugiyama, Hiroyuki", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e2zf_d369_zs5z", "00000000-0000-0000-11AB-77DF2BA07F86", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Acetylcholine Receptors\"", "101584910X266", "101584910X261", "1977", "September 1977", "Summary of work for this project as indicated in the report: \"Our aim is to study the distribution of nicotinic acetylcholine receptors in intact and cultured tissues of the peripheral and central nervous system in relationship to the development and function of synapses.  To this purpose histochemical localization of alpha-bungarotoxin bound to the receptors is used in conjunction with light and electron microscopy.  In the past year we have used alpha-bungarotoxin-horseradish peroxidase conjugate to identify the synoptic sites of nicotinic acetylcholine receptors aggregation on cultured skeletal muscle cells by neuroblastoma-glioma hybrid cells and by substances secreted by those cells.\"", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00001-05 LBG Period Covered: July 1, 1976 through September 30, 1977 Title of Project: Acetylcholine Receptors Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Mathew P. Daniels, Staff Fellow, LBG NHLBI OTHER: Marshall W. Nirenberg, Chief, Lab. of Biochem. Genetics, LBG NHLBI P. Nelson, Chief, Behavioral Biology Branch, BB NICHD C. Christian, Special Fellow, BB NICHD G. Maloney, NIH Postdoctoral Fellow, LBG NHLBI Zvi Vogel, Assistant Professor, Weizmann Institute Cooperating Units (if any): Behavioral Biology Branch, NICHD Neurobiology Unit, Weizmann Institute of Science Lab/Branch: Laboratory of Biochemical Genetics Section:  Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20014 Total Man Years: 3.5 Professional: 2.5 Other: 1.0 Summary of Work: Our aim is to study the distribution of nicotinic acetylcholine receptors in intact and cultured tissues of the peripheral and central nervous system in relationship to the development and function of synapses.  To this purpose histochemical localization of alpha-bungarotoxin bound to the receptors is used in conjunction with light and electron microcopy.  In the past year we have used an alpha-bungarotoxin-horseradish peroxidase conjugate to identify the synaptic sites of nicotinic acetylcholine receptors in the chicken retina and studied the control of nicotinic acetylcholine receptor aggregation on cultured skeletal muscle cells by neuroblastoma-glioma hybrid cells and by substances secreted by these cells. Project Description: Methods Employed:  We have used indirect immunoperoxidase staining of monolayer cultured cells to which alphaBT has been bound, and peroxidase staining of tissue incubated in vivo with peroxidase-labeled alphaBT.  These materials are subsequently examined by light and electron microscopy. Major Findings: (l) Horseradish peroxidase was crosslinked to alphaBT to form a conjugate which retained the specific affinity of alphaBT for nicotinic acetylcholine receptors.  This conjugate bound to 5-7% of the synapses in the inner synaptic layer of the chicken retina.  Amacrine cell and bipolar cell synapses bound conjugate, indicating that some synapses of both types have nicotinic acetylcholine receptors.  (2) Co-culture of mouse muscle fibers with neuroblastoma-glioma hybrid cells (which form synapses with the muscle cells) increased the number of nicotinic acetylcholine receptor clusters 2-4 fold.  A similar increase was obtained by adding cell-free conditioned medium from hybrid cell cultures to the muscle cell cultures. The effect did not depend on the synthesis of new receptors. Significance to Biomedical Research:  Knowledge of ultrastructural distribution of acetylcholine receptors is of clear importance in any attempt to understand the role of neurotransmitters and their receptors in the function and development of the nervous system.  The alpha-bungarotoxin-immunoperoxidase technique already has shown promise for the diagnosis and analysis of mechanisms in human neuromuscular disorders. The results obtained with chick retina (1) represent the first direct demonstration of the synaptic localization of neurotransmitter receptors in the central nervous system, and should lead to a better understanding of neuronal specificity in the CNS. The cultured muscle studies (2) may lead to a better understanding of the mechanism whereby neurons control the distribution of receptors on muscle cells and on other neurons. Proposed Course:  (1) We will extend the study of cholinergic synapses in retina to: a) further analyze the pattern of cholinergic synaptic transmission, b) follow the course of receptor accumulation at synapses during development.  (2) We will attempt to characterize the factor(s) in hybrid cell conditioned medium which promotes the aggregation of receptors on muscle cell membranes and to determine its mechanism of action. Publications: 1.  Ringel, S. P., Bender, A. N., Engel, W. K., Daniels, M. P. and Vogel, Z.: A sequential study of denervation-ultrastructural immunoperoxidase localization of alpha-bungarotoxin.  Trans. Am. Neurol. Assoc. 100: 52-56, 1975. 2.  Bender, A. N., Ringel, S. P., Engel, W. K., Vogel, Z. and Daniels, M. P.: Immunoperoxidase localization of alpha-bungarotoxin (alphaBT) binding: A new approach to the study of myasthenia gravis.  Ann. N. Y. Acad. Sci. 274: 20-30, 1976. 3.  Carpenter, David O., Greene, Lloyd A., Shain, William and Vogel, Zvi: Effects of eserine and neostigimine on the interaction of alpha-bungarotoxin with Aplysia acetylcholine receptors. Mol. Pharmacol. 12:  999-1006, 1976.", "Daniels, Mathew P. ; Vogel, Zvi ; Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nelson, Phillip G. ; Maloney, G. ; Christian, Clifford", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ajgj~ig3u_3qyf", "00000000-0000-0000-E0AF-C2B2F907D3BA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"The Development of Chick Embryo Retina\"", "101584910X267", "101584910X261", "1977", "September 1977", "Summary of work for this project as indicated in the report: \"Some biochemical aspects of chick embryo retina differentiation were studied: 1) An alternate route for GABA synthesis was characterized in the retina; which depends upon the conversion of putrescine to GABA.  2) Glutamic acid decarboxylase specific activity in the retina, increased during the course of embryonic development, either when measured in ovo or in aggregate cultures.  3) The presence of GABA in the culture medium prevented the development of glutamic acid decarboxylase activity in aggregate cultures. 4) The proposed course of this project is to attempt to correlate the biochemical changes observed, with synaptogenesis in the retina.\"", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00011-02 LBG Period Covered: July 1, 1976 through September 30, 1977 Title of Project: The Development of Chick Embryo Retina Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: F.G. De Mello, Visiting Associate, LBG NHLBI Marshall Nirenberg, Chief, LBG, LBG NHLBI Lab/Branch: Laboratory of Biochemical Genetics Section:  Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20014 Total Man Years: 0.5 Professional: 0.5 Summary of Work: Some biochemical aspects of chick embryo retina differentiation were studied: 1) An alternate route for GABA synthesis was characterized in the retina; which depends upon the conversion of putrescine to GABA.  2) Glutamic acid decarboxylase specific activity in the retina, increased during the course of embryonic development, either when measured in ovo or in aggregate cultures.  3) The presence of GABA in the culture medium prevented the development of glutamic acid decarboxylase activity in aggregate cultures. 4) The proposed course of this project is to attempt to correlate the biochemical changes observed, with synaptogenesis in the retina. Project Description: Objectives:  The objective of the project is to study the biochemical step required for synaptogenesis in chick embryo retina. Major Findings:  Two pathways for gamma-aminobutyric acid synthesis were found in chick embryo retina.  The first pathway depends upon the conversion of putrescine to ornithine decarboxylase and the subsequent conversion of ornithine to gamma-aminobutyric acid.  The second route of synthesis is dependent upon the conversion of glutamic acid to gamma-aminobutyric acid, catalyzed by glutamic acid decarboxylase.  Elevation of cAMP levels in neuroblastoma cells was shown to induce ornithine decarboxylase activity.  Thus, in the developing embryo, neurotransmitters which affect cAMP levels may regulate ornithine decarboxylase activity and thereby control the rate of GABA synthesis from ornithine. GABA was found to regulate the specific activity of glutamic acid decarboxylase in cells dissociated from chick embryo retina and cultured in vitro. Significance to Biomedical Research:  These findings show that GABA can be synthesized by a novel pathway and provide new insight on the relationship and regulation of GABA synthesis. Proposed Course:  To determine the effect of retina neurotransmitters and other compounds on retina synaptogenesis. Publications: 1. De Mello, F. G., Bachrach, U. and Nirenberg, M.:  Ornithine and glutamic acid decarboxylase activities in the developing retina.  J. Neurochem. 27: 847-851, 1976.", "Nirenberg, Marshall W. ; DeMello, F. G. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4wja_jgcz~dgsn", "00000000-0000-0000-D229-B507C751132B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Morphine Receptors as Regulators of Adenylate Cyclase\"", "101584910X268", "101584910X261", "1977", "September 1977", "Summary of work for this project as indicated in the report: \"The objectives are to elucidate the mechanisms upon opiates and to define the effects of opiate peptides on cells.\"", "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00002-04 LBG Period Covered: July 1, 1976 through September 30, 1977 Title of Project: Morphine Receptors as Regulators of Adenylate Cyclase Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI OTHER: Arthur Lampert, Guest Worker, LBG NHLBI James Kenimer, Staff Fellow, LBG NHLBI Werner Klee, Research Chemist, LGCB NIMH Cooperating Units (if any): Laboratory of General and Comparative Biochemistry, NIMH Lab/Branch: Laboratory of Biochemical Genetics Section:  Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20014 Summary of Work: The objectives are to elucidate the mechanisms of dependence upon opiates and to define the effects of opiate peptides on cells. Project Description: Endorphin peptides were shown to inhibit adenylate cyclase of NG108-15 cells.  The inhibition constants (Ki) were 12, 40, 63 and 98 nM for methionine enkephalin, leucine enkephalin, Beta-ehdorphin and alpha-endorphin, respectively.  Thus, the endorphins are the most potent peptide inhibitors known and thus, the activations of adenylate cyclase by other species of neurotransmitters or hormones are suppressed.  In effect, the opiate peptides act as pleitropic desensitizers of many kinds of receptors, which in concert with the corresponding ligands, activate adenylate cyclase.  Exposure of cells to methionine enkephalin for 12 to 97 hours results in an increase in adenylate cyclase activity which compensates for the inhibition of enzyme activity by methionine enkephalin.  The cells then have normal cAMP levels and appear tolerant to methionine enkephalin because the increase in adenylate cyclase activity is approximately equal to the inhibition of enzyme activity by the peptide.  However, the cells are dependent upon the opiate to maintain normal cyclic AMP levels.  Withdrawal of methionine enkephalin removes the enzyme inhibition and reveals abnormally high adenylate cyclase activity.  Dual regulation of adenylate cyclase by opiates thus accounts for the phenomena of narcotic dependence and tolerance.  Thus, the endorphin peptides and narcotics are pleitropic regulators of cell responses to neurotransmitters and hormones which are coupled to the activation of adenylate cyclase.  In this way, opiates can alter the perception of neurons to incoming messages which are destined for adenylate cyclase. Reactions mediated by the opiate receptors that inhibit adenylate cyclase are closely coupled to subsequent reactions that gradually increase adenylate cyclase activity of neuroblastoma x glioma NG108-15 hybrid cells.  Opiate-treated cells have higher basal-, PGE1-, and 2-chloroadenosine-stimulated activities than control cells.  However, NaF or guanosine 5'-(Beta,alpha-imido)triphosphate abolish most of the difference in adenylate cyclase activity observed with homogenates from control and opiate-treated cells.  Cycloheximide blocked some, but not all, of the opiate-dependent increase in adenylate cyclase activity.  These results suggest that the opiate-dependent increase in adenylate cyclase is due to conversion of adenylate cyclase to a form with altered activity.  Protein synthesis also is required for part of the opiate effect.  A hypothesis is proposed that the activity of adenylate cyclase determines the rate of conversion of the enzyme from a high to a low activity form or via a new opiate, by inhibiting adenylate cyclase. Highly purified [Leu 5 3] enkephalin and seven derivatives including [Ala2, Leu5]-, [Ser2, Leu5]-, [Aba2, Leu5]-, and [des-Gly2(3), Leu5] enkephalin were obtained by solid phase synthesis and their morphine-like activities in neuroblastoma x glioma cell homogenates were measured.  Changes at the 2, 3, and 5 positions of the enkephalin provided analogues which were all less active than [Leu5] enkephalin.  The results are discussed in terms of recently suggested conformational structures for the enkephalin peptides. No melanocyte stimulating activity was observed for [Leu5] enkephalin, [Ala2, Leu5] enkephalin, or [Ser2, Leu5] enkephalin. Significance to Biomedical Research.  Effects of endogenous opiate peptides on adenylate cyclase activity was defined and molecular mechanisms for the phenomena of narcotic dependence and tolerance were proposed. Proposed Course:  Further studies on the regulation of adenylate cyclase by narcotics and endorphin peptides and the mechanism of coupling inhibition of adenylate cyclase with a subsequent increase in adenylate cyclase activity are in progress. Publications: 1.  Lampert, Arthur, Nirenberg, Marshall and Klee, Werner A.:  Tolerance and dependence evoked by an endogenous opiate peptide. Proc. Natl. Acad. Sci. USA 73:  3165-3167. 2.  Klee, Werner A., Lampert, Arthur and Nirenberg, Marshall:  Dual regulation of adenylate cyclase by endogenous opiate peptides. In:  Kosterlitz, H. (Ed.): Opiates and Endogenous Opioid Peptides.  Amsterdam, Elsevier/North Holland Biomedical Press, 1976, pp. 153-159. 3.  Goldstein, Avram, Cox, Brian M., Klee, Werner A. and Nirenberg, Marshall: Endorphin from pituitary inhibits cyclic AMP formation in homogenates of neuroblastoma x glioma hybrid cells.  Nature 265:  362-363, 1977. 4.  Agarwal, Nirankar S., Hruby, Victor J., Katz, Robert, Klee, Werner and Nirenberg, Marshall:  Synthesis of leucine enkephalin derivatives:  Structure-function studies. Biochem. Biophys. Res. Commun. 76: 129-135, 1977. 5.  Klee, Werner A. and Nirenberg, Marshall:  Mode of action of endogenous opiate peptides.  Nature 263:  609-612, 1976. 6.  Nirenberg, Marshall:  Studies on synapse formation and opiate dependence.  J. Natl. Cancer Inst., in press. 7.  Sharma, Shail K., Klee, Werner A. and Nirenberg, Marshall:  Opiate dependent modulation of adenylate cyclase. Proc. Natl. Acad. Sci. USA, in press.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Lampert, Arthur ; Kenimer, James G. ; Klee, Werner A.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yz4m-c9cr_vxpm", "00000000-0000-0000-29A0-925F2C01EA43", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on effects of monoclonal antibodies", "101584910X269", null, "1982", "18 February 1982", "These pages include a possible outline of a draft for a paper on monoclonal antibodies, including a list of the effects and possible follow-up experiments that Nirenberg will perform.", "Laboratory notes", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "» oS. NBA lak coh ate 7 * BS Nei", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-zm8n.pphk~wfhw", "00000000-0000-0000-1649-72118AABC8F1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics, October 1, 1989 - September 30, 1990", "101584910X270", null, "1990", "19 July 1990", "This draft report provides a narrative of the research and results with homeobox genes; summarizes some of the earlier work with neuroblastoma; and describes some of the other projects that Nirenberg oversaw but did not participate directly in as an investigator.", "Reports, Drafts (documents)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "12", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Dad 12 rH ANNUAL REPORT OF THE LABORATORY OF BIOCHEMICAL GENETICS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE October 1, 1989 through September 30, 1990 Previously, we cloned three neighboring Drosophila homeobox genes designated NK-1, NK-3, and NK-4 that reside in the right arm of the third chromosome at bands 93E1-5, and a fourth homeobox gene, NK-2 that resides in the X chromosome at bands 1C1-5. The nucleotide sequences of approximately 3.5 kb of NK-2 cDNA and genomic DNA were determined. The deduced amino acid sequence of NK-2 protein consists of 723 amino acid residues. NK-2 is a basic protein, with regions rich in alternating acidic and basic amino acid residues, regions rich in seryl-, threonyl-, and prolyl-residues, repetitive alanine residues, PEST sequences, which specify rapid turnover of protein, an acidic domain, a homeobox domain, and an alternating repeat of histidine and alanine residues. Northern analysis revealed one species of NK-2 poly At RNA, 3.2 kb in length, which is most abundant in 3-6 hr embryos. NK-2 mRNA was detected at all later stages of embryonic development and in adult flies, but less NK-2 mRNA was detected in larvae and pupae. In situ hybridization analysis showed that expression of the NK-2 gene is initiated at the cellular blastoderm stage in the ventrolateral portion of the embryo, which later in development, gives rise to the nervous system. In 12 hr and older embryos expression of the NK-2 gene was found both the central nervous system and in cells associated with the midgut. The nucleus of a fertilized Drosophila egg divides thirteen times during the first 130 minutes of development to form the blastoderm, which is a single cell with five thousand nuclei. During the next forty minutes of development plasma membranes are laid down around each nucléegus resulting in the formation of a five thousand cell embryo termed the cellular blastoderm. As the cellular blastoderm is forming, the nuclei in the ventrolateral portion of the blastoderm become committed to the neuroectodermal pathway of differentiation. This formation of the neuroectodermal anlage is the first step in the developmental program that leads to the formation of the nervous system. Both the initial time of expression of the NK-2 gene at the cellular blastoderm stage of development and the initial pattern of expression of NK-2 coincide exactly with the appearance of the neuroectodermal anlage. Since homeobox proteins are known to be regulators of gene expression, we predict that NK-2 protein activates the gene program that commits cells to the neuroectodermal pathway of development and thereby initiates the developmental program for the formation of the nervous system. Both genomic and cDNA NK-1 clones were sequenced and approximately 7500 nucleotide residues of the NK-l gene were determined. The NK-1 gene contains 4 exons; the last intron is located within the region of the DNA that codes for the third helix of the homeobox protein at the same site as the corresponding introns of labial, Abdominal-B, and Distal-less homobox genes. NK-1 protein is comprised of 661 amino acid residues and contains repetitive, alternating histidyl-prolyl residues termed a paired repeat, repetitive histidyl-glutamyl residues, repetitive alanyl residues, a highly acidic domain before the homeobox, and PEST sequences. One major poly At RNA transcript, 2.9 kb in length, was detected in 6-12 hr embryos and thereafter during embryonic development, but not in 0-3 hr embryos. In sity hybridization analysis of embryo sections and whole embryos showed that the expression of the NK-1l gene starts in 7.5-8.0 hr embryos in the ventral nervous system and ultimately results in a bilaterally symmetric, segmentally repeated pattern that consists of a of small subset of neurons on each side in each thoracic and abdominal segment and some unidentified cells in the head. The NK-1l gene also is expressed in a similar pattern in a small subset of striated muscle cells in each thoracic and abdomenal segment. The nucleotide sequence of Drosophila NK-1 homeobox DNA was used to clone genomic DNA from monkey, rat, Xenopus, and salmon DNA. Nucleotide sequence analysis showed that the deduced amino acid sequences of the homeobox domains of monkey and rat are identical to that of the Drosophila NK-1l homeobox, and that salmon and xenopus homeoboxs are novel homeobox that differ from NK-1 by 4 and 9 amino acid residues, respectively. NK-3 and NK-4 are neighboring homeobox genes that are separated by only 7.5 kb. Approximately 3.6 kb of cloned NK~3 cloned cDNA and genomic DNA were sequenced. The NK-3 gene contains a short intron and codes for a homeobox protein 374 amino acid residues in length, which contains charged regions rich in acidic and basic amino acids, and a region rich in serine, threonine, and proline residues. Northern analysis of NK-3 revealed one species of poly At RNA, 1.6 kb in length, that is expressed transiently in 6 to 12 hr embryos but not thereafter. By in situ hybridization NK-3 mRNA was shown to be expressed in a bilaterally symmetric, segmentally repeated pattern in the visceral mesoderm of Drosophila embryos. Sequence analysis of NK-4 cDNA and genomic DNA clones show that the NK-4 gene has 2 introns and that the deduced NK-4 protein is 371 amino acid residues in length. NK-4 is a basic protein with CAX repeats, a homeobox domain, glutamine repeats (M repeats), and regions rich in serine, threonine, and proline. One major species of NK-4 poly At RNA was detected, 1.7 kb in length, which first appears in 3 - 6 hr Drosophila embryos, is less abundant in 6 - 12 hr embryos, and was not detected thereafter. NK-4 was found by in situ hybridization to be expressed only in cephalic, thoracic, and abdominal mesodermal tissue starting at gastrulation (3 hr) when the mesoderm first appears. NK-4 mRNA was not detected after 7.5 hr of embryonic development. Short segments of mouse genomic DNA that correspond to homeobox regions of homeobox genes were selectively amplified by the polymerase chain reaction and were cloned. Nucleotide sequence analysis showed that some of the DNA clones correspond to novel mouse homeobox genes. A mouse genomic DNA library with inserts in the 15 kb range then was screened using the small DNA inserts that had been identified as novel mouse homeobox genes as probes. Genomic DNA clones, were obtained that correspond to four novel mouse homeobox genes. The nucleotide sequences before and after the homeobox regions of two of the genomic DNA clones (CL-8B and CL-101) were determined. RNA transcripts of portions of CL-8B DNA then were used as probes to screen a mouse embryo cDNA library and a CL-8B cDNA clone was obtained. Further work is needed to determine the structures of these homeobox genes and the time and sites of expression of the genes in mouse embryos and adults. A subfamily of homeobox genes has been reported that code for proteins that contain a highly conserved domain, termed a Pou-box, before the homeobox. During the past year, two novel species of Pou-box-homeobox cDNA were cloned and the Pou-box-homeobox regions were sequenced. Both of the novel cDNA's correspond to species of poly At RNA that are expressed in adult mouse brain. A method of selection for cloned mouse genomic DNA fragments that contain functional enhancer or promoter sequences was developed that is based on the observation that the synthesis of polyoma virus DNA in mouse cells is dependent upon viral enhancer or promoter sequences that also are needed for activation of transcription of polyoma genes. An E. coli-mammalian cell shuttle vector, pPyEO, was used that contains the B-lactamase gene and origin of replication from pBR322, and polyoma viral DNA with a library of mouse genomic DNA fragments inserted in the vector in place of the polyoma enhancer region. Thus, recombinant plasmids with DNA inserts that contain enhancer or promoter sequences were expected to replicate in mouse cells, but not recombinants that lack such sequences. Neuroblastoma, glioma, myoblast, or fibroblast cell lines were transfected with plasmid DNA and incubated for several days to allow plasmid replication. Then plasmid DNA was recovered, purified, and cloned in E. coli. Thousands of recombinants were recovered from mammalian cells and all of the cloned DNA inserts that were tested had enhancer activity when ligated to a reporter gene. The results show that the enhancer selection method yields many kinds of cloned enhancer sequences. Marked differences also were found in the activities of some cloned sequences in stimulating the expression of a reporter gene in different cell types. Previously, we showed that prolonged elevation of cyclic AMP levels of NG108-15 neuroblastoma~glioma hybrid cells and other neuroblastoma cell lines results in the appearance in cells of electrically excitable ion channels, and that storage vesicles for acetylcholine increase in abundance, and the number of synapses of NG108-15 cells with striated muscle cells increases. The increase in synapses is due to a cAMP-dependent acquisition by cells of functional L-type voltage-sensitive calcium channels that are required for stimulus-secretion coupling in NG108-15 cells. cDNA clones were obtained that correspond to mRNA from two voltage-sensitive calcium channel Q@j-subunit genes from rat brain. Northern analysis revealed that poly At RNA corresponding to an Q@j-subunit of a voltage-sensitive calcium channel markedly increased in abundance when NG108-15 cells were incubated for several days under conditions that increase cellular levels of CAMP. In addition, cDNA clones were obtained that correspond to species of NG108-15 poly At RNA that increase in abundance when cells are treated with compounds that elevate cellular cAMP. Six kinds of geke cDNA clones were identified by nucleotide sequence analysis. Four of the cDNA clones correspond to different portions of mouse mitochondrial DNA; i.e., pNG-10 corresponds to RNA transcribed from the mitochondrial DNA displacement loop which contains promoters for the transcription of mitochondrial heavy- and light- chain DNA, the origin of replication for mitochondrial heavy chain DNA, and other regulatory signals. pNG-32 cDNA corresponds to mRNA transcribed from the mitochondrial ATPase 6 gene, which codes for a protein that is part of the proton pump-ATP synthase complex. pNG-37 corresponds to mRNA transcribed from the mitochondrial cytochrome oxidase I; whereas, pNG-47 corresponds to mitochondrial 12S ribosomal RNA. Treatment of NG108-15 cells with dibutyryl cAMP for 5 - 8 days results in two to five fold increases in RNA transcripts from both the mitochondrial heavy and light strands of DNA and in a two to three-fold increase in the amount of mitochondrial DNA. Similar results were obtained when adenylate cyclase of NG108-15 cells was activated with prostaglandin El, mediated by PGEl receptors, or by forskolin. In addition, pNG-64 cDNA was shown to correspond to secretogranin I, a secretory vesicle protein. Poly At RNA for secretogranin I was found to increase three-fold in abundance due to treatment of cells for 5 days with dibutyryl cAMP. cDNA clone pNG-57 was shown to correspond to mRNA for ribosomal protein S-10. In previous studies cAMP and phorbol esters that activate protein kinase C were shown to synergistically elevate neuropeptide Y mRNA levels 20 to 200-fold in 4 to 24 hr in PCl12 rat pheochromocytoma cells. Treatment of cells with nerve growth factor (NGF) results in 40 to 100-fold increases in neuropeptide Y mRNA within 1 to 6 days. Glucocorticoids biphasically modulate the stimulations by NGF, potentiating early (3-10 hr) effects of NGF, but inhibiting later stimulations (1-6 days). The results of nuclear run-on transcription assays showed that the apparent rate of neuropeptide Y gene transcription in PCl2 cells is altered by NGF, NGF plus dexamethasone, and cAMP plus phorbol ester. The effects of these compounds on neuropeptide Y mRNA stability were less significant. These results show that neuropeptide Y gene transcription is controlled by multiple, potentially interacting regulators. To identify regions upstream from the neuropeptide Y promoter that regulate gene expression, portions of the upstream region of the neuropeptide Y gene were ligated to plasmids that contained the chloramphenicol acetyltransferase (CAT) gene. In transient expression assays, constructs that contained 144 and 671 nucleotide residues of upstream sequence were weakly responsive to cAMP elevation by forskolin or to NGF, but no enhancement by phorbol ester was noted. The responses were much less than were observed with the endogenous gene, suggesting the additional sequences or variables are important. Glucocorticoids such as dexamethasone potentiate proenkephalin gene expression in several systems including rat brain. We previously showed that glucocorticoids and cAMP synergistically elevate proenkephalin mRNA levels. Nuclear run-on transcription assay results show that the proenkephalin gene transcription rate was not altered by dexamethasone alone, was weakly stimulated by CAMP, and was persistently stimulated by dexamethasone and cAMP together (3-6-fold over 1-24 hr). To search for cooperative glucocorticoid and cAMP regulatory elements, chimeric plasmids were constructed containing the CAT reporter gene under the control of upstream proenkephalin gene sequences in C6 glioma cells transfected with these constructs. Full stimulation of CAT activity by cAMP required sequences between residues -145 and ~192, which are beyond the previously described human proenkephalin cAMP responsive site. Unexpectedly, dexamethasone did not stimulate expression but reduced cAMP stimulated CAT activity; the full negative effect required sequences between —435 and -1000. A DNA fragment from the 5'-upstream region of a rat proenkephalin gene was inserted into a promoterless CAT vector and a Rous sarcoma virus-transformed quail embryo retina cell line was transfected with the DNA. Treatment of the transfected cells for 24 hr with forskolin, prostaglandin El and theophylline, or retinoic acid resulted in 12-, 6-, and 4-fold increases in CAT 10 expression, respectively. In addition, the transactivation protein tax, of human T-cell leukemia virus I was found to activate the proenkephalin gene promoter and resulted ina 3-9-fold increase in CAT expression in C6 glioma cells transfected with the chimeric rat proenkephalin regulatory region-CAT DNA. Ankyrin, a peripheral membrane protein that links certain integral membrane proteins to the spectrin-actin membrane cytoskeleton, was shown by immunofluorescence and immunogold labeling and electron microscopy to be concentrated at the triad junctions of striated muscle cells formed by the transverse tubules and the sarcoplasmic reticulum. Ankyrin thus may play a role in organizing the structure of the triad, or may be involved in linking the triad membranes to the muscle cytoskeleton at specific sites along the sarcomere. Mice with the mdg/mdg genotype die at birth due to failure of excitation-contraction coupling in skeletal muscle that results from a mutation in the gene for the voltage-sensitive calcium channel Q, subunit. Disruptions have been reported in the formation of cross-striated myofibrils and in the organization of the excitation-contraction coupling membrane system in mdg/mdg mice. Sections of striated muscle from 18 day embryo wild type mice or from homozygous or heterozygous mdg mutant mice were incubated with antibodies directed against the Q@1 calcium channel subunit and examined by indirect immunofluorescence. The results Gait Ae 11 show that the expression of the Q, subunit is sharply reduced or absent in homozygous mdg mutants; however, the spatial organization of the sarcoplasmic reticulum and myofibrils is normal, although the intensity of staining is reduced. T-tubule organization also is normal, although the intensity of staining is reduced. These results suggest that the Q, calcium channel subunit is not required for the assembly of the excitation-coupling membrane system, and that the absence of the 1 subunit may indirectly inhibit myotube maturation. In cultured rat myotubes, transverse tubule membranes differ in molecular composition from plasma membranes. The precursors of transverse tubules appear to be intracellular vesicles or tubules that first appear in myoblasts. Intracellular tubular membranes with markers for both transverse tubules and sarcoplasmic reticulum are associated with the Z lines of immature myofibrils in newly formed myotubes. Catabolite gene activator protein (CAP) in the presence of cAMP stimulates transcription from several operons in E. coli. A CAMP-independent variant, in which alanine-144 is replaced by   threonine (CAP-91), is activated, osine, which does not   _ Activate the wild type cap. f fo define the mechanism of the regulation, site-specific mutations in serine-46 of HPr were constructed and then tested for PTS activity. The results indicate that imposition of a negative charge in the region of serine-46 of HPr markedly reduces the activity of HPr as a 12 phosphocarrier. Since high levels of cAMP are toxic to E. coli, difficulties have been experienced in constructing strains that overproduce adenylate cyclase which catalyzes the synthesis of cAMP. A plasmid vector suitable for the expression of lethal genes was constructed and the vector DNA was ligated to genomic DNA for adenlyate cyclase. The level of adenylate cyclase in E. li cells transformed with the recombinant plasmid DNA increased about 7,000 fold, corresponding to 30 percent of the total protein. A relatively simple procedure was devised to purify adenylate cyclase from extracts after hyperexpression to yield nearly homogenous protein.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j9uh_szyr-n2tv", "00000000-0000-0000-7B9A-69890DA068DB", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from John Glenn to Marshall W. Nirenberg", "101584910X271", null, "1982", "6 August 1982", "Senator Glenn conveys his thanks to Marshall Nirenberg for the information he provided in response to an inquiry regarding federal support for scientific and technological research.  Glenn welcomes Nirenberg's analysis of past accomplishments through federal funding and his predictions of future difficulties if such support is discontinued or decreased.  Glenn encloses the results of the survey to which Nirenberg and other Nobel scientists contributed.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "August 6, 1982 Dear Dr. Nirenberg: I want to thank you for the information given in response to my inquiry regarding Federal financial support of scientific and technological research. Your perspective on the impact that federally funded research has had on your work and achievements is most valuable to me in my efforts to emphasize the importance of stable and predictable Federal funding of basic research. I welcome both your analysis of past scientific accomplishments that Federal funding has permitted as well as your predictions for future difficulties that U.S. science will encounter if such support is discontinued or decreased. Enclosed are the results of the survey to which you and other Nobel scientists contributed.  It is my intention to make the aggregate data publicly available. In addition, it might also be useful to have the specific replies released for public view so that the statistics can be verified by anyone caring to do so.  If you would prefer not to have the information which you provided made public in this way, please let me know and I shall respect your wishes. Again, thank you for the facts and views that you have provided me. Best regards. Sincerely, John Glenn Results Of Survey Of American Nobel Prize Winners Concerning Federal Funding For Research Letters requesting data were sent to 54 Nobel Prize winners representing all the Americans who won the Prize in Physics, Chemistry, or Physiology and Medicine during the years 1967-1981.  Twenty-eight scientists responded by letter, and nineteen responses were obtained by telephone.  Seven scientists did not respond: The total response rate was 87%. Scientists were asked if they had received federal support in the course of their research that led to their receipt of the Nobel Prize, and if so, to offer a brief summary of the type of support received.  Furthermore, they were asked to indicate the importance of the role that federal financial support played in the conduct of their research. Respondees fell into two distinct categories: Those who received at least half their research support from the federal government and those who received little or no direct support. Strength Of Federal Support Table 1: Summary of data on support from federal government for Nobel Prize winning research (for awards granted during the period 1967-1981). Substantial Direct Support (50-100% of funds from gov't): 83% (39/47)* Little or No Direct Support: 17% (8/47)** * Of this number, 28 scientists (60% of respondees) received from 90-100% of the financial support for their research from the government.  This category includes 6 scientists who are full time government employees: 4 are employed by NIH, and 2 are employed by the Veterans Administration ** Of the 8 scientists in this category: a. 4 scientists were industrially supported; 3 work for Bell Laboratories (AT&T) the fourth works for General Electric.  Two industrially supported scientists stressed that they received indirect federal support through the use of federally funded facilities or through reliance on previously conducted research of other scientists who had been federally funded.  Another scientist offered the opinion that he would be unable to duplicate his research his today without federal funds. b. Two scientists had carried out most of their prize-winning research prior to World War II when federal funds for research were not available. c. One scientist started his prize-winning work before coming to the U.S. and continued it here as a hobby. d. The work of one prize-winning scientist was indirectly related to his main research efforts (which were federally funded). Necessity Of Federal Support Federally funded scientists were asked to evaluate the necessity of federal financial support to the conduct of their research.  Thirty-eight of the thirty-nine scientists who received substantial direct federal support indicated that federal funds were vitally necessary to their work. Sources Of Federal Funds Although scientists were not requested to do so, may federally funded respondees mentioned specific agencies as sources of their support.  This information is tabulated below. Table 2: Exhibits the percentages of federally funded scientists who mentioned any of the following sources of funds as financially instrumental in their research. [TABLE = \"Sources Of Federal Funds\"] [TABLE = \"Categorization Of Combined Sources\"] * This categorization exhibits percentages of scientists who mentioned the given combined sources of federal funding.", "Glenn, John ; United States. Congress. Senate", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2a2z-e4qz_a5z7", "00000000-0000-0000-B221-D9D20A5159C1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Frank H. Ruddle to Marshall W. Nirenberg", "101584910X272", null, "1971", "17 March 1971", "Ruddle updates Nirenberg on the work of other scientists engaged in neuroblastoma related research, such as John Minna and Art Morris, and expresses an interest in obtaining protocols for research.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Frank H. Ruddle.", "Copyright may apply", null, null, "March 17, 1971 Dear Marshall: It was good seeing you and hearing about your work with the neuroblastoma line at the NRP Conference.  John Minna expressed an interest in learning about our techniques for identifying cell line specific chromosomes utilizing heterochromatin staining techniques.  Mr McMorris has sent to him a preprint of an article in press on this subject by Ruddle & Chen.  We have also sent him our protocol for visualization of phosphoglucomutase electrophoretic differences between Neuro and L-strain cell populations. As you know, Art McMorris in our laboratory has some hybrid cell lines between neuro and mouse L cell lines.  We are particularly interested in testing these hybrid clones for differentiated functions - especially neuro associated enzymes.  It would be a great help to us if we could obtain your protocols for tyrosine hydroxylase, choline acetylase, and glutamate decarboxylase.  If it is too much trouble to send these, I can stop by possibly next Thursday and pick them up. Yours sincerely Frank H. Ruddle", "Ruddle, Frank H. ; Yale University", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-uiyx.pdpn~mx4w", "00000000-0000-0000-F34F-5406E3A0D599", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from M. J. Jaffe to Marshall W. Nirenberg", "101584910X273", null, "1971", "21 February 1971", "Jaffe asks Nirenberg about his latest research with acetylcholine and neuroblastoma.  He also provides details from his own research, suggests additional research possibilities, and offers the prospect of future collaborations.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "21 February, 1971 Dear Dr. Nirenberg: Thank you for sending me a reprint of \"Acetylcholinesterase in neuroblastoma cells\".  I am particularly interested to see the inverse relationship between AChE activity and cell division.  My laboratory is concerned with the problem of sensory physiology, and we have been studying several developmental systems that also have interesting electrical properties.  I have shown that phytochrome, the light receptor that mediates all photoperiodic phenomena in higher plants, is itself regulated by ACh (as in the enclosed reprint).  Since ACh has now been implicated in several animal systems as a developmental regulator (e.g. Gustafson and Toneby, Exp. Cell Res. 62: 102-117 (1970)), I am curious as to whether or not you have measured the ACh concentration in your cells when they are or are not.  As you can see in figures 6 and 7 of the enclosed reprint, the ACh levels are especially high in buds and secondary roots, the only organs in which cell division may be said to be actively occurring.  Whether or not this is due to decreased AChE activity, or increased acetylcholine synthesis remains to be seen.  We have just now succeeded in demonstrating true AChE activity, and are beginning to look for choline acetyltransferase activity in the secondary roots. Although ACh is best known for its role as a neuro-transmitter, it seems to be found in all living cells including prokaryotic cells.  In addition to its control of membrane permeability (Table VI, and much animal work), might it not be that its primary function evolved to regulate cell division, and that its role in nerves, etc. is, in fact, regressive, with the advent of an auxiliary system to protect against division? I hope ultimately to be able to explain the mode of action of ACh in our system, and have taken the approach of studying its interaction with each organelle in turn.  In addition to the obvious effect of ACh on the cell membrane, we have also demonstrated a clear cut and unexpected type of mitochondria regulation, and have preliminary evidence that the nuclear envelope is strongly effected by ACh.  If you are interested, I shall send you reprints of those papers when they appear. Very truly yours, M. J. Jaffe Asst. Prof.", "The Ohio State University ; Jaffe, M. J.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-h5gj_ucih.a4s6", "00000000-0000-0000-B9AF-FB02EA201DAD", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Donald West King to Marshall W. Nirenberg", "101584910X274", null, "1967", "26 April 1967", "King asks Nirenberg for help in developing a Department of Pathology at Columbia Medical School.  He requests the names of three people who might be interested in professorial appointments in Biology.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Donald West King.", "Copyright may apply", null, null, "April 26, 1967 Dear Professor Nirenberg: I am moving to Columbia in September and need your help in developing a Department of Pathobiology at the Medical School.   I should like to make three full professorial appointments in Biology (e.g., genetics, ultrastructure, differentiation, protein, nucleic acid, lipid, or carbohydrate chemistry).   I am writing hoping that you will give me the names of three people who might be interested in the New York environment.  Good space, complete freedom, seats at the Lincoln Center and an opportunity to have postdoctoral fellows and graduate students, as well as to teach undergraduates at Columbia College are available. Thank you for your kindness in this matter. Sincerely, Donald West King", "King, Donald West ; University of Colorado Medical Center", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hwz2~rzbs-gt2y", "00000000-0000-0000-C007-62D6E7EAAC3A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from K. A. Green to Marshall W. Nirenberg", "101584910X275", null, "1989", "10 January 1989", "Green requests some of Nirenberg's neuroblastoma cells for research in the Department of Biology and Preclinical Medicine at the University of St. Andrews in Scotland.  He offers to obtain the cells from Dr. Bradford at the Department of Biochemistry at the Imperial College of Science and Technology in London, rather than having them sent from Bethesda, MD.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of K. A. Green.", "Copyright may apply", null, null, "10 January 1989 Dear Dr Nirenberg, I am writing to request a gift of some of your neuroblastoma clone NS-20Y.  We may be able to obtain this line with permission from Dr Bradford at the Department of Biochemistry, Imperial College of Science and Technology, London, where you have also donated them.  This might be more convenient than sending them from the USA. We are currently starting a new research project. I enclose the relevant pages of our grant application.  We originally planned to use the astrocytoma clone D384 as a source of D1 dopamine receptors.  We have subsequently found, however, that NS-20Y cells also contain D1 receptors and may be a better cell line to use (Monsma and Sibley, 1988, Soc. Neurosci. Abs. 14, 165.12).  This will enable better comparison with our source of D2 receptors (NCB-20) and should hopefully enable monitoring of both channels by electrophysiologioal means in the Xenopus oocytes. With your permission we will contact Dr Bradford and request some of their NS-20Y cells. Thanking you, Yours sincerely, Dr K.A. Green", "University of St. Andrews ; Green, K. A.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cp6m.etkm.fh37", "00000000-0000-0000-5EF5-B3D277293A8D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Eric A. Barnard to Marshall W. Nirenberg", "101584910X276", null, "1989", "9 January 1989", "Barnard provides Nirenberg with a report on the use of cell lines by the Medical Research Council (MRC) at the University of Cambridge Medical School in England.  He also addresses the issue of ownership rights for the cells obtained from Nirenberg but developed at MRC.  The advice of Nirenberg is requested.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Reproduced with permission of the Medical Research Council of Great Britain.", "Copyright may apply", null, null, "9 January 1989 Dear Marshall It is quite a long time since we were in contact.  Happy New Year!  I do hope all is well with you. I am writing to give you a report on our use of the hybrid cell lines which you so kindly provided to me in 1986.  You may recall that after I spoke to you at the Katchalski Conference in England and we arranged that we could use the brainstem-neuroblastoma hybrid cell lines which you had made and stored, we received these and they were worked on here jointly by Michael Hanley and myself, and also by a Hungarian lab. who are collaborating with us and send workers here for that purpose.  Michael has been in touch with you since, but the time has come for me to summarize to you what has been found overall with them, since (a) we are about to write up results using those lines and (b) a question has arisen about a commercial organization acquiring one of them, which I need your guidance on. Two of the lines have been examined in detail, which we named BS1 and BS2 (BS = brain stem).  These were freed from contaminating cells and the hybrid neurons were grown in permanent cultures, which have proved fully stable and monotypic.  BS1 has no opioid receptors but does have some other receptors which are not yet well defined.  BS2 has been characterized extensively: it has a variety of receptors on it, including bradykinin and NMDA.  It has opioid receptors, kappa and delta.  It also constitutive voltage-activated Na+, K+ and Ca2+ (monitored by Ba2+) channels.  (All of these results are mentioned here on a confidential basis.) We intend to use these cells now in some final experiments prior to publication.  I felt that at this stage I should summarize the joint results, and check with you about the publication.  We would, of course, fully acknowledge you as the originator of the cells and send you manuscripts when submitted.  I believe this is what was said at the time, but I want to check that you are quite happy about this or have any other wishes. Secondly, originally I sent you a letter (June 23, 1986) stating, as requested, that we would not pass on these cells or derivatives of them to anyone else without your agreement.  I have not given them outside this Unit, other than to the Hungarian laboratory mentioned above - who cleared this with you in person at the time, and who are doing the work strictly in collaboration with myself and have in turn given the same guarantee.  However, a US pharmaceutical company - following up on a contact they had with someone in this Unit - want to acquire BS2 for use in testing drugs on the NMDA (and perhaps other) receptors and for molecular biology on the NMDA receptor.  This was not envisaged by myself when I got the cell lines from you, and I would like any such commercial development to be conducted in a proper manner, and after your agreement, on whatever terms are appropriate.   Does NIH own the rights to the cells themselves?  It is true that the recognition of the receptors on them was made here, so that MRC has an interest also, but since the cell line was created by you at NIH, I would be glad to receive advice on what would be required from your end, if we were to provide them in that context. Very best wishes Eric", "Medical Research Council (Great Britain) ; Barnard, Eric A.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-em5r~ucpv.53pc", "00000000-0000-0000-6069-C5003B1C07CC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Eric J. Nestler to Marshall W. Nirenberg", "101584910X277", null, "1989", "9 February 1989", "Nestler shares findings from Yale University School of Medicine's work on neuroblastoma cells.  They found that chronic morphine treatment increases the levels of specific enzymes and proteins in the brain.  Nestler sees this work as a support and an extension of Nirenberg's novel proposal for a role for cyclic AMP in mediating opiate addiction in the 1970s.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Eric J. Nestler.", "Copyright may apply", null, null, "February 9, 1989 Dear Dr. Nirenberg: Based on your earlier work on neuroblastoma cells, we have been studying the involvement of the cyclic AMP system in opiate addiction in the rat locus coeruleus.  Similar to your earlier findings, we have found that chronic morphine treatment increases levels of certain G-proteins, adenylate cyclase activity, cyclic AMP-dependent protein kinase activity, and certain phosphoprotein substrates for the protein kinase in this brain region.  Interestingly, these effects were all specific to the locus coeruleus, as they were not observed in the other brain regions examined.  Enclosed please find reprints that describe some of our work on opiate action in the locus coeruleus.  I thought you might be interested in them, as they support and extend your novel proposal of a role for the cyclic AMP system in mediating opiate addiction elaborated over ten years ago. Sincerely, Eric J. Nestler, M.D., Ph.D. Assistant Professor of Psychiatry and Pharmacology", "Yale University ; Nestler, Eric J.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jm5b_x3jp.qvkg", "00000000-0000-0000-77AC-48B6F604262C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Haruhiro Higashida to Marshall W. Nirenberg", "101584910X279", null, "1988", "8 April 1988", "Higashida updates Nirenberg on his research project on hybrid cells at the University of Kanazawa School of Medicine in Japan.  He provides Nirenberg with abstracts of work and data from experiments.  This letter concludes with a request for more cells, qualified by the statement \"these cells will only be used as a part of my own experiments.\"", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Haruhiro Higashida.", "Copyright may apply", null, null, "April 8, 1988 Dear Marshall: I'm spending a very busy time, because every tasks are quite new to me as a laboratory head, though these are not distressful. Unfortunately I haven't had an invitation letter from the Cold Spring Harbor Laboratory for this year's symposium on Molecular Biology on Signal Transduction, I have two abstracts in the meeting as a coauthor with S. Numa and D. Brown.  Two abstracts are enclosed for your interest. The research project on N x L hybrid cells are going well.  The enclosed is a piece of data which shows the IP3(1,4,5) formation is a signal for ACh-induced hyperpolarization.  We are planning to define which muscarinic subtype is present in these NL (308, 309, & 1F) hybrid cells. As you know Toshi Nagastu has cloned the tyrosine hybroxylase gene, he is interested in seeing the gene expression of species-difference in clonal cell lines as well as different tissues.  I've provided him N1E115 and NX31T cells which really have TH molecules in them (see enclosed photos).  I remember that you said you have several adrenergic clones of chick retina and neuroblastomas (including N18-RE-105). Please send me N18-RE-105 cells and if possible, one or two other retina hybrid cells in your list for TH-gene-expression experiments, under the understanding that these cells will only be used as a part of my own experiments participated. With my best regards. Sincerely yours, Haruhiro Higashida", "Kanazawa Daigaku. Igakubu ; Higashida, Haruhiro", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p8wm-37b2.86eq", "00000000-0000-0000-C440-3C11A82F5F64", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics [summary of laboratory projects]", "101584910X281", "101584910X280", "1984", "September 1984", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Annual Report of the Laboratory of Biochemical Genetics National Heart, Lung, and Blood Institute October 1, 1983 through September 30, 1984 Populations of relatively undifferentiated NG108-15 neuroblastoma-glioma hybrid cells or NS20-Y neuroblastoma cells can be shifted a more differentiated state by increasing the levels of cellular cAMP for several days.  In previous studies the differentiated cells were shown to possess functional voltage sensitive channels for Na+, K+, and Ca2+, a Ca2+-dependent K+ channel that is not voltage-sensitive, long neurites, and small clear vesicles and large dense-core vesicles; whereas, these components were absent or were reduced in undifferentiated cells.  Differentiated cells also exhibit higher specific activities of choline acetyltransferase and acetylcholinesterase, secrete more acetylcholine when stimulated, and form more synapses with striated muscle cells than do undifferentiated cells. During the past year, poly A+ RNA was obtained from differentiated NG108-15 and NS20-Y cells, cDNA was synthesized and then cloned using plasmid pBR322 as the vector.  cDNA corresponding to species of poly A+ RNA that are more abundant in differentiated cells than in undifferentiated cells were purified by repetitive hybridization with poly A+ RNA from undifferentiated cells; single-stranded nucleic acids then were separated from double-stranded nucleic acids.  The species of cDNA that increase in abundance as cells differentiate were cloned.  Some clones will be used as probes to study the mechanisms of cAMP-dependent regulation of mRNA in neuroblastoma cells. Prolonged elevation of cellular cAMP results in an increase in at least one species of protein that is part of the voltage-sensitive calcium channel complex.  Concomitantly, cells acquire functional voltage-sensitive calcium channels.  Voltage-sensitive calcium channel proteins were purified extensively.  The cDNA libraries that have been constructed will be screened for clones that correspond to the channel proteins. Our previous analysis of glycoproteins synthesized by NG108-15 cells grown in the presence of PGE1, an activator of adenylate cyclase, or in the absence of PGE1, was extended during the past year.  The cells were incubated with [35S] -methionine solubilized, and the glycoproteins fractionated by wheat germ agglutinin-, ricin-, or lentil lectin-affinity column chromatography and then by 2-dimensional polyacrylamide gel electrophoresis.  35S-Glycoproteins detected by autoradiography were compared with those detected by silver staining.  Both methods of analysis showed that elevation of intracellular cAMP levels for several days results in the expression of new glycoproteins, the disappearance of others, changes in the apparent abundance of some glycoproteins, and shifts in the pI of some glycoproteins.  However, silver staining revealed many glycoproteins that were not detected by autoradiography, including additional glycoproteins that were expressed only by PGE1-treated cells.  These results suggest that elevation of cAMP levels of NG108-15 cells for several days affects the expression of genes for some glycoproteins and alters the post-translational modification of other glycoproteins.   Current studies focus on the purification of sufficient amounts of the regulated glycoproteins to obtain partial amino acid sequences far the proteins and to obtain antibodies that recognize the proteins.  The cDNA libraries that have been generated will be screened for cDNA that corresponds to some regulated species of glycoproteins. Whether a peripheral neuroblast will give rise to sympathetic or parasympathetic neurons during differentiation is determined by mechanisms that regulate the expression of the genes for tyrosine hydroxylase and choline acetyltransferase, respectively.  An extracellular protein and calcium ions are known to be involved in the regulation of these genes, but the mechanisms of regulation are unknown.  Previously, choline acetyltraxsferase from rat brain was purified to essential homogeneity and 4 monoclonal antibodies that recognize the enzyme were obtained.  A large NG108-15 cDNA library was constructed using the bacteriophage expression vector, [lambda]gtll.  Possible DNA clones that direct the synthesis of choline acetyltransferase in E. coli have been detected, but further work is needed to establish the identity of the clones. A monoclonal antibody was obtained previously that recognizes a large dorsal-ventral concentration gradient of a protein in plasma membranes of chicken retina cells.  The amount of protein detected is a function of the position of the cells in retina with-respect to the dorsal-ventral axis of the retina.  The protein is synthesized by proliferating neuroblasts and by nondividing neurons and the gradient is formed as the retina is formed.  The protein was detected on all cells examined in dorsal and middle retina.  Cells that were dissociated from retina and cultured in vitro express the amount of gradient protein that would be expected of cells in the intact retina depending upon the original position of the cells in the retina.  These results suggest that the gradient is established by an irreversible, clonally inherited mechanism and thereafter, the gradient is perpetuated independently by each cell. Monoclonal antibody that recognizes the gradient protein, or hybridoma cells synthesizing the antibody, were injected into the amniotic cavity of chick embryos in ovo from the second to the fifth day after Fertilization and into the vitreal space of chick embryo eyes to determine whether the antibody affects the development or the spatial organization of the retina.  The retinas of embryos were continuously exposed to antibody throughout development from the second to the twentieth day after fertilization.  Injection of antibody to the gradient protein into the eye resulted in a marked reduction of isynapses and neurites in the inner synaptic layer of the retina; whereas, antibodies synthesized by parental P3X63 Ag8 myeloma cells had no effect. RNA was isolated from 14 day chick embryo retinas and a large cDNA library was constructed in [lambda]gtll that can be used to direct the synthesis of proteins specified by the cDNA in E. coli.  The library currently is being screened for recombinants that direct the synthesis of the gradient protein.  Injection of poly A+ RNA from retina into Xenopus laevis oocytes resulted in the synthesis of the gradient protein.  This assay can be used for the purification of mRNA for the gradient protein.  The cDNA library also is being screened for transducin subunits in collaboration with A. Spiegel. Seventy-six hybridoma cell lines were generated that synthesize monoclonal antibodies that bind to 8 day chick embryo optic tectum.  Fifteen hundred hybridoma lines were generated from spleen cells of mice immunized with the cervical-thoracic spinal cord and dorsal root ganglia of 8 day chick embryos.  Some of the hybridoma lines synthesize antibodies that recognize antigens that are restricted to fiber tracts or neuronal cell body regions of the spinal cord. Additional information was obtained about other antigens that are recognized by monoclonal antibodies.  For example, antigen 13H9 was shown to be a protein with an approximate Mr of 180,000.  The antigen is associated with cell membranes of all chick retina cells but has not been detected on neurons or glia in other parts of the nervous system.  The antigen defines a functional set of cells in the nervous system. 18B8 antigens are first expressed by ganglion neurons and then by other types of neurons in retina.  The antigens are found on cell soma initially, but later in development antigens disappear from cell soma and can be seen in a highly stratified, multi-laminar pattern in the inner synaptic layer of the retina and in a circular \"organelle\" in the outer synaptic layer. The antigens are expressed by approximately 10% of the cells in retina.  In collaboration with Victor Ginsburg and his colleagues, the antigens were shown to be novel gangliosides of unknown structure that contain disialyl residues whose abundance and structure change during development; the location of the gangliosides in retina also changes during retinal development.  Most of the antigens are associated with the inner and outer synaptic layers of retina in late embryo and adult retina.  In addition, the antigens for many other monoclonal antibodies were characterized and in some cases were partially purified. A heat-stable, acidic, soluble, bovine brain protein was found that induces neurite outgrowth from chick embryo cerebral cortical neurons at nM concentrations in defined medium.  The Neurite Extension Factor (NEF) rapidly stimulates the phosphorylation of a protein with an apparent M, of 90,000 in the absence of calcium ions or cyclic nucleotides.  Phoslsphopeptide mapping results show that the 90,000 M, protein is related to an 87,000 Mr, protein that is a major substrate for C kinase in brain. Further information has been obtained on the aggregation of nicotinic acetylcholine receptors on cultured myotubes induced by neuronal factors. In experiments using image intensification to directly observe changes in receptor distribution, and electron microscopy to study changes in the subsurface cytoskeleton and extra-cellular matrix, we have demonstrated discrete steps in the assembly of receptor aggregates from diffuse receptors.  The transition from microaggregates of acetylcholine receptors, which appear first, to large dense aggregates is dependent on temperature and involves an increase in the stability of the aggregate. The fine structure was studied of regions of myotubes containing microaggregates, of nicotinic acetylcholine receptors that form within 90 minutes of exposure to embryonic brain extract.  A mixture of rhodamine conjugated [alpha]-bungarotoxin and peroxidase conjugated toxin-was used so that the formation of receptor microaggregates could be observed directly and so that the distribution of acetylcholine receptors on myotube membranes also could be determined.  Small receptor aggregates are converted to larger aggregates that are more stable than the smaller ones.  Microaggregates form in the presence of embryonic brain extract and accumulate at temperatures between 18 degrees and 23 degrees but do not form larger aggregates.  At 36 degrees C aggregates rapidly form from microaggregates.  Microaggregates are destabilized rapidly when brain extract is removed or when sodium azide is added to inhibit ATP formation.  In contrast, aggregates remain stable for several hours under these conditions.  Azide reversibly blocks the information of both microaggregates and aggregates at 36 degrees C. Electron micrographs of microaggregates reveal characteristic mounds in the cell surface, subtended by loosely organized cytoplasmic filaments.  The receptor aggregates have, in addition, an increased association with basal lamina and a characteristic dense filamentous structure below the cell membrane. The regulation of the gene coding for preproenkephalin, the precursor of the opioid peptides methionine-and leucine-enkephalin, was investigated.   cDNA was cloned in the Pst I site of pBR322.  A full-length cDNA clone corresponding to rat striatum preproenkephalin mRNA was found and sequenced.  The primary structure of the rat preproenkephalin protein deduced from the nucleotide sequence of the cDNA (269 amino acid residues, Mr 30932) is similar to bovine and human preproenkephalin (78% and 82% matched amino acid residues, respectively) and contains 4 copies of Met-enkephalin, 1 of Leu-enkephalin, 1 of Met-enkephalin-Arg-Gly-Leu, and 1 of Met-enkephalin-Arg-Phe.  Southern blot analysis of rat genomic DNA with a probe prepared from the rat preproenkephalin clone suggests that the rat contains a single gene for preproenkephalin,  The relative abundances of rat preproenkephalin mRNA are as follows:  striatum 100, hypothalamus 11.2, pons + medulla 10.8, spinal cord 10.3, cerebellum 6.1, midbrain 5.9, frontal cortex 4.6, hippocampus 2.0, thalamus 1.6.  Electroconvulsive shock treatment (1 set per day) of rats for 10 days elicited increases of 78% and 0-14% in the relative abundances of preproenkephalin mRNA of the hypothalamus and striatum, respectively. Preproenkephalin mRNA was detected in NG108-15 mouse neuroblastoma x rat glioma poly A+ RNA by Northern blot hybridization.  The-abundance was 1/650 of that of the rat striatum and was increased 3.5 fold by treatment of the cells with the glucocorticoid hormone, dexamethasone, for 4 days. This cell line can be used for the study of preproenkephalin gene expression. Large cDNA libraries were prepared from cat dorsal root ganglion poly A+ RNA and rat spinal cord poly A+ RNA, for the isolation of clones containing cDNA for the precursors of tachykinin neuropeptides. Histidyl-proline diketopiperazine (cyclo(His-Pro)), a metabolite of the thyrotropin releasing hormone, has been reported to inhibit prolactin secretion from the pituitary, elicit anti-depressant effects, alter cyclic nucleotide levels, and alter body temperatures.  A search for receptors for cyclo(His-Pro) revealed specific binding sites for cyclo(His-Pro) in particulate fractions derived from bovine adrenal cortex or liver, but not in fractions derived from brain or pituitary.  A single class of binding sites was found with a Kd of 900 nM and a maximum number of sites of 92 pmol per mg protein. The binding was stereospecific and the histidine moiety of the peptide was a major determinant of the binding.  The binding sites for cyclo(His-Pro) were inactivated by incubation of particulate fractions with trypsin or at 100 C.  No metabolism of cyclo(His-Pro) was detected. Some strains of E. coli accumulate toxic levels of methylglyoxal that inhibit cell growth. One such strain was isolated and shown to synthesize a mutant form of the cAMP receptor protein and to lack the gene for adenylate cyclase.  Growth of the cells on glucose-6-phosphate, but not glucose, resulted in premature growth arrest due to the accumulation of methylglyoxal. The specific activity of phosphofructokinase in the mutant cells was elevated. The mechanism of growth arrest in the mutant cells was suggested to involve an increase in the synthesis of triose phosphate via giycolysis with spillover of metabolites into a pathway leading to the formation of methylglyoxal.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xggs_tevg_dckt", "00000000-0000-0000-90C6-1DEEC4803BB1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X282", "101584910X280", "1984", "September 1984", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00009-10 LBG Period Covered: October 1, 1983 - September 30, 1984 Title of Project: Cell Recognition and Synapse Formation Principle Investigator: Marshall Nirenberg, Chief, LBG, NHLBI David Trisler, Staff Fellow, LBG, NHLBI Dana Hilt, Staff Fellow, LBG, NHLBI Maria Giovanni, Staff Fellow, LBG, NHLBI Hemin Chin, Guest Worker, LBG, NHLBI Karl Krueger, Staff Fellow, LBG, NHLBI Patricia Bray, Biologist, LBG, NHLBI Hsi-Ping Li, Fogarty Fellow, LBG, NHLBI Gerald Grunwald, Staff Fellow, LBG, NHLBI Cooperating Units (if any): William Strauss, Staff Fellow, LDN, NICHD Victor Ginsberg, Biochemist, LBP, NIADDK Lab/Branch: Laboratory of Biochemical Genetics Section: Section of Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20205 Total Man Years: 12 Professional: 10 Other: 2 Summary of Work: Undifferentiated NG108-15 neuroblastoma-glioma hybrid cells or NS20-Y neuroblastoma cells can be shifted to a differentiated state by increasing levels of cellular cAMP for several days.  cDNA libraries were constructed for species of mRNA that increase in abundance during  differentiation.  Elevation of cAMP levels of NG108-15 cells for several days results in the expression of new glycoproteins, the disappearance of others, changes in the apparent abundance of some glycoproteins and shifts in the p1 of some glycoproteins.  The abundance of a voltage-sensitive calcium channel glycoprotein increases during differentiation.  A large NG108-15 cDNA library was constructed in [lambda]gtll, for use in cloning cDNA for choline acetyltransferase.  A monoclonal antibody that recognizes a plasma membrane protein in chick retina cells that is distributed in a dorsal-ventral gradient in retina was injected into chick embryos in ovo.   A marked reduction in synapses and neurites in the inner synaptic layer of the retina was observed.  cDNA was synthesized frown 14 day chick embryo retina RNA and cloned in [lambda]gtll for use in cloning cDNA for the gradient protein.  Many monoclonal antibodies were obtained that recognize antigens in the developing nervous system and some of the antigens were characterized.  Several antibodies recognize novel gangliosides.  A protein was purified from bovine brain that induces neurite outgrowth at nM concentrations and stimulates the phosphorylation of proteins in the absence of Ca2+ or cAMP. Project Description Objectives To identify and characterize molecules involved in synapse formation and function and to elucidate mechanisms that regulate gene expression for proteins that are required for synapses. Major Findings Populations of relatively undifferentiated NG108-15 neuroblastoma-glioma hybrid cells or NS20-Y neuroblastoma cells can be shifted a more differentiated state by increasing the levels of cellular cAMP for several days.  In previous studies the differentiated cells were shown to possess functional voltage sensitive channels for Na+, K+, and Ca2+, a Ca2+-dependent K+ channel that is not voltage-sensitive, long neurites, and small clear vesicles and large dense-core vesicles; whereas, these components were absent or were reduced in undifferentiated cells.  Differentiated cells also exhibit higher specific activities of choline acetyltransferase and acetylcholinesterase, secrete more acetylcholine when stimulated, and form more synapses with striated muscle cells than do undifferentiated cells. During the past year, poly A+ RNA was obtained from differentiated NG108-15 and NS20-Y cells, cDNA was synthesized and then cloned using plasmid pBR322 as the vector.  cDNA corresponding to species of poly A+ RNA that are more abundant in differentiated cells than in undifferentiated cells were purified by repetitive hybridization with poly A+ RNA from undifferentiated cells; single-stranded nucleic acids then were separated from double-stranded nucleic acids.  The species of cDNA that increase in abundance as cells differentiate were cloned.  Some clones will be used as probes to study the mechanisms of cAMP-dependent regulation of mRNA in neuroblastoma cells. Prolonged elevation of cellular cAMP results in an increase in at least one species of protein that is part of the voltage-sensitive calcium channel complex.  Concomitantly, cells acquire functional voltage-sensitive calcium channels.  Voltage-sensitive calcium channel proteins were purified extensively.  The cDNA libraries that have been constructed will be screened for clones that correspond to the channel proteins. Our previous analysis of glycoproteins synthesized by NG108-15 cells grown in the presence of PGE1, an activator of adenylate cyclase, or in the absence of PGEl, was extended during the past year.  The cells were incubated with [35S]-methionine for 18 hours, solubilized, and the glycoproteins fractionated by wheat germ agglutinin-, ricin-, or lentil lectin-affinity column chromatography and then by 2-dimensional polyacrylamide gel electrophoresis.  35S-Glycoproteins detected by autoradiography were compared with those detected by silver staining.  Both methods of analysis showed that elevation of intracellular cAMP levels for several days results in the expression of new glycoproteins, the disappearance of others, changes in the apparent abundance of some glycoproteins, and shifts in the pI of some glycoproteins.  However, silver staining revealed many glycoproteins that were not detected by autoradiography, including additional glycoproteins that were expressed only by PCE1-treated cells.  These results suggest that elevation of cAMP levels of NG108-15 cells for several days affects the expression of genes for some glycoproteins and alters the post-translational modification of other glycoproteins.   Current studies focus on the purification of sufficient amounts of some regulated glycoproteins to obtain partial amino acid sequences of the proteins and to obtain antibodies that recognize the proteins.  The cDNA libraries that have been generated will be screened for cDNA that correspond to some regulated species of glycoproteins. Whether a peripheral neuroblast will give rise to sympathetic or parasympathetic neurons during differentiation is determined by mechanisms that regulate the expression of the genes for tyroslne hydroxylase and choline acetyltransferase, respectively. An extracellular protein and calcium ions are known to be involved in the regulation of these genes, but the mechanisms of regulation are unknown.  Previously, choline acetyltransferase from rat brain was purified to essential homogeneity and 4 monoclonal antibodies that recognize the enzyme were obtained.  A large NG108-15 cDNA library was constructed using the bacteriophage.expression vector, [lambda]gtll.  Possible cDNA clones that direct the synthesis of choline acetyltransferase in E. coli have been detected, but further work is needed to establish the identity of the clones. A monoclonal antibody was obtained previously that recognizes a large dorsal-ventral concentration gradient of a protein in plasma membranes of chicken retina cells.  The amount of protein detected is a function of the position of the cells in retina with respect to the dorsal-ventral axis of the retina.  The protein is synthesized by proliferating neuroblasts and by nondividing neurons and the gradient is formed as the retina is formed.  The protein was detected on all cells examined in dorsal and middle retina.  Cells that were dissociated from retina and cultured in vitro express the amount of gradient protein that would be expected of cells in the intact retina depending upon the original position of the cells in the retina.  These results suggest that the gradient is established by an irreversible, clonally inherited mechanism and that once established, the gradient is perpetuated independently by each cell thereafter. Monoclonal antibody that recognizes the gradient protein, or hybridoma cells synthesizing the antibody, were injected into the amniotic cavity of chick embryos in ovo from the second to the fifth day after fertilization and into the vitreal space of chick embryo eyes to determine whether the antibody affects the development or the spatial organization of the retina. The retinas of embryos were continuously exposed to antibody throughout development from the second to the twentieth day after fertilization.  Injection of antibody to the gradient protein into the eye resulted in a marked reduction of synapses and neurites in the inner synaptic layer of the retina; whereas, antibodies synthesized by parental P3X63 Ag8 myeloma cells had no effect. RNA was isolated from 14 day chick embryo retinas and a large cDNA library was constructed in [lambda]gtll that can be used to direct the synthesis of proteins specified by the cDNA in E. coli.  The library currently is being screened for recombinants that direct the synthesis of the gradient protein.  Injection of poly A+ RNA from retina into Xenopus laevis oocytes resulted in she synthesis of the gradient protein.  This assay can be used for the purification of mRNA for the gradient protein.  The cDNA library also is being screened for transducin subunits in collaboration with A. Spiegel. Seventy-six hybridoma cell lines were generated that synthesize monoclonal antibodies that bind to 8 day chick embryo optic tectum.  Fifteen hundred hybridoma lines were generated from spleen cells of mice immunized with the cervical-thoracic spinal cord and dorsal root ganglia of 8 day chick embryos. Some of the hybridoma lines synthesize antibodies that recognize antigens that are restricted to fiber tracts or neuronal cell body regions of the spinal cord. Additional information was obtained about other antigens that are recognized by monoclonal antibodies.  For example, antigen 13H9 was shown to be a protein with an approximate M, of 180,000.  The antigen is associated with cell membranes of all chick retina cells but has not been detected on neurons or glia in other parts of the nervous system.  The antigen defines a functional set of cells in the nervous system. 18B8 antigens are first expressed by ganglion neurons and then by other types of neurons in retina.  The antigens are found on cell soma initially, but later in development antigens disappear from cell soma and can be seen in a highly stratified, multi-laminar pattern in the inner synaptic layer of the retina and in a circular \"organelle\" in the outer synaptic layer. The antigens are expressed by approximately 10% of the cells in retina.  In collaboration with Victor Ginsburg and his colleagues, the antigens were shown to be novel gangliosides of unknown structure that contain disialyl residues whose abundance and structure change during development; the location of the gangliosides in retina also changes during retinal development.  Most of the antigens are associated with the inner and outer synaptic layers of retina in late embryo and adult retina.  In addition, the antigens for many other monoclonal antibodies were characterized and in some cases were partially purified. A heat-stable, acidic, soluble, bovine brain protein was found that induces neurite outgrowth from chick embryo cerebral cortical neurons at nM concentrations in defined medium.  The Neurite Extension Factor (NEF) rapidly stimulates the phosphorylatfon of a protein with an apparent Mr of 90,000 in the absence of calcium ions or cyclic nucleotides.  Phosphopeptide mapping results show that the 90,000 Mr protein is related to an 87,000 Mr protein that is a major substrate for C kinase in brain. Significance to Biomedical Research New information was obtained concerning synaptogenesis and synaptic functions. Publications 1.  Nirenberg, M., Wilson, S. P., Higashida, H., Rotter, A., Kreuger, K., Busis, N., Ray, R., Kenimer, J., Adler, M., and Fukui, H.:  Synapse Formation by Neuroblastoma Hybrid Cells.  In: Molecular Neurobiology. Cold Spring Harbor Symposia on Quantitative Biology XLVIII: 707-715. (1983). 2.  Nirenberg, M., S. Wilson, H. Higashida, A. Rotter, K. Krueger, N. Busis, R. Ray, J. G. Kenimer, and M. Adler.:  Modulation of Synapse Formation by Cyclic Adenosine Monophosphate.  Science 222: 794-799. (1983). 3.  de Blas, A., Adler, M., Shih, M., Chiang, P. K., Cantoni, G. L., and Nirenberg, M.:  Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.  Proc. Natl. Acad. Sci. 81: 4353-4357. (1984). 5.  Trisler, D., Grunwald, G.B., Moskal, J., Darveniza, P., and Nirenberg, M.: Molecules that identify cell types or position in the retina. In: Neuroimmunology. Behan, P.O. and Spreafico, F. (Eds.). New York, Raven Press, 89-97. (1984). 6. Nirenberg, M., Krueger, K. Rotter, A., Wilson, S., and Higashida, H.: Regulation of Synapse Formation by Cyclic AMP: In: The Symposium of the International Society for Developmental Neurosciences. In Press. 7. Fredman, P., Magnani, J. L., Nirenberg, M., and Ginsburg, V.: Monoclonal antibody A2B5 reacts with many gangliosides in neuronal tissue.  Archives of Biochemistry and Biophysics.   In Press. 8.  Strauss, W. L. and Nirenberg, M.:  Inhibition of choline acetyltransferase by monoclonal antibodies. Journal of Neuroscience. In Press.", "Nirenberg, Marshall W. ; Giovanni, Maria ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Grunwald, Gerald ; Krueger, Karl ; Bray, Patricia ; Trisler, G. David ; Li, Hsi-Ping ; Hilt, Dana ; Chin, Hemin", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wvfa~ytzi~yvwy", "00000000-0000-0000-9F05-D0A60FAAAF62", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marvis Matthews to Marshall W. Nirenberg", "101584910X283", null, "1966", "22 May 1966", "Matthews praises Nirenberg for the altruistic nature of his research and requests his opinion on three distinct methods of treatment for cancer: Glenn Kittler's laetrile control, Herbert Bailey's Krebiozen treatment, and Max Gerson's dietary methods.  Mention is made of John Beard's book on enzyme treatment for cancer and the work of Ernst Krebs, Sr. and Jr. on developing a urine test for the detection of cancer.  Matthews asks for Nirenberg's advice on avenues that she might pursue toward getting a fair trial for these therapies.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "May 22, 1966 Dear Dr. Nirenberg, Today I read the article in All Florida Magazine about your research into the fundamental \"Building Blocks\" of life. It is gratifying to know that some people are still engaged in research to better mankind, rather than to obliterate us. A related subject is of vital interest to me. In 1964 I was treated for inoperable breast cancer by means of surgery and X-Ray therapy. By God's grace, this was successful, and I believe that there will be no recurrence. Many are not so blessed. Because I wish to help, I have been reading as much as possible on the subject. Among other books, I have read Glenn Kittler's \"Laetrile Control for Cancer\", Bailey's book, \"K\", about Krebiozen, and Haught's book about Dr. Max Gerson's dietary methods and cures of cancer, lupus, etc. Unless these authors are making many false statements, all three of these methods have been proved effective, non-toxic and have been used for several years with good success! I am amazed and puzzled to know that they are not generally used and available in our country and would like to see this accomplished immediately. In Mr. Kittler's book, mention is made of Dr. John Beard's work and his book, \"The Enzyme Treatment of Cancer\". I would very much like to read this, and wonder if you could tell me where to get a copy. You must be familiar with the work of Drs. Ernst Krebs, Sr. and Jr., and how Dr. Krebs, Sr. decided that Dr. Beard must be wrong, it was so simple. However, after long and thorough research on the subject of enzymes and their effect on cancer, Dr. Krebs, Sr. decided that Dr. Beard was RIGHT! Then he and his son, Dr. Ernst Krebs, Jr. spent most of their lives experimenting and studying, and finally developed Laetrile, and a urine teat for the detection of cancer. Also a non-mysterious and non-terrifying definition and explanation of this disease which has baffled, tortured and killed man for countless generations. If you are not familiar with this work, which also goes into the \"building block\" dept., which explains about the trophoblast cell, without which no birth could occur, and which, I believe, conclusively proves that the trophoblast cell and the cancer cell are one and the same thing, please read Mr. Kittler's little 60 cent paperback book which you will find at Natural Health Food Stores. An indication that Drs. Krebs and Dr. Beard are on the right track came to me in my own case. My own doctor told me that he must remove my unaffected ovaries because the hormone that they produce (estrogen) would completely defeat the work of the X-Ray. Dr. Krebs also found that trophoblasts are formed by cell division when contacted by estrogen!  I have seen cancer cells referred to in an unrelated book as \"blasts\". A doctor here in town told me that they use drugs on only one kind of cancer, the chorionepithelioma. (He didn't know that I had just swallowed Mr. Kittler's book and had a little idea of what he was talking about.) If they use drugs on that kind, why not on other types? Which drug? Your opinion will be appreciated. I firmly believe that an intelligent use of any or all three of these methods could make the present methods of surgery and radiation, (practically unchanged during the past 20 years), practically obsolete. The need is to get the professional organizations in authority over doctors and their practices to give these methods a fair and truthful trial. In 1946 a bill was submitted to Congress, #S1875, whose purpose was to empower the President of the US to get a meeting together in a convenient location, of all the accredited research workers on the subject of cancer cure and treatment, to see if something could be done to improve the situation.  And to appropriate sufficient money to do this. So far as I know, after some hearings, this bill was put away. Could it not be revived? What would be the procedure? Except for writing to our congressmen can you suggest any way to try to get a fair trial for these three methods, or any one of them? Your help will be greatly appreciated. Thanks and God bless you. Sincerely, Marvis Matthews P.S. According to my findings, these three methods have in common the following: 1 They are within the financial reach of any ordinary person. 2 They remove the terror and mystery from the word \"cancer\", thus encouraging periodic check-up and early detection. 3 Afford a painless death for terminal cases. Am I right?", "Matthews, Marvis", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9vuh-mgyt-zfq5", "00000000-0000-0000-58B4-6CF3AA613B13", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Brian D. Pate, Simon Fraser University to Marshall W. Nirenberg", "101584910X284", null, "1967", "24 October 1967", "Pate informs Nirenberg that the Annual Conference of the Chemical Institute of Canada will feature a panel discussion on \"The Technical, Economic, and Political Consequences of  Man's Control  of his Own Evolution,\" involving scientists, economists, and clerics.  He suggests that the idea for the panel stemmed from Nirenberg's Science editorial on a similar subject.  Nirenberg is invited to attend and appear on the panel.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Simon Fraser University.", "Copyright may apply", null, null, "24th October, 1967. Dear Dr. Nirenberg, I am writing to you in connection with the 1968 Annual Conference of the Chemical Institute of Canada.  This is to be held in Vancouver, British Columbia between June 2nd and June 5th, 1968 and I am responsible for organizing the program. We intend to have as a prominent feature of the Conference a panel discussion on \"The Technical, Economic and Political Consequences of Man's Control of His Own Evolution\" with a panel composed of chemists, biochemists, economists, political scientists, clerics, etc.  This arose from a suggestion of Dr. J. Ross Colvin of Biophysics at the National Research Council of Canada following your editorial in Science (August 1967) on a similar subject.  We hope to have Canada-wide broadcast coverage and I feel that the event would be a very interesting and important one. The Institute and the local committee would be very pleased if you would accept our invitation to attend the Conference and appear on the panel for this discussion.  We would be pleased to defray your travel expenses to and from Bethesda, Maryland and your expenses in Vancouver.  There is also an honorarium. The panel discussion we have tentatively set for the evening of Monday, June 3rd.  I hope very much this date will be convenient and that you will be willing to participate in this event. Yours sincerely, Brian D. Pate Professor and Head, Department of Chemistry Simon Fraser University Program Chairman 1968 CIC Anuual Conference P.S.  If your editorial in Science was reprinted I would be very pleased to receive a copy.", "Simon Fraser University ; Pate, Brian D.", null, null, null, "National Heart Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tsqb.p8vy_7p8p", "00000000-0000-0000-C022-638F45F286C8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Acceptance speech for Gairdner Foundation Award", "101584910X285", null, "1967", "[1967?]", "The Gairdner Foundation is a non-profit corporation devoted to the recognition of outstanding achievement in biomedical research worldwide.  Nirenberg won the award in 1967.  In this first page of his draft he discusses the importance of scientific freedom as an ideal to cherish.", "Speeches, Drafts (documents)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Dr. Michener, Dr. [ . . . ], Mr. Gairdner, honored guests, ladies and gentlemen. In ancient times in the Orient an artist always used an assumed name to sign the pictures he painted.  The reason was that the artist then could never either be rewarded or punished by others for his work -- the artist then would always be free -- for no one, other than the artist, would know who to punish or who to reward.  Now this is an interesting concept relevant to art and science today. Certainly the concepts of freedom and creation for the sake of creation are ideals to cherish.  But on the other hand if enduring beauty is the [ . . . ] to truth and most important [ . . . ] willingness of led individual to accept responsibilities of his work, even good scientist [ . . . ] long and hard to validate his work, to check it, to take pride in his feelings or workmanship.  For his colleagues judge his scientific ability on the basis of what he creates or what he tries to create. The basic values of the scientific community are simple and good.  In essence, it is a devotion to truth, and to integrity to one's craft.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-w6w7.7v8f.83v3", "00000000-0000-0000-BF90-23F23E4E89B5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Lab Chiefs' Meeting", "101584910X286", null, "1996", "11 March 1996", "This agenda from a meeting of NIH Laboratory Chiefs includes Nirenberg's handwritten notes regarding administrative and managerial details.. NOTE: The handwriting is cut off on the left and bottom of the original image.", "Notes,Lists (document genres)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, ". Uiwar dy nell . $8 SS 43. 00 npn Wn nord es de: {raced on fto - ne 3 aa thane rchrbind: J a J iy ‘ hs us LAB CHIEFS’ MEETING (Me fprmovret GODIN, 4) of ih S: 2°) — 2Q i, hone . 20 J . March 11, 1996 Abrus 8 rca ponte Heatly Riva > | 5X Guest- | Mr. John L. Barnhart (handout) lbky SO ; Division of Safety 4o Wx beret: _ : ELK, Kom : . 2. Guest - Cindy Walczak - Computer Support 9 RE 2d Sy hdfc . (LRG) eben ee peak AS . 3. Clinical Center Update - Smits Report; Reinvention Laboratory,- “ee ne to MIRE: ) ‘ pee omel” ON?\" / . rebel pr G7 b2kg Z 22. GOB. Employee Apprais for 1996 ~ 10 LOT f Comple . hee ob Animal Care & Use Concerns within NIH (handout) Administrative Updates — Honoraria Ban (handout) ce Letter from FAXON (handout) id. —— €. Ke atiogs Historic Artifact Identification is Fa pe Travel Reimbursement ~~ theta pawhc Shalt £4 VALERIE     Visiting Fellow Program - J-1 Visas — Eb, Unarere of artes beh eS 1h ae ft ME ~> NIH Fellows Committee - need representative Am idetly “WD Crank fuer NIH Fellows’ Travel Award Program (handout) SEE: mm Leg obetn, aan Career Transition Program Ct ow Ol LE Gk,", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ti5s-auyz.g4bn", "00000000-0000-0000-D706-38142A05C192", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Lilli S. Chertoff, Committee of Concerned Scientists, Inc. to Marshall W. Nirenberg", "101584910X287", null, "1975", "6 June 1975", "Chertoff informs Nirenberg that Jack Cohen has told her of Nirenberg's intention to write a letter on behalf of Dr. Mikhail Shtern, an imprisoned Soviet Jewish scientist.  Background material is enclosed, along with a draft letter for Nirenberg to use.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "5", "pages", "Text", "English", "Reproduced with permission of Lilli S. Chertoff.", "Copyright may apply", null, null, "June 6, 1975 Dear Dr. Nirenberg: Jack Cohen has told me of your kind intention to write a letter on behalf of Dr. Shtern. I am enclosing herewith some brief background material on the case, together with a draft letter you can use as you see fit. Very briefly, Dr. Shtern is an endocrinologist who practiced many years in his town of Vinnitsa. Shortly after he applied for permission to emigrate to Israel with his family, he was accused of poisoning children, accepting bribes, and similar outrageous charges. The case came to trial accompanied by the usual difficulties with witnesses -- some patients refused to testify, at great personal risk to themselves, and others willingly gave false testimony. He was found guilty and sentenced to eight years at hard labor. There is absolutely no point in talking about the travesty of justice which took place in the case; it occurs daily in the Soviet Union. The only thing we can ask for with any hope of success is clemency and commutation of sentence.  He is a terribly sick man and the conditions in the labor camp have already aggravated his condition. He cannot possibly survive very long.  His second son has now been given permission to leave and in fact has been ordered to leave in a few days. That will leave Mrs. Shtern quite alone and helpless. Our most recent information is that he is preparing an appeal for presentation to the USSR Supreme Court. It is doubtful that they will take jurisdiction in the case, or that any appeal will be considered.  However, we must pursue every avenue still open to us, to plead for his release. We are most grateful for your concern, and for your willingness to translate that concern into action. Will you please send me a copy of the letter you send to Podgorny? Sincerely yours, Lilli S. Chertoff Working Draft President Nikolai V. Podgnorny The Kremlin Moscow RSFSR, USSR Sir: I am writing to you, as the supreme voice of authority in the Soviet Union, to plead for the release of Dr. Mikhail Shtern. Dr. Shtern by this time is a sick and tormented man, prematurely aged by his experiences.  He suffers from severe health problems and I fear for his life under the regimen of the labor camp. Certainly, he will never survive to serve his eight-year sentence. The Soviet Union today is one of the world's great powers. Its place among the nations is secure and does not depend on keeping one frail, sick man in prison. The prestige of the Soviet Union abroad can only be enhanced by a humanitarian and generous act of clemency in his case. I and many of my colleagues in science have welcomed the reduction in tensions between our two countries as a result of the policy of detente. We are in the forefront of exchanges in science between the USSR and the United States. But those exchanges cannot take place without the collaboration and support of American scientists, and their enthusiasm for such cooperation is not encouraged when they read about Dr. Shtern or other Soviet scientists whose desire to emigrate to Israel has resulted in severe punishment. I urge you to grant amnesty to Dr. Shtern, and allow him and his wife to leave so that they may join the remaining members of their family and live out their remaining years in peace. Sincerely yours, Pertinent Addresses In U.S. And U.S.S.R. For Protest (Please retain for future reference) President Gerald Ford The White House Washington, D.C. 20500 Secretary of State Henry Kissinger Department of State Washington, D.C. 20525 Ambassador Anatoly Dobrynin Embassy of the U.S.S.R. 1125 16th Street NW Washington, D.C. 20036 Procurator General Roman Rudenko Pushkinskaya Street 15A Moscow RSFSR USSR Chairman Vladimir A. Kirillin State Committee on Science and Technology The Kremlin Moscow RSFSR USSR M. Keldysh USSR Academy of Sciences Moscow RSFSR USSR Secretary General Leonid Brezhnev The Kremlin Moscow RSFSR USSR President Nikolai V. Podgorny The Kremlin Moscow RSFSR USSR Premier Aleksei Kosygin The Kremlin Moscow RSFSR USSR Senator Henry Jackson United States Senate Washington, D.C. 20510 Your own Congressman Your own Senator The head of any academic or scientific institution in the USSR known to you, or with whom you have professional contact. Committee of Concerned Scientists, Inc. Profile:  Dr. Mikhail Shtern Born: 1918 From: Vinnitsa Address: USSR Ukrainian SSR Vinnitsa Osipenko 7/9 Tel. 28813 Occupation:  Endocrinologist, chief of a polyclinic Status:  Married. Wife:  Ida, born 1917. Sons: Viktor, born 1941 (physicist, now working as a postman); Avgust, 1945 (Ph.D., biology, now tutoring) Arrested: 5/25/74 Investigation Completed: 10/18/74 Charges:  a) \"Bribery\"; b) possibly, \"attempted homicide\" Trial: Date to be announced; due end of November, 1974. In September 1973, the Shtern family took the first step in the emigration process when they received a vyzov (invitation) from Mrs. Shtern's sister in Israel. Immediately following this, the entire family became the target of systematic harassment; unofficial searches; calls to local government offices, interrogations.  This preliminary campaign culminated in the arrest of Dr. Mikhail Shtern on May 25, 1974 during a KGB search of the Shtern apartment. Four days later the family was informed that an investigation had been opened in preparation for court action against Dr. Shtern. The Vinnitsa Procurator's Office stated explicitly that the accusation was in retaliation for the entire family's desire to emigrate to Israel, while the Chief of Investigation for the Ukrainian Republic commented that he didn't \"like, people going to Israel.\" Apparently acting on orders sanctioned by the Regional Communist Party, the procurator attempted to accumulate evidence to support bribery charges.  In view of the weakness of the evidence, however, additional charges were sought and \"found.\" According to Mrs. Shtern, on the 9th of September, Maria Grigorievna Soloveichuk, residing in the village of Kitaigorod, Ilienetsky Raion, Vinnitsa Oblast, came to the Vinnitsa Oblast Dispensary and demanded to know what \"Dr. Shtern had poisoned her son with.\"  As Mrs. Soloveichuk informed the dispensary workers, interrogators at the Ilienetsky Municipal Procurator's Office had asked her and other residents of the village, in particular Elena Timoshenko, for information which would prove that the physician Shtern, had \"taken bribes' and sold drugs.\" The authorities explained that they needed this information to possibly save the \"lives of children deliberately poisoned by Shtern.\" Not satisfied with the explanation given by the dispensary workers, Mrs. Soloveichuk went to the Shtern's apartment. Upon hearing the entire story, and learning how evidence was being initiated and gathered, Mrs. Shtern, (Dr. Shtern was still in custody) requested that Mrs. Soloveichuk tell this to the  Vinnitsa Oblast Procurator.  Mrs. Soloveichuk attempted to comply, but was directed instead to the administrator of the investigative department, Procurate Potapov.  Mrs. Shtern's request to have the informants recite their charges in her presence, and their version of how the investigation was conducted, was denied.  Instead, the local man merely \"conversed\" with Mrs. Soloveichuk privately and then sent her home. Despite endless searches and confiscation of property worth 3,000 rubles, authorities found no evidence to support their charges.  The bribery charges alone carry a maximum penalty of 20 years. N.B. Vinnitsa is a remote Ukrainian town whose isolation has served as cover for recent anti-Jewish outbreaks.  In 1973, Isaak Shkolnik, an unskilled laborer, was tried and sentenced to 7 years on charges of \"treason.\"  In recent weeks, one of the few remaining activists in the town, Mikhail Mager, has been continually harassed and under surveillance. Appeal by the Shtern Family to the Medical Profession and to all concerned people: In September, 1973 our family received an invitation from our mother's sister, living in Israel; immediately after receipt of this invitation we all began to undergo systematic and continuous harassment and persecution.  On May 12th, 1974, our flat was broken into by unknown persons, for an unknown purpose; at this time all members of the family were called to different state and local government offices, and all retained in these various places for more than 4 hours.  On May 25th, 1974, our father was arrested; the arrest was carried out by officials of the K.G.B.: 10 men burst into the apartment in the most brutal manner, without any warning or ringing of the bell or any previous notification whatsoever.  On May 29th, 1974, during the investigation, the procurator of the Vinnitsa Office openly stated that the reason for the preparation of an accusation against our father was connected with our desire emigrate to live in Israel. When it became clear that the attempts of the K.G.B. to institute, a political or even a civil case were doomed to failure as they had no evidence of any kind, of either political activity or of bribery or corruption of any sort, the case was handed over to the Procurator's office with instructions to prepare some sort of case so that our father could be brought to trial.  When the accusation was presented to him, a further search was undertaken, and all his personal documents were removed, including his diploma of Kandidat Nauk, original manuscripts of scientific articles, a list of scientific works and so on; At the request of the Vinnitsa Procurator, exit visas for the younger son and his wife have been held back. In spite of the obvious absurdity and the open dishonesty of the accusation being prepared and presented against our father, a doctor with more than 30 years experience in medical practice, the official governmental organs have been persisting for the last 4 months with their efforts to press the charge of \"attempting to poison little children who were under his care as a doctor.\" Officials of the investigating authorities are browbeating witnesses, compelling them to give false testimony, accusing the doctor whom they have always trusted and respected of the intention to poison their children.  This was stated by M. Soloveichuk and E. Timoshenko on September 9th, 1974.  These two are relatives of former patients of our father. Thus as it has not been found possible or convenient to bring a charge of violation of the criminal law code, or of abuse of his rights as an official (by the taking of bribery), they have now decided to bring a charge against this doctor of \"attempting murder,\" highly reminiscent of the infamous \"Doctors' Plot\" case of 1952. It is up to the consciences of all honest people not to allow this to take place.  We appeal to the Medical Profession and beg you to help us to save our father, whose whole aim in life has always been to help to save the lives of others. Signed by all members of the Shtern family, September 26th, 1974", "Chertoff, Lilli S. ; Committee of Concerned Scientists, Inc.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mj6k-trbe_44wz", "00000000-0000-0000-7C16-AFBA065A7479", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to Nikolai V. Podgorny", "101584910X288", null, "1975", "16 June 1975", "In this copy of the letter Nirenberg sent to the Russian President, he asks that Shtern be released and treated for severe health problems.  He emphasizes reducing tensions between the U.S. and U.S.S.R. while noting that the prestige of the Soviet Union would only be enhanced by a humanitarian act in his case.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "June 16, 1975 Sir: I am writing to you, as the supreme voice of authority in the Soviet Union, to plead for the release of Dr. Mikhail Shtern. Dr. Shtern by this time is a sick and tormented man, prematurely aged by his experiences.  He suffers from severe health problems and I fear for his life under the regimen of the labor camp.  Certainly, he will never survive to serve his eight-year sentence. The Soviet Union today is one of the world's great powers.  Its place among the nations is secure and does not depend on keeping one frail, sick man in prison.  The prestige of the Soviet Union abroad can only be enhanced by a humanitarian and generous act of clemency in his case. I and many of my colleagues in science have welcomed the reduction in tensions between our two countries as a result of the policy of detente.  We are in the forefront of exchanges in science between the USSR and the United States.  But those exchanges cannot take place without the collaboration and support of American scientists, and their enthusiasm for such cooperation is not encouraged when they read about Dr. Shtern or other Soviet scientists whose desire to emigrate to Israel has resulted in severe punishment. I urge you to grant amnesty to Dr. Shtern, and allow him and his wife to leave so that they may join the remaining members of their family and live out their remaining years in peace. Sincerely yours, Marshall Nirenberg", "Nirenberg, Marshall W.", null, null, null, "Union of Soviet Socialist Republics ; Podgorny, Nikolai V.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ihzp.eduq~acwd", "00000000-0000-0000-7218-808430AF26A9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Frank Press, National Academy of Sciences to Marshall W. Nirenberg", "101584910X289", null, "1987", "6 January 1987", "Press, President of the National Academy of Sciences, provides an update on the release from exile of Soviet scientist Andrei Sakharov and remains hopeful that formal relations with the Soviet Academy are possible.  Press notes that in press interviews Sakharov \"attributed his release from exile to the role of Western academies,\" making Sakharov's return to Moscow and his scientific work a \"reason for rejoicing.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Frank Press.", "Copyright may apply", null, null, "January 6, 1987 Members of the Academy Dear Colleagues: Two weeks ago I informed Academician Guri Marchuk, the new president of the Soviet Academy of Sciences, of our delight at the good news that our foreign associate, Academician Andrei Sakharov, and his wife, Dr. Elena Bonner, were being permitted to return to Moscow and that Sakharov would resume his scientific work.  (As you may have read in the press reports, President Marchuk visited Sakharov in Gorky.)  Academician Yevgeny P. Velikhov, a vice president of the Soviet academy and one with whom I have personally discussed the situation of Sakharov and his wife, was with Vladimir F. Petrovsky, a deputy foreign minister, when the official announcement was made at a press conference.  Dr. Velikhov was quoted as saying that he would welcome Academician Sakharov's return to active research at the academy's Institute of Physics.  Dr. Sakharov has always retained his membership in the Soviet academy, although he was reportedly stripped of all other honors when sent into internal exile in Gorky in January 1980.  Members of the academy in good standing are entitled to a number of privileges, including special medical care, which the Sakharovs deserve. Dr. Bonner stressed on several occasions last spring, when she was a guest at the NAS Annual Meeting, that our appeals in Dr. Sakharov's behalf should continue to focus on his wish to return to Moscow and to resume his scientific work.  In his press interviews, Dr. Sakharov attributed his release from exile to the role of Western academies.  It is both gratifying and heartening, therefore, to know that our many efforts, about which I have written you earlier, have been rewarded, and that this wish has been fulfilled.  I am hopeful that the decision to reestablish formal relations with the Soviet academy and to use these renewed channels of communication to encourage Soviet scientific leaders to address human rights concerns will continue to show results.  Time will tell. I know that along with the numerous and repeated appeals, both public and private, that were made by the officers of this Academy, many individual members have steadfastly continued their own efforts in Dr. Sakharov's behalf.  The leaders of Western governments have also played an important role.  Thus, I know that Dr. Sakharov's long-awaited return to Moscow and his scientific work gives us all reason for rejoicing. Yours sincerely, Frank Press President", "Press, Frank ; National Academy of Sciences (U.S.)", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-se57-8j83.fhnm", "00000000-0000-0000-67E1-FD9306F7EDA6", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Kohzo and Noriko Nakayama to Marshall W. Nirenberg", "101584910X290", "101584910X364", "1990", "[December 1990]", "The Nakayamas update Nirenberg on Kohzo's latest work at the Cancer Research Institute at Kanazawa University.  Nirenberg receives thanks for the wonderful times Kohzo spent as a postdoctoral student in Nirenberg's lab.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Kohzo Nakayama.,Reproduced with permission of Noriko Nakayama.", "Copyright may apply", null, null, "No1 Have a blessed christmas [sic] and a happy new year. Dear Marshall and Perola. We live a happy life in Kanazawa city except small apartment, crowded traffic jam and missing wonderful friends in NIH. Kohzo jointed to the new lab. and has started a new project, c-rel, which is a vertebrate homolog of drosophila dorsal gene. There is high homology between c-rel and NF-kB which is a transcription factor suggesting that there is rel/NF-kB gene family in vertebrate. Now Kohzo just has cloned NF-kB and c-rel cDNA by PCR. Kohzo is looking for the new members of rel family and will try to in situ hybridization and histochemistry as a first step. Kohzo hopes rel family give us a good hint to understand a problems of dorsal-ventral polarity formation including a development of nervous system. In addition to this project, Kohzo has got drosophila (Oregon R) and started the small scale culture. We hope we can continue the drosophila work, when Kohzo can get a grant (Now. my Lab is really poor.). [END PAGE ONE] [BEGIN PAGE TWO] No 2. Noriko got a new job in hospital as a pharmacist and she is going to joint to my Lab. in the evening as a research student. It takes her about ten years to get a ph D. degree (I can't understand Japanese education system at all). Anyway, Noriko hopes to continue research. We believe it most important. We really hope we can have a next chance to live in America again. We hope everything is wonderful for you in 1991 with our best regards               sincerely yours Kohzo and Noriko P.S.1. We wish we can read our draft about Nk-2 homeobox gene. 2. Enclosed is Japanease [sic] candy (?). Please try it.", "Nakayama, Noriko ; Kanazawa-Daigaku ; Nakayama, Kohzo", null, null, null, "Nirenberg, Perola ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qb5u~uutt-36yj", "00000000-0000-0000-5A56-57416AD5418C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Robert Hofstadter, Henry Taube, William Fowler, and George Wald to Marshall W. Nirenberg", "101584910X291", "101584910X310", "1984", "6 September 1984", "This recalls the statement signed by Albert Einstein, Bertrand Russell, and seven other Nobel laureates in 1955 renouncing thermonuclear weapons and promoting the establishment of peaceful means of settling international disputes.  The authors ask Nirenberg to join a new statement, part of a five day conference involving more than one hundred environmental and peace organizations in Washington, D.C.  The statement, an elaboration of the Einstein-Russell \"Manifesto\" of 1955, is included with the letter.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Henry Taube.", "Copyright may apply", null, null, "6 September, 1984 Dear Dr. Nirenberg: Nearly three decades ago Bertrand Russell and Albert Einstein, along with seven other Nobel laureates, published a statement calling for the renunciation of thermonuclear weapons and the establishment of peaceful means for settling international disputes.  Since that time, the prospects for global nuclear war have become even more dire.  Recent studies on the possibilities of a nuclear war triggering a nuclear winter now confirm earlier fears that nuclear war might well mean an end to life on this planet.  Despite the increasing danger, national policy-makers are devoting ever more money to armaments and continue to ignore pressing environmental needs.  Another warning by the world's intellectual leaders -- in the spirit of the Einstein-Russell \"Manifesto\" -- is long overdue. We are writing to ask you to join a new statement that will be issued in the third week of September.  Originally drafted by David Brower of the Friends of the Earth and Russell Peterson of the National Audubon Society and the Global Tomorrow Coalition, this statement is part of a five-day-long conference involving more than one hundred environmental and peace organizations.  The Conference begins September 19, 1984 in Washington, D.C. and is the second in a series of biennial conferences sharing the theme \"Conservation and Security in a Sustainable Society.\" A draft of the new statement (a Policy and Action Statement) is enclosed, along with a Legislative Action Agenda spelling out some short-term steps for action.  We urge you to read the statements and consider becoming a signatory.  Some of us endorse only the Policy and Action Statement, while others of us endorse both.  Please use your own judgment about whether you wish to sign one or both. As Russell and Einstein understood, the endorsement of scientists of international stature has the power to endow such a declaration with universal respect and recognition.  With your assistance and the assistance of other Nobel laureates, we hope to provide an effective, widely-publicized call to action to prevent nuclear war and environmental decay. We hope you will join us in endorsing this effort.  Although the statements cannot be panaceas, they perhaps can alert the public to the need for new understanding, responsibility, and commitment.  It is hard to imagine an election year when such an alert was more needed. Since time is short, we would greatly appreciate if you -- or a representative -- could call Laura Nelson, the statement coordinator, as soon as possible at (415)328-5137. Otherwise, she will try to reach you.  The statement will be going to press within the week, and your endorsement would be most useful before that date. However, if necessary, endorsements up until the first day of the Conference, September 18, are possible. Robert Hofstadter, Stanford University Henry Taube, Stanford University William Fowler, U.C. San Diego George Wald, Harvard University", "Taube, Henry ; Fowler, William ; Wald, George ; Hofstadter, Robert, 1915-1990", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-d7kv-q34y_bcie", "00000000-0000-0000-0077-884FAF071061", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Israel Halperin to Marshall W. Nirenberg", "101584910X292", null, "1989", "15 June 1989", "This form letter, sent to all persons who joined in the statement of support of the international campaign to end all abduction and torture in Chile, includes an updated bulletin from March-April 1989 detailing the nature of the campaign.  The bulletin details human rights violations and includes the names of supporters of the campaign, including 133 Nobel laureates.  A handwritten note from Halperin to Nirenberg asks for permission to include his name with the other Nobelists.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "10", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "[handwritten note consists of a portion of a larger printed letter] Dr. Nirenberg, please see note below Israel Halperin Dr. Nirenberg, This Chile Campaign to end all torture in Chile is a concrete step in the program to end torture everywhere. If you permit your name to be added to the list of 134 Nobel Laureates who support this Chile Campaign this will NOT imply any further commitment by you of any kind whatsoever. Will you permit this?", "Halperin, Israel ; International Campaigns for Human Rights", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6tta~vp3w.4vyp", "00000000-0000-0000-7D65-4F8CFF1F74C9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The School of Public Health Sciences, Inaugural Convocation", "101584910X293", null, "1986", "21 March 1986", "The State University of New York's School of Public Health Sciences Inaugural Convocation program includes a brief statement of Nirenberg's achievements with the genetic code and his work in training a generation of outstanding researchers, earning him an Honorary Degree of Doctor of Science.", "Programs (documents)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "8", "pages", "Text", "English", "Reproduced with permission of State University of New York. Albany.", "Copyright may apply", null, null, "The School of Public Health Sciences     Inaugural Convocation March 21, 1986 Convocation Colloquia New York State Museum Auditorium Cultural Education Center Empire State Plaza, Albany 10:00 a.m. - 11:00 a.m. Dr. Marshall W. Nirenberg Nobel Laureate Chief, Laboratory of Biochemical Genetics National Institutes of Health, Bethesda, Md. **Positive and Negative Regulation of Species of mRNA by. Cyclic AMP” 11:00 a.m. - 12:00 a.m. Dr. Rollin D. Hotchkiss Professor Emeritus, Rockefeller University Research Professor, State University of New York at Albany “‘Cycles in Genetics and the Academic Metabolism’’ Convocation Ceremony 3:00 p.m. New York State Museum Auditorium, Cultural Education Center, Empire State Plaza Academic Procession Concourse, Empire State Plaza Schenectady Pipers Invocation The Reverend Laman H. Bruner, Jr. Rector, Saint Peter’s Episcopal Church, Albany ‘National Anthem Opening Welcome Vincent O’Leary, President State University of New York at Albany Greetings The Honorable Thomas M. Whalen, III, Mayor, City of Albany Dr. Henrik N. Dullea, Director of State Operations & Policy Management, on behalf of Governor Mario Cuomo Dr. Gordon M. Ambach, Commissioner, New York State Department of Education Mr. Donald M. Blinken, Chairman of the Board of Trustees, State University of New. York Dr. Robert L. Friedlander, President and Dean, Albany Medical College Dr. W. Jean Dodds, Chief, Laboratory of Hematology, Wadsworth Center for Laboratories and Research, New York State Department of Health Professor of Hematology, School of Public Health Sciences Presentation of Honorary Degrees Dr. Rollin D. Hotchkiss, Doctor of Science Dr. Marshall W. Nirenberg, Doctor of Science Address Dr. David Axelrod, Commissioner, New York State Department of Health Benediction The Reverend Laman H. Bruner, Jr. Recession Reception String quartet Members of the Procession Dean David O. Carpenter, Mace Bearer Dr. David Axelrod President Vincent O’Leary The Honorable Thomas M. Whalen, Ii Dr. Henrik N. Dullea Dr. Gordon M. Ambach Mr. Donald M. Blinken Dr. Robert L. Friedlander Dr. W. Jean Dodds Dr. Lawrence H. Flesh Dr. Judith A. Ramaley Honorary Degree Recipients Dr. Marshall W. Nirenberg Dr. Rollin D. Hotchkiss Members of the University Council Mr. Alan V. Iselin The Reverend Laman H. Bruner, Jr. Ms. A, Rita Chandellier Glavin Mr. Richard A. Hanft Mrs. Betty H. Hay Mr. Peter D. Kiernan Mrs. Athena C. Kouray Mrs. Martha W. Miller Mr. Lewis A. Swyer Mr. Michael Miller Distinguished Guests Dr. Magda Gabor-Hotchkiss The Honorable Richard J. Conners The Honorable Geraldine Daniels Regent Shirley Brown Dr. Donald J. Nolan Trustee Edgar A. Sandman Dr. Jerome B. Komisar Dr. Joseph Burke Dr. Alden N. Haffner President Joseph J. Bulmer President Charles J. Meder Dr. Adrian G. Marcuse Ms. Lorna McBarnette Dr. Linda Randolph Mr. Lee J. VanDeCarr Rev. Carl B. Taylor Vice President Lawrence H. Poole State University of New York at Albany University Senate Chair Kendall A. Birr Dr. Walter Gibson Mr. John Hartigan Dr. Frank Pogue Dr. John W. Shumaker Dr. Lewis P. Welch Provost Warren FE. Iichman Dean Richard Halsey Dean Robert Koff Dean Terence Thornberry Dean John Webb Dean Daniel Wulff Acting Dean Richard Edwards Members of the Faculty, School of Public Health Sciences Rajender Abraham, Ph.D. Kenneth Aldous, Ph.D. John Aronson, Ph.D. Corrado Baglioni, M.D. Shelton Bank, Ph.D. Marlene Belfort, Ph.D. Ronald Bellisario, Ph.D. Donald Berns, Ph.D. Monica Bishop, M.D. Joseph Bohorquez, M.D. Brian Bush, Ph.D. David Carpenter, M.D. Thomas Carter, Ph.D. James Catalfamo, Ph.D. Everly Conway de Macuric, Ph.D. Anthony DeCaprio, Ph.D. Rudolf Deibel, M.D. Herbert Dickerman, M.D., Ph.D. W. Jean Dodds, D.V.M. George Eadon, Ph.D. Michael Fasco, Ph.D. John Fenton, Ph.D. Lorraine Flaherty, Ph.D. Joachim Frank, Ph.D. John Galivan, Ph.D. John Gierthy, Ph.D. Nisan Gilboa, M.D. Morris Gordon, Ph.D. Leo Grady, Ph.D. Howard Gruft, Ph.D. Katherine Henrikson, Ph.D. Liaquat Husain, Ph.D. Laurence Kaminsky, Ph.D. John Kaplan, Ph.D. Gordon Kaye, Ph.D. James Kim, D.V.M., D.Sc. Brajesh Kothari, Ph.D. S. Anand Kumar, Ph.D. Charles Kunz, Ph.D. Robert Laffin, Ph.D. David Lawrence, Ph.D. Alberto Macario, M.D. John Mackiewicz, Ph.D. Albert Magro, Ph.D. Frank Maley, Ph.D. Gladys Maley, Ph.D. Carmen Mannella, Ph.D. Raphael Mannino, Ph.D. David Martin, Ph.D. Joseph Mascarenhas, Ph.D. Joseph Mazurkiewicz, Ph.D. James McSharry, Ph.D. Anne Messer, Ph.D. Terry Miller, Ph.D. Douglas Mitchell, Ph.D. Gerald Mizejewski, Ph.D. Volker Mohnen, Ph.D. Daniel Nealon, Ph.D. Patrick O’Keefe, Ph.D. . Enzo Paoletti, Ph.D. Donald Parson, M.D., D.Sc. Kenneth Pass, Ph.D. Ian Porter, M.D. Robert Rej, Ph.D. G-Yull Rhee, Ph.D. Conly Rieder, Ph.D. Thomas Rosano, Ph.D. William Samsonoff, Ph.D. Muriel Schauble, M.D. Richard Seegal, Ph.D. Jacquelin Shaffer, Ph.D. William Shain, Ph.D. Mehdi Shayegani, Ph.D. Jay Silkworth, Ph.D. Walter Stahl, Ph.D. Lawrence Sturman, M.D., Ph.D. John Swann, Ph.D. Harry Taber, Ph.D. James Turner, Ph.D. Martin Wahlen, Ph.D. Ann Willey, Ph.D. Jonathan Wolpaw, M.D. Meyer Wolin, Ph.D. Au-Ya Shih Wu, M.D., Ph.D. Andrew Yencha, Ph.D. Members of the Faculty, State University of New York at Albany Citations Rollin D. Hotchkiss Your remarkable scientific career, marked by half a century of contributions to molecular genetics, qualifies you as a founding father of that field. You were the first to isolate, in pure form, an antibiotic, an important accomplishment that stimulated the development of a life-saving class of drugs. Well ahead of ctiiezs, you brought forth to the scientific community. vital information demonstrating that DNA molecules form the basis of genetic material, a profound discovery that sparked one of the most exciting periods in the history of science. For all of your brilliant and highly innovative work in molecular genetics, which continues today, for your boundless curiosity, your devotion to truth and rigor in thinking, and your warm and genuine interest in others, the State University of New York is deeply honored to confer upon you, Rollin D. Hotchkiss, the Honorary Degree of Doctor of Science. Marshall W. Nirenberg Your supreme achievement in deciphering the genetic code places you among the pioneers of modern molecular biology. By breaking this code, and subsequently devising an ingenious method to unlock the sequence of the code, you have deciphered the Rosetta Stone of the biological world. Your enormous scientific talents now extend to the study of the nervous system, providing revolutionary insights into the fundamental workings of the brain. | As a recipient of the Nobel Prize in Medicine, you represent an exemplary model of a scientist who has attained the pinnacle of your profession and yet is still capable of standing in awe and wonder at the magic of the universe. For your unparalleled contributions and your work training a generation of outstanding researchers, the State University of New York is privileged to confer upon you, Marshall W. Nirenberg, the Honorary Degree of Doctor of Science.", "State University of New York. Albany", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-w99j.ne7r.2xjb", "00000000-0000-0000-FBCB-D81576E82084", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Edwin C. Whitehead and Paul G. Rogers to Marshall W. Nirenberg", "101584910X294", null, "1986", "6 June 1986", "The Chairman and the Vice Chairman of the Friends of the National Library of Medicine inform Nirenberg that he has been awarded the National Library of Medicine Medal as a living Nobel laureate in medicine and biology.  As one America's most distinguished scientists, Nirenberg was selected as part of the celebration of the 150th anniversary of the National Library of Medicine.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Susan Whitehead.", "Copyright may apply", null, null, "June 6, 1986 Dear Dr. Nirenberg: To celebrate the 150th Anniversary of the National Library of Medicine, the Library wishes to award the National Library of Medicine Medal to living American Nobel Laureates in medicine and biology.  We hope that you will be willing to accept this honor. The Friends of the National Library of Medicine are planning a commemorative event in Washington for September 16 and 17.  The highlight of this two-day gala will be a reception and dinner, during which the medals will be formally presented. We have asked President Reagan to be the presenter and the affair will be attended by members of Congress and distinguished members of the American scientific community. We believe it is fitting and proper that the National Library of Medicine, as one of America's   great medical institutions, present this medal to you, one of America's most distinguished scientists. 1986 marks an important milestone for the Library and the commemorative medal will honor this occasion. We are writing this letter now to be sure the date can be reserved on your calendar.  We will be sending you further details.  Not only should this be a most historic moment, but also a gala celebration party, for you, your colleagues and peers, and the Library. We would very much appreciate an indication of your ability to attend. Sincerely, Edwin C. Whitehead, Chairman Paul G. Rogers, Vice Chairman", "National Library of Medicine (U.S.). Friends ; Rogers, Paul G. ; Whitehead, Edwin C.", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3gs3_y8mk~yx72", "00000000-0000-0000-A831-006062DFA55D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Toby M. Horn to Marshall W. Nirenberg", "101584910X295", null, "1987", "2 March 1987", "Horn invites Nirenberg, on behalf of the science faculty and biology students, to honor Thomas Jefferson High School for Science and Technology in Virginia by returning to discuss his work once again.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Toby M. Horn.", "Copyright may apply", null, null, "March 2, 1987 Dear Dr.  Nirenberg: On behalf of the Science Faculty and biology students, I am writing to ask you to honor our school again this year by coming to talk about your work. We have a new group of 400 ninth-graders, and 20 seniors in AP Biology and Chemistry who would certainly enjoy meeting you. Since we now also have videotape cameras, we could tape your presentation if you agreed. If you can visit again, may I suggest a Wednesday of Thursday afternoon towards the end of April or May.  Our activity periods occur between 3 and 4 PM. A telephone reply would be welcomed. Yours truly, Toby M Horn,  Biotechnology Lab 941-3621 x 266", "Thomas Jefferson High School for Science and Technology ; Horn, Toby M.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-px92-pnf2.tcdq", "00000000-0000-0000-5819-FF871A298CB8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Elie Wiesel to Marshall W. Nirenberg", "101584910X296", "101584910X311", "1987", "15 July 1987", "Wiesel invites Nirenberg to a conference of Nobel laureates to be held in Paris in January 1988.  The purpose of the conference, to address the principle moral and political problems challenging mankind and explore solutions to them, is detailed in the conference outline which is included.  Wiesel states that the \"conference grows out of the belief that each of us feels a profound responsibility to confront those issues that affect all humanity.\"  Nirenberg is invited because of the faith that \"science and human genius can be used on behalf of the humankind.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Elie Wiesel.", "Copyright may apply", null, null, "July 15, 1987 Dear Dr. Nirenberg: I am writing to invite you to a conference of Nobel Laureates to be held in Paris, January 18-22, 1988. The purpose of the conference is to address the principal moral and political problems that challenge humankind as we approach the next century and to explore creative solutions to them.  A formidable agenda, I know. The conference grows out of the belief that each of us feels a profound responsibility to confront those issues that affect all humanity.  That children are starving, that wars continue to be fought, that oppressive regimes flourish, that the potential for nuclear destruction exists -- for us, none of these problems is an abstraction. We know that science and human genius can be used on behalf of humankind. That is why President Francois Mitterand and I jointly invite you to be our guest at a conference which would enable each of us -- regardless of our particular discipline or area of expertise -- to discuss, formulate and communicate our ideas about these critical issues. We intend to open in plenary session on Monday, January 18, 1988, and through seminars, formal and informal discussions, to search for new ways to approach these important issues.  Our hope is that our deliberations will bear fruit and inspire a dialogue with those in positions of power. Your participation will add breath and depth to our discussion. President Mitterand and I both hope you will be able to attend. I am enclosing a copy of the conference proposal and would welcome your suggestions and comments.  Details about travel and hotel arrangements will be sent at a later date. In the meantime, please return the enclosed response card indicating your acceptance in principle of our invitation. I know your time is precious and you have many commitments. And yet, I am hopeful that we shall meet in Paris next January. We have many important matters to discuss. Yours sincerely, Elie Wiesel", "Wiesel, Elie, 1928-2016 ; Elie Wiesel Foundation for Humanity", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bcj7~ma6n~xs4x", "00000000-0000-0000-F739-9A758D241490", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Frank Press, National Academy of Sciences to Marshall W. Nirenberg", "101584910X297", null, "1987", "June 1987", "In this form letter, President of the National Academy of Sciences Frank Press informs readers that the NAS is redistributing \"Science and Creationism: A view from the National Academy of Sciences,\" a discussion reaffirming the position that creationism is not science, in response to the U.S. Supreme Court decision on a Louisiana law that calls for \"balanced treatment.\"  The letter ends with the statements: \"Classroom science teachers do not teach the geography of a flat earth.  Neither should they teach creationism.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Frank Press.", "Copyright may apply", null, null, "June 1987 Dear Reader: In 1984, when the National Academy of Sciences first distributed Science And Creationism: A View From The National Academy Of Sciences, my letter to the reader mentioned the seemingly unending question of whether science, creationism, or both should be taught in the science classroom.  Three years later, the question has reached the level of a United States Supreme Court decision on a Louisiana law that calls for \"balanced treatment.\" In the face of this legal decision, the National Academy of Sciences is redistributing Science And Creationism to reaffirm its position that creationism is not science.  To teach creationism and science as equally sound and valid alternative scientific theories is both misleading and inaccurate. Our booklet describes the nature of scientific inquiry and its application to understanding the evolution of the universe and life on earth.  We ask that you examine the findings, study the facts supporting the ideas, question the verifiability, and examine the predictive powers -- all criteria at the heart of science.  That is the ethos of science and scientists:  questioning existing information; testing facts; discarding ideas that cannot be supported by what has been learned; and building on ideas that are supported. Classroom science teachers do not teach the geography of a flat earth.  Neither should they teach creationism. Frank Press", "Press, Frank ; National Academy of Sciences (U.S.)", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rrbm~nvyb-wqiu", "00000000-0000-0000-FC89-FA2A6F3DD479", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Open Letter to Congress", "101584910X298", null, "1986", "12 March 1986", "This petition expresses the concerns of scientists and engineers about the Strategic Defense Initiative (SDI) commonly known as \"Star Wars\".  It maintains that statements from the Reagan administration give an erroneous impression of virtually unanimous support for the initiative from the scientific and technological community, while the program has actually grown without the appropriate technological and policy scrutiny.", "Petitions", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "March 12, 1986 Dear Colleague: The undersigned are part of an ad-hoc group of scientists concerned about the Strategic Defense Initiative (SDI). Attached find an Open Letter to Congress which is being circulated to scientists and engineers at industrial and government laboratories. We urge you to sign the letter and to circulate it among your colleagues. We plan to collect signatures for a period of roughly three months and to send the signed petitions to each member of the U.S. Congress in the Spring of 1986. All efforts will be made to ensure that no representations as to the views of the signatories are made in the press or anywhere else, beyond the actual text of the letter. Please return signed petitions to the above address. Only persons currently or formerly affiliated with industrial or governmental laboratories should sign. If you have further questions, please call (201)-467-7629. Original signed by: Philip W. Anderson*, AT&T Bell Laboratories (retired) John Backus, IBM Fellow, IBM Almaden Research Center Owen Chamberlain*, Lawrence Berkeley Laboratory Ernest D. Courant, Brookhaven National Laboratory Albert V. Crewe, Former Director, Argonne National Laboratory Gerhart Friedlander, Brookhaven National Laboratory (retired) Pierre C. Hohenberg, AT&T Bell Laboratories Jack L. Kerrebrock, Former Associate Administrator, Office of Aeronautics and Space Technology, NASA J. Carson Mark, Former Theoretical Division Leader, Los Alamos Scientific Laboratory (retired) Edwin M. McMillan*, Former Director, Lawrence Berkeley Laboratory (retired) Marshall W. Nirenberg*, National Institutes of Health Thressa C. Stadtman, National Institutes of Health Kenneth L. Thompson, AT&T Bell Laboratories Robert W. Wilson*, AT&T Bell Laboratories NOTE: institutional affiliations for identification purposes only *Nobel laureate Biographies of Sponsors Philip W. Anderson born: December 13, 1923 Ph.D. 1949 Physics, Harvard University. Naval Research Laboratory, 1943-1945. AT&T Bell Labs, 1949-1984; Consultant 1984-present. Princeton University, 1975-present. Nobel Prize in Physics, 1977 (magnetism and disordered systems). National Medal of Science, 1982 (President Reagan). Member, National Academy of Sciences. John Backus born: December 12, 1924 MS 1950 Mathematics, Columbia University. IBM 1950-present, IBM Fellow, 1983-present. Inventor of FORTRAN (1957). AM Turing Award 1977 '(highest computer science honor). National Medal of Science 1975 (President Ford). Member, National Academy of Sciences. Member, National Academy of Engineering. Owen Chamberlain born: July 10, 1920 Ph.D. 1949 Physics, University of Chicago. Los Alamos Scientific Laboratory, 1942-1946. Lawrence Berkley Laboratory, 1934-present. Nobel Prize in physics, 1959 (discovery of anti-proton). Member, National Academy of Sciences. Ernest D. Courant born: March 26, 1920 Ph.D. 1943 Physics, University of Rochester. Brookhaven National Laboratory, 1960-present. Member, National Academy of Sciences. Albert V. Crew born: February 18, 1927 Ph.D. 1950 Physics, University of Liverpool, England. Director, Argonne National Laboratory, 1961-1967. Currently: Professor, University of Chicago. Member, National Academy of Sciences. Gerhart Friedlander born: July 28, 1916 Ph.D. 1942 Nuclear Chemistry, University of California, Berkeley. Staff Member and Group Leader, Los Alamos Scientific Laboratory, 1943-1946. General Electric Research Laboratory, 1946-1948. Brookhaven National Laboratory, 1948-present; Chairman, Chemistry Department, 1968-1977. Member, National Academy of Sciences Pierre C. Hohenberg born: October 3, 1934 Ph.D. Physics, Harvard University AT&T Bell Labs (currently Head of Theoretical Physics Department). Vice President for Physical Science, New York Academy of Sciences 1986-. Member NSF Materials Research Advisory Committee 1981-present. Jack L. Kerrebrock born: February 6, 1928 Ph.D. 1956, California Institute of Technology Associate Administrator, Office of Aeronautics and Space Technology, National Aeronautics and Space Administration, 1981-1983. Associate Dean of Engineering, M.I.T., 1983-present. Oak Ridge National Laboratory, 1956-58 Member, National Academy of Engineering. Currently serving on: USAF Scientific Advisory Board; National Commission on Space. J. Carson Mark born: July 6, 1913. Ph.D. 1938 Mathematics, University of Toronto. Los Alamos Scientific Laboratory 1945-1973. Theoretical Division Leader 1947-1973 in charge of new weapons design. Currently:  consultant at Los Alamos and on many government boards, including (since 1976) Advisory Committee on Reactor Safeguards of the Nuclear Regulatory Commission. Edwin M. McMillan born: September 18, 1907 Ph.D. 1938 Mathematics, University Lawrence Berkley Laboratory, 1934-1973; Director, 1971-1973. Los Alamos Scientific Laboratory, 1942-1945. Trustee, Rand Corporation, 1959-1969. Nobel Prize in Chemistry, 1951 (discovery of Plutonium and other elements). Member, National Academy of Sciences. Marshall W. Nirenberg born: April 19, 1927 Ph.D. 1957 Biochemistry, University of Michigan. National Institute of Health, 1957-present. Nobel Prize in Medicine, 1968 (deciphering the genetic code). Member, National Academy of Sciences. Thressa C. Stadtman born: February 12, 1920 Ph.D. 1949 Microbiology, University of California, Berkeley. National Institute of Health, 1950-present; Section chief, 1966-present. Member, National Academy of Sciences. Kenneth L. Thompson born: February 4, 1943 M.S. 1966 Electrical Engineering, University of California, Berkeley. AT&T Bell labs, 1966-present. Co-Author, UNIX operating system. A.M. Turing award (highest computer science honor). Member, National Academy of Engineering. Member, National Academy of Sciences. Robert W. Wilson born: January 10, 1936 Ph.D. 1962 Physics, California Institute of Technology. AT&T Bell Labs, 1963-present; Head of Radio Physics Research Department, 1976-present. Nobel Prize in Physics, 1978 (co-discoverer of cosmic background radiation from the Big Bang). Member, National Academy of Sciences. An Open Letter to the U.S. Congress June 19, 1986 We, the undersigned scientists and engineers currently or formerly at government and industrial laboratories, wish to express our serious concerns about the Strategic Defense Initiative (SDI), commonly known as \"Star Wars\". Recent statements from the Administration give the erroneous impression that there is virtually unanimous support for this initiative from the scientific and technical community. In fact the SDI has grown into a major program without the technical and policy scrutiny appropriate to an undertaking of this magnitude. We therefore feel that we must speak out now. The stated goal of the SDI is developing the means to render nuclear weapons \"impotent and obsolete\". We believe that realization of this dream is not feasible in the foreseeable future. The more limited goal of developing partial defenses against ballistic missiles does not fundamentally alter the current policy of deterrence, yet it represents a significant escalation of the arms race and runs the serious risk of jeopardizing existing arms control treaties and future negotiations. Furthermore, in view of the international economic competition faced by the US., it should be asked whether the country can afford the diversion of resources, especially scientific and technical manpower, that the SDI entails. The Congressional Office of Technology Assessment has raised serious questions concerning the scope and scale of the present SDI effort. We urge the Congress to heed these concerns and to limit the SDI to a scale appropriate to exploratory research, while assessing the costs, the risks and the potential benefits of the program in comparison with alternative strategies for strengthening the overall security of the nation. Top priority must be given to this task before the momentum inherent in a program of such magnitude makes this venture irreversible.", "Hohenberg, Pierre C. ; Kerrebrock, Jack L. ; Nirenberg, Marshall W. ; Mark, J. Carson ; Courant, Ernest D. ; Crewe, Albert V. ; Friedlander, Gerhart ; Stadtman, Thressa C. ; Anderson, Philip W. ; Thompson, Kenneth L. ; Backus, John ; Wilson, Robert W. ; Chamberlain, Owen ; McMillan, Edwin M. (Edwin Mattison), 1907-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-63hf~t46g~vez9", "00000000-0000-0000-FD84-BB2B0BF9FE0B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Douglas Mattern, World Citizens Assembly to Marshall W. Nirenberg", "101584910X299", null, "1977", "5 December 1977", "Mattern enclosed a copy of the resolution \"To End the Arms Race\" adopted by the World Citizens Assembly meeting in Paris, France.  More than 600 representatives from 25 countries participated.  The WCA sought endorsement of the resolution by influential individuals and organizations in preparation for the United Nations General Assembly on Disarmament meeting scheduled for 1978.. NOTE: The text is cut off on page 3 of the original.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "3", "pages", "Text", "English", "Reproduced with permission of Douglas Mattern.", "Copyright may apply", null, null, "December 5, 1977 Enclosed is a major resolution (To End the Arms Race) adopted by the World Citizens Assembly (WCA) meeting in Paris, France from July l-5, 1977.  The WCA was attended by 600 people from 25 countries.  The resolution was written by Commission I after two days of extensive discussion with representatives of peace organization from Western, Socialist, and Third World countries. The focus of the resolution is the United Nations General Assembly on Disarmament scheduled for 1978. With the arms race continuing its macabre course, it is imperative that this crucial U.N. Special Session achieve positive results. The WCA resolution appeals to all peoples, all governments, all non-government organizations to demonstrate their support; to arouse public opinion sufficient to inspire their governments to positive action. To help further this goal, the WCA is seeking endorsement of this resolution by influential individuals and organizations.  Early in 1978, the resolution (with endorsements) will be presented to leaders of governments, and directly to the United Nations.  We would be honored to add your name to the list of prominent global citizens endorsing this resolution.  More than 35 Nobel Laureates have already added their name, along with many other outstanding people and organizations.  The deadline for receiving endorsements is January 31, 1977. The coming months of activities, leading to and including the U. N. Special Session on Disarmament, could provide a turning point away from the hopeless trend of the arms race toward fundamental steps to build a world community which offers humanity a future.  We must not fail this historic opportunity. All endorsers will receive a copy of the final document when it is readied for presentation. Sincerely, Douglas Mattern Secretary General, WCA Chairman, Commission I World Citizens Assembly \"To Build A World Community\" The second World Citizens Assembly meeting in Paris, France with 600 delegates from 25 countries, officially adopted the following resolution on July 5, 1977.  This resolution was written by Commission I after days of extensive discussion with representatives from Western, Socialist, and Third World countries. Commission I, To End the Arms Race \"The Future Of Mankind Depends On The Capacity Of The Present Generation To Realize This Interdependence And Take Urgent Action At The International Level To Achieve The Necessary Progress In Disarmament.\" Kurt Waldheim - Secretary General - United Nations Whereas the priority task facing the world community is to stop the arms race; to achieve drastic reductions in military expenditures, together with programs converting the arms industry into peaceful production; and Whereas it is essentially through ending the arms race that a new world economic order can be funded which will provide a more equitable distribution of the world's wealth and resources, and assure an improved quality of life for the people of the earth; and Whereas negotiations between the major powers, including the SALT agreements, have failed to slow the arms race, and the world arms trade continues to escalate; and Whereas concrete steps toward controlled disarmament can lead to a strengthened United  Nations with the authority to settle disputes between nations through the framework of world law; and Whereas the central purpose of the United Nations Charter is to end the scourge of war, the corollary is to implement this principle by all peaceful means; Therefore Be It Resolved that the World Citizens Assembly, meeting with 600 delegates from 25 countries in Paris, July, 1977, appeals to all peoples, all governments, and all NGOs to support the United Nations General Assembly on Disarmament in 1978; to arouse public opinion through persuasion and direct action, in order to insure that this most crucial Assembly attains positive results; Further Be It Resolved that the World Citizens Assembly, seeking a significant input at the Special Assembly on Disarmament, appeals to the Secretary General and the organizers of the Special Session to permit the full participation of peoples' organizations as well as governments in this historic opportunity to save humanity. List Of Endorsers Of The WCA Resolution (as of 12/l/77) Nobel Laureates Dr. Hannes Alven, 1970 (Physics) Dr. Christian Anfinsen, 1972 (Chemistry) Dr. Kenneth Arrow, 1972 (Economics) Lord Phillip Noel-Baker, 1959 (Peace) Sir Frank Burnet, 1960 (Medicine) Dr. Andre Cournand, 1956 (Medicine) Dr. Christian de Duve, 1974 (Medicine) Dr. Paul Flory, 1974 (Chemistry) Dr. Ragnar Granit, 1967 (Medicine) Dr. Odd Hassel, 1969 (Chemistry) Dr. Dorothy Crawfoot-Hodgkin, 1964 (Chemistry) Dr. Alfred Hershey, 1969 (Medicine) Dr. Gerhard Herzberg, 1971 (Chemistry) Dr. Albert Szent-Gyorgi, 1937 (Medicine) Dr. Francois Jacob, 1965 (Medicine) Dr. Brian Jospehson, 1973 (Physics) Dr. Alfred Kastler, 1966 (Medicine) Dr. Konrad Lorenz, 1973 (Medicine) Dr. Salvador Luria, 1969 (Medicine) Dr. Andre Lwoff, 1965 (Medicine) Mr. Sean McBride, 1974 (Peace) & Lenin Peace Prize Dr. Rudolf Mossbauer, 1961 (Physics) Dr. Robert Mulliken, 1966 (Chemistry) Mr. Gunner Mydral, 1975 (Economics) Dr. Severo Ochoa, 1959 (Medicine) Dr. George Palade, 1974 (Medicine) Dr. Linus Pauling, 1954 (Chemistry) 1962 (Peace) Dr. Max Perutz, 1962 (Chemistry) Dr. Reichstein, 1951 (Medicine) Dr. Hugo Theorell, 1955 (Medicine) Dr. Jan Tinbergen, 1969 (Economics) Dr. Nikolaas Tinbergen, 1973 (Literature) International Peace Bureau, 1910 (Peace) Dr. Harold C. Urey, 1934 (Chemistry) Dr. Wassily Lenotief, 1973 (Economics) Other outstanding endorsers (partial list) Frank Allaun, MP, England; Chairman Labor Action for Peace. R.K.R. Alston, Pres. UNA, Australia Soviet Peace Committee, USSR. Mr. A.T. Ariyaratne, President Sarvodaya Sharadana Movement, Sri Lanka. Ernest DeMaio, U.N. Representative of World Federation of Trade Unions. Dr. Frank Barnaby, Director, Swedish International Peace Research Institute Major Boehm, President, WILPF-USA. Harry Bridges, President Emeritus, International Longshoremen's Union. Center of Economic and Social Studies of the Third World, Mexico. Dr. Jerome Davis, Gandhi Peace Prize. Reva King, President, SERVAS Int. Rev. Eugene Blake, former Chairman, World Council of Churches. Samuel Day, Jr., Editor of the Bulletin of the Atomic Scientists Rev. Gerald Grant, President, Exec. Council, World Association of World Federalists. The Honorable Hubert Humphrey, U.S. Senator, former U.S.  Vice-president. Victor Lloyd, Director, SANE Elena Gil Izquierdo, President, Movement for Peace and Sovereignty of the People, Cuba Bhupendra Kishore, Asian Secretary, Service Civil International. Margaret Mead, Anthropologist. Dr. Luther Evans, former Director-General UNESCO. Ahmad Subardjo Djoyoadisurjo Former Foreign Minister, Indonesia Mrs. Kathleen Tacchi-Morris, President Women for Disarmament, England. Eeve-Liisa Myllymaki, President Finnish Peace Research Association. Pastor Martin Niemoller, Lennin Peace Prize. Sheila Oakes, General Secretary, National Peace Council, England. Mr. Alan Paton, Civil rights leader, South Africa. Aurelio Peccei, Founder, Club of Rome. H.E. Mr. Rossides, Ambassador of Cyprus to the United Nations. H.E. Mr. Frank Boaten. Ambassador of Ghana to the United Nations. Dr. Cahit Talas, Chairman, Turkish UN", "Mattern, Douglas ; World Citizens Assembly", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bsng-jjaw.5zx6", "00000000-0000-0000-4132-DEF37DA219D5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Shail K. Sharma to Marshall W. Nirenberg", "101584910X278", null, "1976", "24 January 1976", "This letter from Sharma at the All India Institute of Medical Sciences provides evidence of the collaborative nature of much of Nirenberg's work.  Sharma discusses the publication of jointly authored papers, possible summer work at NIH, and the burden of administrative responsibilities.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Shail K. Sharma.", "Copyright may apply", null, null, "January 24, 1976. Dear Marshall, I wrote you 2 letters but I have not received any reply.  Hope you do not mind my writing to you again about the papers.  You can understand that after getting addicted, withdrawal is becoming very difficult.  I heard about the addiction paper from Werner that you may send it soon.  I am sorry I left the acetylcholine paper in the middle, but if you think data is not publishable please let me know.  In any case, if you decide to send the papers, I hope you will send me the first draft before it is sent to the Journal.  I had also written to you tin my earlier letter that I will be happy to come for 3 months in summer if I can do some more work on regulation of adenylate cyclase activity, and in correlation with morphological parameters.  I have not done any experiments yet.  Most of my time goes in ordering chemicals and instruments. With best wishes and regards, Yours sincerely, Shail K. Sharma", "All-India Institute of Medical Sciences ; Sharma, S. K. (Shail K.)", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ky8n-knk9.x5fi", "00000000-0000-0000-A221-52465ED2447C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Christian B. Anfinsen to Marshall W. Nirenberg", "101584910X300", null, "1989", "26 July 1989", "Anfinsen requests Nirenberg's support for an effort to defend the international treaty outlawing biological weapons.  The treaty is reported to be threatened \"not by some small signatory nation openly violating its provisions, but as a result of recently stepped-up military interest at home and abroad in biomedical technologies and their chemical and biological potential.\"  Anfinsen urges Nirenberg to sign a pledge, included with the letter, against military use of biological research at home and abroad.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "3", "pages", "Text", "English", "Reproduced with permission of Libby Anfinsen.", "Copyright may apply", null, null, "July 26, 1989 Dear Marshall Nirenberg, I am writing to request your support for an effort of great importance to all biologists and chemists. As you may already know, the international treaty outlawing biological weapons is currently threatened. This treaty, the 1972 Biological Weapons Convention, is the strongest disarmament treaty in existence, banning not only the deployment and stockpiling of biological weapons, but their development as well. Ironically, the treaty is threatened not by some small signatory nation openly violating its provisions, but as a result of recently stepped-up military interest at home and abroad in biomedical technologies and their chemical and biological potential. I urge you to join me and many other scientists by sponsoring an effort to make our voices heard on this important and disturbing issue. An ad hoc group of colleagues is now circulating a pledge against the military use of biological research both in the U.S. and other countries. By signing this pledge, scientists promise not to engage knowingly in research or teaching that would further the development of chemical or biological arms. This campaign, conducted with the aid of the non-profit organization the Council for Responsible Genetics, has already garnered the signatures of more than 800 of our colleagues in the U.S., but we hope to reach many more. Ultimately, a group of internationally prominent scientists will present the signatures we collect to the next review conference on the treaty (in 1991) along with a call for strengthened verification measures. This effort is particularly important now. Because the biological weapons convention does not now include verification measures, advances in our fields have led to increased mistrust between nations and suspicion that biotechnology may be exploited for offensive purposes under the guise of defensive research. This mistrust can be seen in the policies of the last administration in the U.S. which quadrupled-funds for research in the Army's biological defense program. And suspicion can be seen in stepped-up research efforts in other countries as well. The concern of the signatory nations to the Biological Weapons Convention was evident when the convention was reviewed three years ago. They mandated consideration of a new verification protocol at the 1991 review conference. We need your help now to maintain international confidence in the treaty and to insure that the opportunity to strengthen it in 1991 will not be lost. Your name as a sponsor of the pledge will help us to organize support among the professional scientific community, making a clear statement of opposition to the military use of biomedical research. Please respond to me on this at the above address at your earliest convenience. I am enclosing a sheet to facilitate the process. Thank you for your help, Sincerely yours, Christian Anfinsen The Pledge against the military use of biological research We, the undersigned biologists and chemists, oppose the use of our research for military purposes. Rapid advances in biotechnology have catalyzed a growing interest by the military in many countries in chemical and biological weapons and in the possible development of new and novel chemical and biological warfare agents. We are concerned that this may lead to another arms race. We believe that biomedical research should support rather than threaten life. Therefore, We Pledge not to engage knowingly in research and teaching that will further the development of chemical and biological warfare agents.", "Anfinsen, Christian B. (Christian Boehmer), 1916-1995", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bjt3-2yqc_vbhm", "00000000-0000-0000-0CF2-50D82DA485D7", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Rita Levi-Montalcini to Marshall W. Nirenberg", "101584910X301", "101584910X312", "1995", "13 December 1995", "The 1986 Nobel Prize winner in Physiology or Medicine, Rita Levi-Montalcini, updates Nirenberg on a meeting of outstanding scientists and intellectuals in Italy to draft a \"Declaration of Human Duties.\"  The twelve principle duties are provided in an attached brochure, which also contains the names of those subscribing to the document.  The group called for such things as respecting human dignity and diversity, protecting nature, and promoting peace.  Levi-Montalcini suggests that Nirenberg's approval and support would be \"of invaluable importance in the success of this project.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Rita Levi-Montalcini.", "Copyright may apply", null, null, "Rome, December 13, 1995 Dear Prof. Nirenberg, The importance of the International Human Rights Law as an instrument for the protection of the fundamental rights of every individual is today recognized worldwide.  However, at the same time, the need for a corresponding Declaration of Human Duties has become increasingly evident. On the invitation of the University of Trieste, a number of outstanding scientists and intellectuals from many countries met in Trieste in December 1992 to draft a Declaration of Human Duties. Following a lively discussion, a Declaration of 12 principal Human Duties was accomplished and approved by all the participants in a second meeting in Trieste in 1993. A number of International Organizations have since given their full support of this Declaration. Enclosed you will find a brochure containing the list of duties and the names of the persons and international organizations subscribing to this document. As a fellow Nobel Laureate, your approval and support of this document, along with any suggestions you might have, would be of invaluable importance for the success of this project.  A new global consciousness and human solidarity is imperative in the 21st century as we move beyond the consideration of Rights to the more inclusive concept of Duties for the protection of each other and the earth. In the hope to hear from you at your earliest convenience, I remain Cordially yours, Rita Levi-Montalcini", "Levi-Montalcini, Rita ; International Council of Human Duties", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jvsc_fk6v-urnp", "00000000-0000-0000-37E0-EC6E4CE9A741", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Memorandum from Philip S. Chen, Jr. to the National Institutes of Health's Scientific Directors", "101584910X302", null, "1981", "21 January 1981", "This memo discusses restrictive agreements on the sharing of cell lines.", "Memorandums", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Date: January 21, 1981 To: Scientific Directors From: Deputy Director for Science Subject: Restrictive Agreement on Sharing of Cell Lines It has recently come to my attention that at least one university, and possibly more, are requiring scientists from other institutions (including NIH) to sign restrictive agreements with the university before the university will allow its scientists to make available cell lines they have developed. Among other things, the agreement requires a scientist requesting a cell line to agree on his/her own behalf and on behalf of his/her institution:  (1) not to share the cell line with anyone; and (2) to waive all claims against the university and to defend and indemnify the university from all claims and damages asserted by third parties arising from the requester's use, storage, and handling of the cell line. As you may already know, NIH intramural scientists do not have the authority to agree to these conditions.  Many of our scientists have co-workers in the lab who are working on the project(s) for which the cell line is needed and must necessarily use the cell line along with the requester.   In addition, no one at NIH has authority to waive any claim the Government may have against anyone or to agree on behalf of the Government to defend and indemnify any person or institution against damages arising out of the use, storage, or handling of a cell line.  By signing an agreement to do so, an NIH scientist may leave himself/herself open to personal liability in the event that such claims and damages should in fact occur. Whenever possible, therefore, NIH scientists in need of cell lines should obtain them from sources that do not impose formal restrictions on use of the lines. In those cases in which the only source of a necessary cell line is an organization that asks first for a restrictive agreement, the NIH scientist should respond to that organization by indicating that:  (1) the cell line will be used only for those projects on which that scientist's own laboratory is working, and any requests received by the scientist for the cell line will be referred to the original organization providing the line; and (2) because the Congress has not authorized anyone at NIH to waive claims the Government may have or to defend and indemnify any person or institution against claims or damage, the scientist is unable to sign the waiver/indemnification agreement.  Hopefully, such an approach will be sufficient to mollify organizations that initially request more restrictive agreements.  If not, please let me know, and we will pursue the matter further. Phillip S. Chen, Jr. for  Robert Goldberger, M.D.", "Goldberger, Robert E., 1937- ; Chen, Philip S., Jr", null, null, null, "National Institutes of Health. Scientific Directors", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-zpkx_pcw3.dnss", "00000000-0000-0000-875D-44EEE2152E73", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics [Summary of laboratory projects]", "101584910X304", "101584910X303", "1982", "September 1982", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "ANNUAL REPORT OF THE LABORATORY OF BIOCHEMICAL GENETICS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE October 1, 1981 through September 30, 1982 Monoclonal antibodies were obtained that affect transsynaptic communication, inhibit intercellular adhesion, recognize antigens associated with the synaptic layers of retina, or identify certain types of cells in retina. Five antibodies were obtained that bind to antigens that are restricted to the synaptic layers of retina; three of the antigens are restricted to the outer synaptic layer, one to the inner synaptic layer, and one to both the inner and outer synaptic layers of retina. The latter antibody also markedly inhibits intercellular adhesion of retina cells. Two antibodies are specific for retina ganglion neurons, seven for rods and cones, and seven for Muller cells. Other antibodies recognize various sets of neurons; such as horizontal, bipolar, and -amacrine neurons of the inner nuclear layer of retina. Monoclonal antibodies also were obtained which recognize neuroblastoma~hybrid cell antigens. Five antibodies were found that increase the frequency of miniature endplate potentials of myotubes innervated by the hybrid cells; whereas, six antibodies markedly decrease the frequency of Miniature endplate potentials. The antibodies that affect rates of acetylcholine secretion from hybrid cells do not alter the cell membrane potential. In previous studies we showed that a protein in avian retina, termed Top, is distributed in a large dorsal~ventral gradient. The protein thus defines a dorsal-ventral axis of the retina and identifies the relative positions of cells in retina with respect to this axis. During the past year the protein was purified extensively; the apparent M. of the protein is 47,000 and the isoelectric point is approximately 4.1. A monoclonal antibody directed against Top in retina also recognizes a protein in chick cerebral cortex and thalamus. The protein in brain was purified and characterized; the M, and isoelectric point of the protein from brain were the same as that found for Top from retina. At this stage, we know relatively little about the regulatory mechanism which relates the number of molecules of the protein detected per cell with the relative positions of the cell in the retina, except that cells dissociated from retina that were cultured in vitro continue to express the amount of the protein that would be expected based on the original position of the cell in the intact retina. Further studies on both the structure of the protein and the mechanism regulating the expression of the protein are in progress.   The species of transmitter synthesized by a neuron determines, at least in part, the type of neuron and the kinds of synaptic connections that are formed. A project was initiated to serve as a foundation for future studies on mechanisms that regulate the expression of choline acetyltransferase genes. Choline acetyltransferase from rat brain was purified approximately 100,000-fold and mice then were injected with purified enzyme preparations. Spleen cells from the immunized mice were fused with P3X63 Ag8 myeloma cells; four of the hybridoma cell lines that were obtained were found to synthesize antibodies directed against choline acetyltransferase. The current objective is to use the antibodies for the purification of mRNA coding for choline acetyltransferase in order to clone the corresponding molecules of cDNA by recombinant DNA methods. . pik dk prop d cgih %, 5 — Sixty five monoclonal antibodies were obtained which bind specifically to membrane preparations from NG108-15 neuroblastoma-glioma cells. Six antibodies were found which bind to a greater extent to membranes from differentiated NG108-15 cells than to membranes from undifferentiated NG108-15 cells. Two-dimensional gel electrophoresis also revealed several proteins which are more abundant in membranes from differentiated cells, compared to membranes from undifferentiated cells. We previously showed that the acquisition of functional voltage-sensitive calcium channels by NG108-15 cells enables the cells to form synapses with striated muscle cells. A 1,4-dihydropyridine analog, {3H]-nitrendipine, which reportedly binds specifically to voltage-sensitive calcium channels, was used as a probe for voltage-sensitive calcium channels of NG108-15 cells. (3H]-Nitrendipine was shown to bind specifically to membranes prepared from differentiated NG108-15 cells; the dissociation constant, determined by Scatchard analysis, was approximately 2 x 10710 mM. The number of specific binding sites for [?H]-nitrendipine per average cell was shown to increase 10-fold, from 1,600 to 16,000, when cells were treated for 3 or more days with PGE, which activates adenylate cyclase and elevates cellular cAMP levels. These results suggest that prolonged elevation of cellular cAMP levels results in an increase in the number of voltage-sensitive calcium channels per cell and that the ability of the cells to form synapses is regulated by the rate of synthesis. and/or turnover of voltage-sensitive calcium channels. NG108-15 cells contain large, dense-core vesicles and small, clear vesicles. Cells with endogenously synthesized [3H]-acetylcholine were lysed and the vesicles that were released were separated fron cell membranes and then fractionated by sucrose density centrifugation. Two well-separated peaks of acetylcholine were found, which comprised greater than 90% of the intracellular {3H]-acetylcholine found. Similar results were obtained with a mutant cell line which lacks large dense-core vesicles, but has large vesicles with amorphous electron-lucent cores. These results suggest that acetylcholine in NG108-15 cells is stored in large dense-core vesicles and small clear vesicles and that the clonally inherited change in vesicle morphology in the varient cell line does not affect the ability of the vesicles to store acetylcholine. A protein was obtained from bovine brain which markedly stimulates neurite outgrowth by cultured neurons dissociated from chick embryo cerebral cortex. The protein was purified approximately 200-fold. The native protein is a dimer with an apparent M, of 75,000 which on reduction dissociates into subunits. with an M, of 37,000. The protein, estimated to be 7 90% pure, induces neurite outgrowth at a concentration of approximately 1 nM. A rabbit antiserum to the neurite extension factor, termed NEF, was obtained which blocks the activity of the protein in stimulating neurite extension. Neurite extension protein was detected by immunohistochemical methods in neurons in some, but not all, regions of adult rat brain. These results suggest that NEF is required for neurite extension by some neurons in the central nervous system. NG108-15 cells release a protein into the medium which induces the formation of clusters of nicotinic acetylcholine receptors on myotube plasma membranes. Laminin, a myotube basement membrane glycoprotein, was shown to potentiate the aggregation of the acetylcholine receptors. Basement membrane proteins of myotubes such as laminin, collagen types IV and V, and heparin sulfate proteoglycan were detected on the surface of muscle fibers at early stages of receptor aggregation in vivo. The basement membrane proteins associated with acetylcholine receptor aggregates were relatively resistant to extraction by detergent. Exposure of myotubes to brain extracts resulted in marked aggregation of acetylcholine receptors on myotubes and the appearance of cytoskeletal specializations under myotube plasma membranes. These results suggest that the binding of laminin to the muscle cell surface and the formation of cytoskeletal structures beneath the cell membrane may promote the formation - of nicotinic acetylcholine receptor aggregates and/or stabilize the receptor aggregates. Polyadenylated mRNA was purified from adrenal medulla and brain and was used to direct the cell-free synthesis of preproenkephalin, a precursor of opioid enkephalin pentapeptides. The apparent M, of preproenkephalin was found to be 30,000. The bovine precursor was show to contain the amino acid sequences of both methionine- and leucine-enkephalin. Preproenkephalin was converted to a smaller protein, termed proenkephalin, with an apparent M, of 28,000. Monoclonal antibodies directed against Met-enkephalin and Met~enkephalin [ArgPhe] were obtained. A rat brain cDNA library was prepared by recombinant DNA techniques which will be screened for rat preproenkephalin clones and other clones of interest. cDNA hybridization probes then will be used to study the regulation of enkephalin gene expression. In a related project, corticotropin-releasing factor was shown to stimulate the secretion of beta-endorphin and corticotropin from clonal AtT~20 mouse pituitary tumor cells and to increase the activity of adenylate cyclase of the cells. Histidyl-proline diketopiperazine, after injection into rats, was cleared from the circulation with biphasic kinetics (tis2 = 1.25 and 33 min). Unmetabolized histidyl-proline diketopiperazine appeared rapidly in the urine. The longer half-time of clearance of the peptide of 33 min suggested a tissue reservoir of the peptide. Histidyl-proline diketopiperazine was found to accumulate in adrenal, liver, and kidney. Therefore, specific binding of the diketopiperazine to membrane preparations was examined. Such binding was observed in membrane preparations derived from adrenal and liver.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6q6y-fjfj~sheq", "00000000-0000-0000-74C2-9D52780B806C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X305", "101584910X303", "1982", "September 1982", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00009-08 LBG Period Covered: October 1, 1981 - September 30, 1982 Title of Project: Cell Recognition and Synapse Formation Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged on the Project: PI: Marshall Nirenberg Chief, LBG, LBG, NHLBI OTHER: Howard Burrows, Guest Worker, LBG, NHLBI Hiroyuki Fukai, Visiting Fellow, LBG, NHLBI Gerald Grunwald, Guest Worker, LBG, NHLBI Karl Krueger, Guest Worker, LBG, NHLBI Joseph Moskal, Staff Fellow, LBG, NHLBI William Strauss, Staff Fellow, LBG, NHLBI David Trisler, Staff Fellow, LBG, NHLBI Ilan Spector, Visiting Associate, LBG, NHLBI Lab/Branch: Laboratory of Biochemical Genetics Section: Section of Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20205 Total Man Years: 12 Professional: 9 Other: 3 Summary of Work: Monoclonal antibodies were obtained that affect transsynaptic communication, inhibit intercellular adhesion, recognize antigens associated with the synaptic layers of retina, or are expressed by specific cell types in retina such as ganglion neurons, rods and cones, or Muller cells.  Five antibodies were obtained that increase the rate of acetylcholine secretion from neuroblastoma-hybrid cells, whereas six antibodies decrease the rate.  Another antibody recognizes a 47,000 Mr protein isolated from retina cell membranes, which is distributed in a dorsal-ventral topographic gradient in avian retina.  A similar protein also was purified from chick cerebral cortex and thalamus. In addition, monoclonal antibodies were obtained to choline acetyltransferase from rat brain.  Evidence was obtained which suggests that acetylcholine of NG108-15 neuroblastoma-glioma hybrid cells is stored in large dense-core vesicles and small clear vesicles and in abnormal large vesicles which lack dense-cores which are produced in a varient cell line. Project Description: Objectives: To identify and characterize molecules involved in synapse formation and function. Major Findings: Monoclonal antibodies were obtained that affect transsynaptic communication, inhibit intercellular adhesion, recognize antigens associated with the synaptic layers of retina, or identify certain types of cells in retina.  Five antibodies were obtained that bind to antigens that are restricted to the synaptic layers of retina; three of the antigens are restricted to the outer synaptic layer, one to the inner synaptic layer, and one to both the inner and outer synaptic layers of retina.  The latter antibody also markedly inhibits intercellular adhesion of retina cells.  Two antibodies are specific for retina ganglion neurons, seven for rods and cones, and seven for Muller cells.  Other antibodies recognize various sets of neurons; such as horizontal, bipolar, and amacrine neurons of the inner nuclear layer of retina. Monoclonal antibodies also were obtained that recognize neuroblastoma-hybrid cell antigens.  Five antibodies were found that increase the frequency of miniature endplate potentials of myotubes innervated by the hybrid cells; whereas, six antibodies markedly decrease the frequency of miniature endplate potentials.  The antibodies that affect rates of acetylcholine secretion from hybrid cells do not alter the cell membrane potential. In previous studies we showed that a protein in avian retina, termed Top, is distributed in retina in a large dorsal-ventral topographic gradient.   The protein thus defines a dorsal-ventral axis of the retina and identifies the relative positions of cells in retina with respect to this axis.  During the past year the protein was purified extensively and characterized; the apparent Mr, of the protein is 47,000 and the isoelectric point is approximately 4.1. A monoclonal antibody directed against Top in retina also recognizes a protein in chick cerebral cortex and thalamus.  The Mr and isoelectric point of the protein isolated from brain were the same as that found for Top from retina. At this stage, we know relatively little about the regulatory mechanism which relate the number of molecules of the protein detected per cell with the relative positions of cells in retina, except that cells dissociated from retina that were cultured in vitro continue to express the amount of the protein that would be expected based on the original position of the cell in the intact retina.  Further studies on both the structure of the protein and the mechanism regulating the expression of the protein are in progress. The species of transmitter synthesized by a neuron determines, at least in part, the type of neuron and the kinds of synaptic connections that are formed. A project was initiated to serve as a foundation for future studies on mechanisms that regulate the expression of choline acetyltransferase genes.  Choline acetyltransferase from rat brain was purified approximately 100,000-fold and mice then were injected with purified enzyme preparations.  Spleen cells from the immunized mice were fused with P3X63 Ag8 myeloma cells and four of the hybridoma cell lines that were obtained were found to synthesize antibodies directed against choline acetyltransferase.  Current studies focus on using the antibodies for the purification of mRNA coding for choline acetyltransferase in order to clone the corresponding molecules of cDNA by recombinant DNA methods. Sixty-five monoclonal antibodies were obtained which bind specifically to membrane preparations from NG108-15 neuroblastoma-glioma cells.  Six antibodies were found which bind to a greater extent to membranes from differentiated NG108-15 cells than to membranes from undifferentiated NG108-15 cells. Two-dimensional gel electrophoresis also revealed several proteins which are more abundant in membranes from differentiated cells, compared to membranes from undifferentiated cells. NG108-15 cells contain large, dense-core vesicles and small, clear vesicles. Cells with endogenously synthesized [3H]-acetylcholine were lysed and the vesicles that were released were separated from cell membranes and then fractionated by sucrose density centrifugation.  Two well-separated peaks of acetylcholine were found, which comprised greater than 90% of the intracellular [3H]-acetylcholine found.  Similar results were obtained with a mutant cell line which lacks large dense-core vesicles, but has large vesicles with amorphous electron lucent cores.  These results suggest that acetylcholine in NG108-15 cells is stored in large dense-core vesicles and small clear vesicles and that the clonally inherited change in vesicle morphology in the varient cell line does not affect the ability of the vesicles to store acetylcholine. Significance to Biomedical Research: New information was obtained concerning synaptogenesis and synaptic function. Publications: 1.  De Bias, A. L., Busis, N. A. and Nirenberg, M.: Monoclonal antibodies to synaptosomal membrane molecules. In:  McKay, R., Raff, M., and Reichardt, L. (Ed.):  Monoclonal Antibodies Against Neuronal Antigens, Cold Spring Harbor Reports in the Neurosciences, Vol. 2, Cold Spring Harbor Laboratory, 1981, pp. 181-191. 2.  Trisler, G. D., Schneider, M. D., Moskal, J. R. and Nirenberg, M.: A gradient of molecules in avian retina with dorsoventral polarity. In:  McKay, R.,  Raff, M. and Reichardt, L., (Ed.):  Monoclonal Antibodies Against Neuronal Antigens, Cold Spring Harbor Reports in the Neurosciences, Vol. 2, Cold Spring Harbor Laboratory, 1981, pp. 231-257. 3.  Kenimer, J. and Nirenberg, M.:  Desensitization of adenylate cyclase to prostaglandin El or 2-chloroadenosine. Mol. Pharm. 20, 585-591, 1981. 4.  Trisler, G. D., Schneider, M. D. and Nirenberg, and Nirenberg, M.: Topographic gradient of cell-membrane molecules in avian neural retina detected with monoclonal antibody.  In: Helfer, S. R. and Sheffield, J. B. (Ed.): Ocular Size And Shape Regulation During Development, Springer-Verlag, New York, 1981, pp. 141-161. 5.  Nirenberg, Marshall.  Synapse Plasticity. In:  Proceedings of the Symposium On Trends In Bioassay Methodology: In Vivo, In Vitro And Mathematical Approaches. NLH Publication No. 82-2382, 1981, pp 201-207. 6.  De Bias, A. L., Ratnaparkhi, M. V. and Mosimann, J. E.: Estimation of number of the monoclonal hybridomas in a cell fusion experiment.  J. Immunol. Methods. 45 109-115 (1981). 7.  Yoboi, J., Hirashima, M., Hirata, F., De Blas, A. L. and Ishizaka, K.: Lymphocytes bearing Fc receptors for IgE.   VII. Possible participation of phospholipase A2 in the glycosylation of IgE-binding factors.  J. Immunol. 127, 476-482 (1981). 8.  Fishman, M. C. and Spector, I.:  Potassium current suppression by quinidine reveals additional calcium currents in neuroblastoma cells.  Proc. Natl. Acad. Sci., USA, 78 5245-5245 (1981). 9.  Trisler, G. D., M. D. Schneider, J. R. Moskal and M. Nirenberg:  Molecules that define a dorsal-ventral axis of retina can be used to identify cell position. In:  Clayton, R. M. and Truman, D. E. S. (Ed.): Stability and Switching in Cellular Differentiation.  Plenum Press, New York, 1982, pp. 123-127. 10.  Trisler, G. David, Michael D. Schneider and Marshall Nirenberg: A topographic gradient of molecules in retina can be used to identify neuron position. In:  Patterson, P. H. and Purves, D. (Ed.): Readings In Developmental Neurobiology.  Cold Spring Harbor Laboratory, 1982, pp. 513-517. 11.  Thompson, Jeffrey M., Eisenbarth, George S., Ruffolo, Jr., Robert R. and Nirenberg, Marshall.  Synapse Selection Based On Differences In Synapse Turnover.  Developmental Neuroscience, In Press (1982). 12.  Spector, Ilan, Sachet, Nava R., Kashman, Yoel and Groweiss, Amiram: Latrunculins:  Novel Marine Toxins That Disrupt Microfilament Organization In Cells. Science, In Press (1982). 13.  Trisler, David:  The Formation of Neural Circuits: Are molecular markers of cell position involved?  Trends In NeuroSciences.  In Press (1982).", "Strauss, William ; Spector, Ilan ; Nirenberg, Marshall W. ; Moskal, Joseph R. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Burrows, Howard ; Fukai, Hiroyuki ; Grunwald, Gerald ; Krueger, Karl ; Trisler, G. David", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-n3dj-qfsw~gkg7", "00000000-0000-0000-B700-C8D03B27C3A7", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics [summary of laboratory projects]", "101584910X307", "101584910X306", "1986", "September 1986", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "4", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "October 1, 1985 through September 30, 1986 Two [lambda]gtll cDNA libraries from human brain were screened with 3 oligodeoxynucleotide probes for recombinants coding for a subunits of G signal transducing proteins, which couple receptors activated by hormones or light to connectors such as adenylate cyclase or cGMP phosphodiesterase.  Fourteen of the 575,000 recombinant clones screened from a human basal ganglia cDNA library and 12 of the 400,000 clones screened from a human cerebral cortex library were detected with 2 or 3 of the 32P-probes used.  DNA inserts from 13 positive clones were sequenced partially; 11 clones were identified as cDNA and 2 clones as [alpha]i.  The DNA insert from one of the as clones was sequenced completely and additional partial sequences were obtained for 10 as clones.  Four species of [alpha]s cDNA were found that differ in nucleotide sequence in the region that corresponds to as amino acid residues 71-88.  The clones differ in the codon for [alpha]s amino acid residue 71 (glutamic acid vs. aspartic acid), the presence or absence of codons for the next 15 amino acid residues, and the presence or absence of an adjacent serine residue.  A mechanism was proposed for generating 4 species of as mRNA by alternative splicing of precursor RNA transcribed from a single gene. cDNA from one of the two human [alpha]i clones was sequenced completely (BG-4), and a partial sequence was obtained for the second clone.  The first nucleotide residue of BG-4 [alpha]i cDNA corresponds to the 14th residue of the bovine [alpha]i coding sequence and the last residue of BG-4 (1261) is in the 3'-untranslated region.  The amino acid sequence derived from the nucleotide sequence of human BG-4 [alpha]i cDNA is highly homologous to bovine and rat [alpha]i sequences reported by others.  In addition, the 3'-untranslated region of BG-4 [alpha]i cDNA is highly homologous to the 3'-untranslated regions of bovine and rat [alpha]i cDNA. The 3'-untranslated nucleotide sequences of human, bovine, and rat as cDNAs also are highly conserved, but differ markedly from [alpha]i 3' untranslated sequences. These results suggest that the 3'-untranslated regions of [alpha]s and [alpha]i genes and/or mRNA are needed for functions that have not been identified thus far. In previous studies we have shown that elevation of cAMP levels of NG108-15 neuroblastoma-glioma hybrid cells or neuroblastoma cells for several days results in 10 -100 fold increases in the activity of voltage-sensitive calcium channels, 15-45 fold increases in spontaneous secretion of acetylcholine at synapses, and 5-15 fold increases in the abundance of synapses with cultured striated muscle cells.  In addition, the number of molecules of the voltage-sensitive calcium channel protein subunit that binds [3H]-nitrendipine increases 12-fold.  We previously obtained about 100 cDNA clones that hybridize to species of mRNA that are more abundant in NG108-15 or NS20-Y cells that had been treated with dibutyryl cAMP for several days then in untreated control cells.  Quantitative studies on the extent of increase in abundance of the species of mRNA that respond to dibutyryl cAMP were performed using the cloned cDNA as probes.  Twenty cDNA clones were obtained that hybridize to species of poly A+ RNA that increase in abundance 10-90 fold due to treatment of cells with dibutyryl cAMP.  Northern blots also were performed and the number of bands of poly A+ RNA that hybridize to each cloned cDNA probe and their chain lengths were determined. Affinity purified antibodies to the a, b, and y protein subunits of voltage-sensitive calcium channels were used to screen a [lambda]gtll cDNA library prepared from poly A+ RNA from rat skeletal muscle.  Approximately 20 recombinant clones were found that were identified tentatively as calcium channel a subunit cDNAs.  Other cDNA clones were obtained that are putative y subunit clones. In previous studies a putative cDNA clone for choline acetyltransferase was found.  We now have determined the nucleotide sequence of the 1118 bp DNA insert.  Partial amino acid sequences of several peptides derived from choline acetyltransferase by the action of peptidases were obtained in collaborative studies by Lou Hirsh and his colleagues in Dallas.  The [lambda]gtll cDNA library was screened again with 2 new oligodeoxynucleotide probes to different regions of choline acetyltransferase and cDNA clones were obtained that were recognized by both probes.  Further studies with these clones are in progress. Antigenic molecules termed TOP, which are distributed in a dorsal > ventral concentration gradient in chicken retina, are expressed early in development (by 48 hr after fertilization) in the optic cup of chicken embryos and continue to be expressed in retina thereafter.  35S-labeled-TOP-antibody complexes were purified by protein A-Sepharose column chromatography and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography.  TOP also was purified from dorsal retina by anti-TOP IgC-Affigel 10 affinity column chromatography.  Both purification methods yielded one major band of protein with an Mr of approximately 47,000.  A protein of M, approximately 47,000 also was purified from chicken embryo brain.  Cultured cells dissociated from 8-day chicken embryo retinas accumulated the amount of TOP expected of cells in the intact retina, depending on the position of the cells in the retina.  TOP accumulations by cells dissociated from dorsal or ventral retina, mixed in different proportions and cocultured were additive.  These results show that TOP is a protein, that the gradient of TOP is established early in development, and that perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusable molecules or on maintenance of interactions between cells.  Synapses and neurites in the retina of developing chick embryos were reduced markedly by injection of anti-TOP antibody into the eye. The addition of bradykinin to NG108-15 cells was shown in previous studies to increase cellular levels of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol.  The newly synthesized IP3 in turn stimulates the release of stored calcium ions into the cytoplasm, thereby activating calcium-dependent K+ channels.  The increased efflux of K+ ions results in cell hyperpolarization.  This is followed by cell depolarization due to inhibition of M channels, thereby decreasing the rate of K+ efflux from cells via M channels.  Additional results now show that inhibition of M channels is due to diacylglycerol and Ca2+ dependent activation of protein kinase C.  Several phosphoproteins were detected by two dimensional gel electrophoresis whose synthesis is dependent upon the addition of bradykinin to cells.  Whereas, injection of inosito1 1,4,5- trisphosphate inside NC108-15 cells results  in the release of stored calcium into the cytoplasm, injection of inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate has little or no effect on calcium mobilization, but instead results in the activation of nonspecification channels .  Calcium ions are not required for the activation of the nonspecification channels.  The nature and significance of these findings warrant further investigation in light of recent reports that inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate are present in some tissues and that inositol 1,3,4,5-tetrakisphosphate is synthesized by phosphorylation of inositol 1,4,5-trisphosphate, catalyzed by an appropriate kinase, and that inositol 1,3,4-trisphosphate is formed by dephosphorylation of inositol 1,3,4,5-tetrakisphosphate. Immunofluorescence staining on cryostat sections prepared from embryonic brain extract-treated myotubes revealed a precise colocalization of a 43,000 Mr, cytoplasmic protein (distinct from actin) with newly-formed ACh receptor aggregates.  This result is consistent with a role for the 43,000 Mr protein in receptor immobilization, as suggested indirectly by studies from other laboratories on fish electric organ and the neuromuscular junction. We previously showed that partially purified and highly purified fractions from the extracellular matrix of the Torpedo electric organ induce ACh receptor aggregation in cultured myotubes with a time course similar to that of embryonic pig brain extract.  We now have found that antiserum against a partially purified fraction from Torpedo (700 units/mg protein) can absorb about 60% of the receptor aggregation activity of brain extract.  Under the same conditions, 90% of the activity in the Torpedo fraction was absorbed. This result is consistent with the presence of immunologically related aggregation factors in electric organ and brain. We previously showed that neural factor induced formation of ACh receptor aggregates on tetrodotoxin-treated myotubes is associated with the localized deposition of basal lamina.  We now find that embryonic brain extract and ciliary ganglion explants induce a widespread deposition of basal lamina on non-tetrodotoxin-treated myotubes.  Ascorbate oxidase blocks this deposition of basal lamina, suggesting that ciliary ganglion and embryonic brain extract contain ascorbate-like factors that promote muscle basal lamina formation.   The extensive induction of ACh receptor aggregates by ciliary ganglion explants was only partially inhibited by ascorbate oxidase, and basal lamina deposition still occurred at the ACh receptor aggregate sites.  These results suggest that the ascorbate-like factor contributes to, but is not primarily responsible for the induction of receptor aggregates.  In addition, they suggest that deposition of basal lamina at receptor aggregates can occur independently of the ascorbate-like factor. We have been studying hormonal and neurotransmitter-dependent mechanisms that regulate the gene for proenkephalin (pEnk), the precursor of the opioid peptides methionine- and leucine-enkephalin, in clonal cell lines of neural origin, as well as in rat brain.  NG10S-15 neuroblastoma-glioma hybrid cells and C6 rat glioma cells contain pEnk mRNA, quantitated by blot hybridization. C6 cells contain a much higher abundance (3-6 pg/ug RNA) but lower enkephalin content than NC-108-15 cells.  Treatment of C6 cells with compounds that activate adenylate cyclase and raise the cAMP concentration (e.g. by a beta-adrenergic receptor agonist such as (-)- norepinephrine or by forskolin) elevate the pEnk mRNA abundance.  Glucocorticoid hormones such as dexamethasone or cortisol, while having no effect alone on the pEnk mRMA level, potentiate the effect of cAMP elevation, producing maximum elevations of 8-fold.  C6 cells contain proenkephalin but do not process this precursor significantly.  Treatment with norepinephrine and dexamethasone raises the content of proenkephalin 11-fold.  Treatment of cells with glucocorticoid and forskolin for 1-6 hr increases pEnk gene transcription at least 2.5 fold.  These results suggest that gluccocerticoids and neurotransmitters that elevate cAMP transcriptionally regulate enkephalin biosynthesis in enkephalinergic cells. Studies have been initiated on the regulation of expression of the gene for proneuropetide Y (pNPY), the precursor of neuropetide Y, a putative regulator in the autonomic nervous system.  pNPY mRNA is relatively abundant in NG108-15 hybrid cells.  Treatment of these cells with gluccorticoids elevates pNPY mRNA 2-fold. Two novel neuropeptides having anti-analgesic activity were recently isolated and sequenced by Dr. H. Y. Yang's group.  Their structures are Ala-Gly-Glu-Gly-Leu-Ser-Ser-Tro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH2 (A18F-:NH2) and Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2. A rat hypothalamus [lambda]gtll cDNA library was screened with 32P-oligodeoxynucleotides corresponding to portions of these peptides and putative A18F-NH2 cDNA clones were obtained. Nearly all prokaryotic genes use the translation initiation codon AUG.  However, there are a few examples where GUG or UUG function as initiation codons in E. coli.  The gene for E. coli adenylate cyclase, is one of the genes that uses the unusual UUG initiation codon.  We have investigated the effect of this unusual initiation codon on the expression of the adenylate cyclase gene by changing the DNA sequence coding for the UUG initiation codon to ATG and GTG, using oligonucleotide-directed mutagenesis.  A comparison of the activities associated with the three codons was made in three different environments: (1) in the normal environment, with the adenylate cyclase gene expressed from its own promoters, (2) in a transcription fusion with the adenylate cyclase gene under the transcriptional control of the phage lambda promoter, and (3) in a gene fusion with the adenylate cyclase gene fused to the E. coli galactokinase gene to generate a fusion protein with galactokinase activity.  In each of the three environments, it was observed that the UUG initiation codon had the lowest efficiency of translation initiation and the AUG initiation codon had the highest efficiency, while the GUG initiation codon was intermediate.  These results may provide a partial explanation for the finding that the cellular concentration of adenylate cyclase is very low. In E. coli cAMP plays a crucial role in regulating the expression of inducible genes.  The levels of this nucleotide are controlled primarily by a catabolite-dependent modulation of adenylate cyclase activity.  Insight into the mechanism of regulation of the activity of this enzyme has come primarily from studies of permeable cells.  Current information suggests that the phosphoenolpyruvate: glucose phosphotransferase system (PTS) is intimately involved in the regulation.  Additionally, potassium and phosphate ions play key roles in modulating adenylate cyclase activity.  A model for interaction of adenylate cyclase with PTS proteins and potassium phosphate to form a regulatory complex was proposed previously by us.  The purpose of the present study was to test the proposed model for adenylate cyclase regulation using a reconstitution approach.  We found that all of the unique features of adenylate cyclase characteristic of the regulatory complex observed in permeable cells were reconstituted in cell-free extracts. The results strongly support the proposal that adenylate cyclase activity is regulated by PTS proteins.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-de5x-7zjy.vxn9", "00000000-0000-0000-E47A-793B409AFBB4", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X308", "101584910X306", "1986", "September 1986", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number Z01 HL 00009-12 LBG October 1, 1985 - September 30, 1986 Cell Recognition and Synapse Formation Principal Investigator: Marshall Nirenberg, Chief, LBG, NHLBI Dana Hilt, Staff Fellow, LBG, NHLBI Hemin Chin, Guest Worker, LBG, NHLBI Karl Krueger, Staff Fellow, LBG, NHLBI Patricia Bray, Biologist, LBG, NHLBI Benjamin Amaladoss, Visiting Fellow, LBG, NHLBI Koh Yano, Visiting Fellow, LBG, NHLBI David Trisler, Staff Fellow, LBG, NHLBI Cooperating Units (if any): Allen Spiegel, Chief, MDB, NIADDK Bruce Schrier, LDN, NICHD Lou Hirsch, Dept. of Biochem., U. of Texas, Dallas, Texas Lab/Branch: Laboratory of Biochemical Genetics Section: Section of Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20205 Total Man Years: 11 Professional: 9 Other: 2 Summary of Work: [lambda]gtll cDNA libraries derived from human brain poly A+ RNA were screened for recombinants that code for [alpha]-subunits of G signal transduction proteins.  Eleven [alpha]s and two [alpha]i clones were characterized.  Four species of [alpha]s cDNA were found.  A mechanism for generating the four species of [alpha]s mRNA by alternative splicing of precursor RNA was proposed. Treatment of NG108-15 neuroblastoma-glioma hybrid cells cAMP for several days results in there appearance of voltage-sensitive calcium channels and other ions channels.  Twenty cDNA clones were obtained that hybridize to species of poly A+ RNA that increase in abundance 10-90 fold due to treatment of cells with dibutyryl cAMP.  Affinity-purified antibodies to the a or y protein subunits of voltage-sensitive calcium channels were used to screen a [lambda]gtll cDNA library.  Twenty putative voltage-sensitive calcium channel a subunit cDNA clones and 29 putative y subunit clones were found. Antigenic molecules termed TOP, which are distributed in a dorsal > ventral concentration gradient in chicken retina, were purified.  TOP was shown to be a protein with an Mr of 47,000.  The gradient of TOP in the retina is formed early in embryonic development.  Thereafter, perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusible molecules or on maintenance of interactions between cells.  Synapses and neuritis in the retina of developing chick embryos were reduced markedly by injection of anti-TOP antibody into the eye. Project Description: Objectives: Our objective is to discover basic mechanisms that regulate the expression of genes. Major Findings: Two [lambda]gtll cDNA libraries from human brain were screened with 3 oligodeoxynucleotide probes for recombinants coding for [alpha] subunits of G signal transducing proteins, which couple receptors activated by hormones or light to effectors such as adenylate cyclase or cGMP phosphodiesterase.  Fourteen of the 575,000 recombinant clones screened from a human cerebral cortex library were detected with 2 or 3 of the 32P-probes used.  DNA inserts from 13 positive clones were sequence partially; 11 clones were identified as [alpha]s cDNA and 2 clones as [alpha]i.  The DNA insert from one of the [alpha]s clones was sequenced completely and additional partial sequences were obtained for 10 [alpha]s clones.  Four species of [alpha]s cDNA were found that differ in nucleotide sequence in the region that corresponds to [alpha]s amino acid residues 71-88.  The clones differ in the codon for [alpha]s amino acid residue 71 (glutamic acid vs. aspartic acid), the presence or absence of codons for the next 15 amino acid residues, and the presence or absence of an adjacent serine residue.  A mechanism was proposed for generating 4 species of [alpha]s mRNA by alternative splicing of precursor or RNA transcribed from a single genre. cDNA from one of the two human [alpha]i clones was sequenced completely (BG-4), and a partial sequence was obtained for the second clone.  The first nucleotide residue of BG-4 [alpha]i cDNA corresponds to the 14th residue of the bovine [alpha]i coding sequence and the last residue of BG-4 (1261) is in the 3'-untranslated region.  The amino acid sequence derived from the nucleotide sequences reported by others.  In addition, the 3'-untranslated regions of BG-4ai cDNA is highly homologous to the 3'-untranslated regions of bovine and rat [alpha]i cDNA.  The 3'-untranslated nucleotide sequences of human, bovine, and rat [alpha]s cDNAs also are highly conserved, but differ markedly from [alpha]i 3'-untranslated sequences.  These results suggest that the 3'-untranslated regions of [alpha]s and [alpha]i genes and/or mRNA are needed for functions that have not been identified thus far. In previous studies we have shown that elevation of cAMP levels of NG108-15 neuroblastoma-glioma hybrid cells or neuroblastoma cells for several days results in 10-100 fold increases in the activity of voltage-sensitive calcium channels, 15-45 fold increases in the abundance of synapses with cultured striated muscle cells.  In addition, the number of molecules of voltage-sensitive calcium channel protein subunit that binds [3H]-nitrendipine increases 12-fold.  We previously obtained about 100 cDNA clones that hybridize to species of mRNA that are more abundant in NG108-15 or NS20-Y cells that had been treated with dibutyryl cAMP for several days then in untreated control cells.  Quantitative studies on the extent of increase in abundance of the species of mRNA that respond to dibutyryl cAMP were performed using the cloned cDNA as probes.  Twenty cDNA clones were obtained that hybridize to species of poly A+ RNA that increase in abundance 10-90 fold due to treatment of cells with dibutyryl cAMP.  Northern blots also were performed and the number of bands of poly A+ RNA that hybridize to each clone cDNA probe and their chain lengths were determined. Affinity purified antibodies to the a, b, and y protein subunits of voltage-sensitive calcium channels were used to screen a [lambda]gtll cDNA library prepared from poly A+ RNA from rat skeletal muscle.  Approximately 20 recombinant clones were found that were identified tentatively as calcium channel a subunit cDNAs.  Other cDNA clones were obtained that are putative [Upsilon] subunit clones. In previous studies a putative cDNA clone for choline acetyltransferase was found.  We now have determined the nucleotide sequence of the 1118 bp DNA insert.  Partial amino acid sequences of several peptidases were obtained in collaborative studies by Lou Hirsh and his colleagues in Dallas.  The [lambda]gtll cDNA library was screened again with 2 new oligodeoxynucleotide probes to different regions of choline acetyltransferase and cDNA clones were obtained that were recognized by both probes.  Further studies with these clones are in progress. Antigenic molecules termed TOP, which are distributed in a dorsal > ventral concentration gradient in chicken retina, are expressed early in development (by 48 hr after fertilization) in the optic cup of chicken embryos and continue to be expressed in retina thereafter.  35S-labeled-TOP-antibody complexes were purified by protein A-Sepharose column chromatography and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography.  TOP also was purified from dorsal retina by anti-TOP IgG-Affigel 10 affinity column chromatography.  Both purification methods yielded one major band of protein with an Mr of approximately 47,000.  A protein of Mr approximately 47,000 also was purified from chicken embryo brain.  Cultured cells dissociated from 8-day chicken embryo retinas accumulated the amount of TOP expected of cells in the intact retina, depending on the position of the cells in the retina.  TOP accumulations by cells dissociated from dorsal or ventral retina, mixed in different proportions and cocultured were additive.  These results show that TOP is a protein that the gradient of TOP is established early in development, and that perpetuation of the gradient does not depend on the continuous presence of an extracellular gradient of diffusible molecules or on maintenance of interactions between cells.  Synapses and neuritis in the retina of developing chick embryos were reduced markedly by injection of anti-TOP antibody into the eye The addition of bradykinin to NG108-15 cells was shown in previous studies to increase cellular levels of inositol-1,4,5-trisphospahte (IP3) and diacyclglycerol.  The newly synthesized IP3 in turn stimulates the release of stored calcium ions into the cytoplasm, thereby activating calcium-dependent K+ channels.  The increased efflux of K+ ions results in cell hyperpolarization.  This is followed by cell depolarization due to inhibition of M channels, thereby decreasing the rate of K+ efflux from cells via M channels.  Additional results now show that inhibition of M channels is due to diacylglycerol and Ca2+ dependent activation of protein kinase C.  Several phosphoproteins were detected by two dimensional gel electrophoresis whose synthesis is dependent upon the addition of bradykinin to cells.  Whereas, injection  of inositol 1,4,5-trisphosphate inside NG108-15 cells results in the release of stored calcium in to the cytoplasm, injection of inositol 1,3,4-triphosphate or inositol 1,3,4,5-tetrakisphosphate has little or no effect on calcium mobilization, but instead results in the activation of nonspecific cation channels.  Calcium ions are not required for the activation of the nonspecific cation channels.  The nature and significance of these findings warrant further investigation in the light of recent reports that inositol 1,3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate are present in some tissues and that inositol 1,3,4,5-tetrakisphosphate is synthesized by phosphorylation of inositol 1,2,5-trisphosphate, catalyzed by an appropriate kinase, and that inositol 1,3,4-trisphosphate is formed by dephosphorylation of inositol 1,3,4,5-tetrakisphosphate. Publications: 1. Higashida, H., Streaty, R.A., Klee, W. and Nirenberg, M.: Bradykinin-activated Transmembrane Singals are Couple via No or Ni to Production of Inositol 1,4,5-trisphosphate, a Second Messenger in NG108-15 Neuroblastoma-glioma Hybrid Cells.  Proc. Natl. Acad. Sci., USA 83, 942-946 (1986). 2. Grunwald, G.B., Gierschik, P., Nirenberg, M. and Spiegel, A.: Detection of a-Trasnducin in Retinal Rods but not Cones.  Science 231, 856-859 (1986). 3. Moskal, J.R., Trisler, D., Schneider, M.D., and Nirenberg, M.: Purification of a Membrane Protein Distributed in a Topographic Gradient in Chicken Retina.  Proc. Natl. Acad. Sci., USA 83, 4730-4733 (1986). 4. Dubois, C., Magnani, J.L., Grunwald, G.B., Spitalnik, S.L., Trisler, G.D., M. Nirenberg, and Ginsburg, V.: Monoclonal Antibody 18B8, Which Detects Synapse-associated Antigens, Binds to Ganglioside Gt3 (II3 (NeuAc)3LacCer).  J. Biol. Chem. 261, 3826-3830 (1986). 5. Dubois, C., J.L. Magnani, G.B. Grunwald, S.L. Spitalnik, D. Trisler, M. Nirenberg, and V. Ginsburg.  1985. Monoclonal antibody 18B8, which detects synapse associated antigens, binds to ganglioside GT3 [II3 (NeuAc)3LacCer].  In Glycoconjugates: Proceedings of the VIIIth International Symposium, E.A. Davidson, J.C. Williams, and N.M. DiFerrante, eds. Vol.2, p. 5545, Praeger Publ., New York. 6. Bray, P., Carter, A., Simons, C., Guo, V., P:uckett, C., Kamholtz, J., Spiegel, A., and Nirenberg, M.: Human cDNA Clones for Four Species of Gas Signal Transduction Protein. Proc. Natl. Acad. Sci., USA (In Press). 7. Trisler, D. Synapse formation in retina is influenced by molecules that identify cell position.  Current topics in Developmental Biology (In Press). 8. Trisler, D. Synapse formation in avian retina is inhibited by antibody to molecules that identify cell position. Amer. Zool. (In Press). 9. Trisler, D., J. Bekenstein, and M.P. Daniels. Antibody to a molecular marker of cell position inhibits synapse formation in retina. Proc. Natl. Acad. Sci., USA, 83: 4194-4198 (1986) 10. Higashida, H. and Brown, D.A.: Two Polyphosphatidyl Metabolites Control Two K+-Currents In A Neuronal Cell.  Nature (In Press). 11. Higashida, H. and Brown, D.A.: Membrane Current Repsonses to Intracellular Injections of Inositol 1,3,4, 5-tetrakisphosphate and Inositol 1,3,4-trisphosphate in NG108-15 Cells. (Submitted to FEBS Letters).", "Nirenberg, Marshall W. ; Amaladoss, Benjamin ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Krueger, Karl ; Bray, Patricia ; Trisler, G. David ; Hilt, Dana ; Yano, Koh ; Chin, Hemin", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e9zz_yuic~kidc", "00000000-0000-0000-96BE-03ACE92FF0E6", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics, October 1, 1977 - September 30, 1978", "101584910X309", null, "1978", "September 1978", "This annual report includes descriptions of the project studying the process of synaptogenesis using clonal cell lines and neurons dissociated from embryos and cultured in vitro.", "Reports, Drafts (documents)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "4", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Two model systems were used to study the process of synaptogenesis: clonal cell lines of neural origin and neurons dissociated from embryos and cultured in vitro. Twenty-three neuroblastoma or hybrid cell lines were tested for their ability to synthesize and release acetylcholine and form synapses with rat striated muscle cells and clonal muscle cells.  Six cell lines form synapses with muscle cells with high frequency; whereas, 17 cell lines were found which have defects in synapse formation.  Of the 19 cell lines found that synthesize and release acetylcholine into the medium, six cell lines form synapses with high frequency, whereas three cell lines form few or no synapses. Rates of acetylcholine synthesis were 34-463 pmol acetylcholine/min/mg/homogenate protein; intact cells incubated with [3H]-choline contained 55-1,600 pmol [3H]-acetylcholine/mg/protein and released 80-6,400 fmol [3H]-acetylcholine /min/mg protein into the medium.  Five kinds of defects were detected with cell lines that form few or no synapses: (1) little or no acetylcholine synthesis, (2) large dense core vesicles present but not clear vesicles, (3) clear vesicles present but not large dense core vesiciles, (4) little or no action potential Ca++ ionophore activity, and (5) a defect in another step required for stimulus-dependent acetylcholine secretion. NG108-15 hybrid cells which form synapses with muscle cells release into the medium a macromolecule which results in aggregation of nicotinic acetylcholine receptors of cultured striated muscle cells.  The factor also is formed by neuroblastoma parental cells, but not by parental glioma cells, fibroblasts, or HeLa cells.  The factor increases the aggregation of acetylcholine receptors without altering the number of receptors, and the action of the factor does not depend on myotube protein synthesis.  The factor was partially purified and was shown to be a heat-labile macromolecule, with a molecular weight of > 10,000.  A role for the acetylcholine receptor aggregation factor in synaptogenesis was hypothesized; namely, that the factor stimulates the accumulation of nicotinic acetylcholine receptors on myotube surface membranes at sites of contact or interaction between neurons and muscle cells. The ability of hybrid cells to form synapses was found to be regulated.  Growth of hybrid cells in the presence of dibutyryl-cAMP increased the concentration of intracellular acetylcholine, the abundance of vesicles, the amount of acetylcholine released from cells in response to excitory stimuli, the efficiency of synaptic communication, and the number of synapses formed.  These effects also were obtained with cAMP, but not with cGMP. The effects of putative neurotransmitters and hormones on intracellular cAMP or cGMP levels, cell membrane potential, and acetylcholine secretion from cells were determined.  At least 10 species of receptors were detected on synapse competent cell lines.  Activation of receptors for serotonin, PGF2a acetylcholine, bradykinin, neurotensin, or angiotensin resulted in secretion of acetylcholine from cells.  Receptor mediated increases in cAMP or cGMP levels had no immediate effect on acetylcholine secretion from cells.  However, growth of hybrid cells for 24 or more hours in the presence of ligands for receptors that are coupled to the activation of adenylate cyclase and/or exposure of cells to other inhibitors of cyclic nucleotide phosphodiesterase resulted in increases in cAMP levels of cells and mimicked all regulatory effects of dibutyryl-cAMP on acetylcholine storage and release from cells.  These results show that cell lines with and without synapse defects can be generated and that receptor mediated reactions that activate adenylate cyclase and elevate cAMP levels of cells regulate the storage and stimulus-dependent secretion of acetylcholine, thereby regulating synapse formation and the flow of information across synapses.  Synapses were turned on or off slowly over a period of days, which suggests that cAMP, directly or indirectly, regulates the acquisition of components that are required for synaptic activity. We previously showed that activation of NG108-15 opiate receptors or muscarinic acetylcholine receptors results in inhibition of adenylate cyclase and that exposure of cells to morphine or carbamylcholine reduces CAMP levels of celis initially, but gradually, over a period of 24-48 hours, cAMP levels increase and return to the control value due to a compensatory, long-lived increase in the specific activity of adenylate cyclase.  Subsequent studies have shown that NG108-15 cells possess presynaptic a2-receptors and that exposure of cells to 1 uM  norepinephrine similarly reduces cAMP levels initially, and that cAMP levels slowly return to the control value over a 10 hour period as the specific activity of adenylate cyclase increases.  The cells then are dependent on norepinephrine to inhibit the elevated adenylate cyclase activity.  Withdrawal of norepinephrine or blockade of the a-receptors results in a 4-9 fold increase in intracellular cAMP.  Approximately 8 hours are required for the elevated enzyme activity to return to the control value. Cyclic AMP levels are elevated during the withdrawal period, and cells are supersensitive to ligands for other species of receptors, such as PGEl, that activate adenylate cyclase.  The demonstration that 3 species of receptors which mediate inhibition of adenylate cyclase also evoke persistent increases in adenylate cyclase activity suggests that the phenomenon is a general one, and that other species of receptors that inhibit adenylate cyclase also may act as dual regulators of the enzyme. The hypothesis that activation of adenylate cyclase may lead, conversely, to a reduction in adenylate cyclase activity also was tested.  Incubation of NG108-15 cells with PGE1 for 12 hours resulted in 60-80% decreases in basal adenylate cyclase specific activity and NaF-, Gpp(NH)p-, 2-Cl-adenosine, and PGE1-stimulated activities.  Basal and PGE1-stimulated adenylate cyclase activities of cells exposed to PGE1 decayed exponentially with half-lives of 6 hours.  On withdrawal of PGE1, adenylate cyclase activity slowly increased and returned to the control value over a period of 24 hours; cyclohexamide inhibited the increase in adenylate cyclase activity > 90%.  These results show that activation of adenylate cyclase leads to a loss of enzyme activity and that the recovery of enzyme activity to the control value requires protein synthesis and approximately 24 hours of incubation. These long-lived, receptor-mediated effects on adenylate cyclase activity, acetylcholine storage, stimulus-secretion coupling, and the demonstration that synapses can be turned on or off by regulating acetylcholine release, have properties that resemble those expected for simple forms of learning and memory, such as habituation, tolerance, dependence, and sensitization but whether synapse plasticity in a cultured cell system is related to behavioral phenomena is not known. Synapse competent cell lines possess serotonin receptors which mediate cell depolarization and stimulate secretion of acetylcholine into the medium.  Serotonin also stimulates adenylate cyclase in homogenates of one, but not other, hybrid cell lines.  The concentration of serotonin required for half-maximal activation of adenylate cyclase (Ka) is 0.5 uM. Hill and Eadie-Scatchard analyses suggest a simple, bimolecular interaction between serotonin and the receptor.  D-Lysergic acid diethylamide (D-LSD) also stimulates adenylate cyclase activity (Ka=12 nM); however, the increase in enzyme activity is less than half that produced by serotonin.  In contrast, D-LSD neither mimics nor antagonizes the effects of serotonin on cell depolarization and acetylcholine secretion.  These and additional studies on serotonin receptor specificity for agonists and antagonists suggest that serotonin receptors that are coupled to activation of adenylate cyclase are postsynaptic serotonin receptors, whereas, those mediating cell depolarization and acetylcholine release are presynaptic serotonin receptors.  One cell line exhibits both pre- and post-synaptic serotonin receptor functions; other cell lines have only presynaptic serotonin receptor function. Cultured neurons dissociated from chick embryo retina and spinal cord also were used to study the process of synapse formation.  Both intact chick retina and cultured retina cells were shown to have high choline acetyltransferase activity and abundant nicotinic and muscarinic acetylcholine receptors.  125I-Labeled a-bungarotoxin and 3-[3H]-quinuclidinyl benzilate, which bind with high affinity and specificity to nicotinic or muscarinic acetylcholine receptors, respectively, were used as probes to determine the properties of the receptors, the number of binding sites, and their distribution within the retina during embryonic development.  Most of the nicotinic acetylcholine receptors and all of the muscarinic receptors of chick retina were localized in layers within the inner synaptic layer of the retina; 11 layers were distinguished within the inner synaptic layer of chick retina on the basis of muscarinic and nicotinic acetylcholine receptor concentrations and acetylcholinesterase activity.  The layers appear in an ordered sequence during development with respect to temporal and positional relationships.  These results and those of others show that neurites of the same type sort out from neurites of other types on the basis of species of receptor, transmitter, or enzyme of transmitter metabolism.  A possible mechanism for generating sets of stratified or columnar neurons with similar properties and relating one set to another by cross-linking neurons of the same type to one another via synaptic connections was proposed. An a-bungarotoxin-peroxidase conjugate was synthesized and used to study the distribution of nicotinic acetylcholine receptors in the developing chick retina at the ultrastructural level.  Five percent of amacrine neuron synapses and 14-20% of bipolar neuron synapses in the inner synaptic layer of the retina were labeled; high concentrations of nicotinic acetylcholine receptors are restricted to these synaptic sites.  These results suggest that some bipolar neurons and amacrine neurons in chick retina synthesize acetylcholine and synapse with ganglion neurons or amacrine neurons which possess nicotinic acetylcholine receptors. The specificity of synapse formation by dissociated chick embryo retina neurons was examined by culturing retina cells with inappropriate synaptic partner cells, such as striated muscle cells which possess nicotinic acetylcholine receptors.  The results show that neurons are generated in chick embryo retina that are able to form synapses with striated muscle cells and then lose the ability to form synapses with a half-life of 21 hours.  These neurons first appear in chick retina on the sixth day of embryo development, and are most abundant on the eight day, comprising perhaps 8% of the retina cell population.  Almost all myotubes were innervated after coculturing retina and muscle cells for only 2 hours.  However, the mismatched synapses between retina neurons and muscle cells are transient and slowly disappear over a period of 8 days.  Neurons lose the ability to form new synapses by the 16th day, but not the ability to synthesize and secrete acetylcholine. Cultured retina neurons also form synapses in abundance with other retina neurons (approximately 1 x log synapses/mg of protein), and synapses between retina neurons were found after all synapses between retina neuron and muscle cells had been terminated. Preparations of neurons from spinal cord, which presumably contain motor neurons-that normally innervate striated muscle cells, also formed synapses with muscle cells in vitro but the number of synapses remained constant during subsequent culture.  Thus, spinal cord neuron either form stable, long-lived synapses with muscle cells or attain a steady state wherein rates of synapse formation and termination are equal.  These results show that populations of cholinergic neurons from retina and spinal cord differ in the rate of synthesis of synapses with muscle cells and probably also the rate of termination and that populations of synapses can be selected on the basis of differences in synapse turnover rates.  The results suggest that part of the specificity of synaptic circuits may be acquired after synapses form by a process of selection. A factor extracted from chick embryo retina and spinal cord was found to agglutinate rabbit erythrocytes in vitro.  The amount of agglutinin activity varies markedly during embryonic development in the spinal cord, rising to a peak on the 10th day of embryonic development and then decreasing 7-fold by the time of hatching.  The factor was first detected in 10 day embryo retina and increased in concentration until, the 16th day in ovo.  Despite differences in hemagglutination activity and the patterns of development, both the retina and spinal cord lectins exhibit the same specificity for saccharides.  Lactose was the most potent inhibitor hemagglutination found (half-maximal inhibition with 2 x 10m^-5 M lactose). To identify retina molecules required for synaptogenesis or communication across the synapse, we have used the technique recently introduced by Milstein and coworkers of monospecific antibody synthesis by clonal spleen cell x myeloma hybrid cell lines formed by fusion of clonal mouse myeloma cells with mouse spleen cells immunized against retina cells.  Large quantities of", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wk38~6q7n_7zap", "00000000-0000-0000-5CC1-676D04DC1B14", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Policy and Action Statement for the Second Biennial Conference on the Fate of the Earth", "101584910X310", "101584910X291", "1984", "[1984?]", null, "Manifestoes, Drafts (documents)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "11", "pages", "Text", "English", "Reproduced with permission of Friends of the Earth.", "Copyright may apply", null, null, "Preamble In 1955, ten years after the first nuclear bombs killed a quarter of a million people in Hiroshima and Nagasaki, Albert Einstein and Bertrand Russell, along with seven other Nobel laureates, urged the people of the world to recognize that nuclear weapons threatened the continued existence of humankind and to renounce these weapons forever: \"We appeal, as human beings, to human beings:  Remember your humanity and forget the rest.  If you can do so, the way lies open to a new paradise; if you cannot, there lies before you the risk of universal death.\" Today, nearly thirty years and 50,000 warheads later, the world continues speeding toward nuclear apocalypse, which we now know would be worse than anything Einstein and Russell ever imagined.  Recent studies by leading American, European, and Soviet scientists have concluded that even a limited nuclear war involving less than one percent of existing arsenals could produce enough smoke and soot to block out more than 99 percent of the Northern Hemisphere's sunlight, plunging the planet for many months into a dark, lethal nuclear winter.  These findings have made it clear that nuclear wars, perhaps even all wars, now imperil the continued existence of both human life and a living planet. What nuclear war could do in 50 to 150 minutes, an exploding population assaulting the Earth's life-support systems could do in 50 to 150 years.  Today, aquifers are being exhausted, farms turned into dustbowls, tropical rainforests destroyed, and limited fossil energy and mineral resources depleted. This environmental assault is particularly devastating to the more than one billion people faced with chronic hunger, deteriorating land, increasing shortages of firewood and other fuels, inadequate housing, and unsanitary drinking water. In the years ahead, unless humanity changes its ways, these people will be joined by billions more competing for dwindling or degraded resources.  If these expanding multitudes continue pursuing economic growth without seeking environmental sustainability, we may all soon be forced to cope with catastrophic food shortages, pollution disasters, and severe climate perturbations. These ominous problems are solvable, but only if we recognize their interconnections. On his death bed, Aurelio Peccei, founder of the Club of Rome, wrote that peace must be understood not only as the prevention of violent conflict between nations \"but also in the relationship between human society and nature.\"-  A recent 31 nations conference issued a similar communique that \"the preservation of the environment and the maintenance of peace are important to one another.\" The connections between nuclear war and environmental destruction are manifold.  In a world where increasing competition over resources such as oil, fisheries, or fertile land could spark conflicts capable of escalating from conventional to nuclear weapons, peace will require us to adopt more sustainable patterns of resource use. In a world where nations squander six hundred billion dollars each year on weapons and armies, sustainability will require us to divert these military-directed funds into ambitious global plans for family planning, environmental restoration, rural development, and pollution control.  And resolution of these problems will require that national attempts to conquer the environment and vanquish security threats must be supplanted by new approaches aimed at international stewardship and cooperation. For the sake of peace, of the Earth, and of its children, we call upon the world's people -- in all branches of their governments, in business, in academia, in churches, and in the press -- to awaken to and to redress these urgent problems.  The prospects of nuclear winter and environmental catastrophe have brought humankind to the most critical crossroads of its existence.  We can no longer afford to go about business as usual.  Nothing less than new, bold policies for restoring rationality to national security and promoting a sustainable global economy will ensure our survival. I.  Restoring Rationality to National Security We first call for all nations to recognize that a rational national security policy must seek to freeze and reduce weapons stockpiles, to prepare the economy for peacetime production, and to seek, ultimately, the universal abolition of nuclear weapons.  Even if complete disarmament may lie decades away, we urge that it become an explicit goal to guide short-term policy.  In the same way that people in the past have rejected inhumane activities like slavery, human sacrifice, and child labor, we must have the moral courage to reject nuclear deterrence, a policy by which nations stand ready to slaughter the human race in the name of national security. As the American Catholic Bishops have stressed, we must make a moral about-face and accept deterrence only as a step on the way toward progressive disarmament.  But to make deterrence even conditionally acceptable, we must make dramatic changes in strategic doctrine and in policies regarding conversion, nonproliferation, intervention, and international law. First, we recommend the elimination of all strategies for fighting, surviving, and winning so-called limited nuclear wars.  Accordingly, we urge all nuclear nations to adopt a \"no first use\" posture accompanied by a strengthening of nonnuclear deterrents to war.  Because almost any nuclear exchange can escalate into a nuclear winter, there is no conceivable gain that could outweigh the likely costs of nuclear war. Second, we call for negotiations or independent initiatives aimed at the universal elimination of all nuclear weapons that give the false perception that nuclear wars are fightable, survivable, or winnable, including weapons with very high accuracy, short flight times, multiple warheads, or explicit war-fighting purposes.  In addition, the development of all nonnuclear technologies such as space-based particle beam weapons and anti-satellite weapons should be abandoned and outlawed. Third, we recommend substantial reductions in the total number of nuclear and conventional weapons deployed.   To effect such reductions, we call upon our leaders to seek, first, through independent initiatives designed to set the stage for fruitful negotiations, a bilateral freeze on the development, testing, product ion, and deployment of all nuclear weapons. Fourth, to ensure we can afford an outbreak of peace, we urge the development of sound conversion programs by both government and private industry to help retrain defense workers and retool plants involved in military production. The resultant redirection of human and material resources to sustenance rather than to destruction is essential to a sound global economy based on conservation. Fifth, we urge stronger national and international policies to halt the spread of nuclear weapons to nonnuclear nations by tightening controls on the export of all nuclear materials and technology, increasing the inspection and enforcement powers of the International Atomic Energy Agency, and implementing wherever cost-effective renewable, nonproliferative energy- supplies, including efficiency improvements.  Furthermore, member nations of the Nuclear Non-Proliferation Treaty should prepare to use the 1985 review conference to bring holdout nations like China, France, and India within the treaty. Sixth, to reduce the chance that nuclear weapons will be used in conventional conflicts, we call upon all nations, especially those possessing nuclear weapons, to strengthen international treaties and institutions aimed at respecting one another's territorial sovereignty. We particularly encourage nations to influence one another, not through military intervention, but through nonmilitary avenues of cultural exchange, trade, and persuasion.  Accordingly, we urge the superpowers to work with their allies in replacing their nuclear umbrellas with regionally based, nonnuclear defense arrangements. We finally urge the endorsement of the McCloy-Zorin principles for secure, phased disarmament, agreed to by American and Soviet negotiators in 1961, and to seek what the United Nations Charter has always envisioned but never implemented: an effective international collective security system as a reliable, legitimate hedge against all global aggression. Finally, to help make disarmament conceivable in the decades ahead, we seek the long-term transfer of some national power and legitimacy to democratic global organizations to promulgate international laws and adjudicate international conflicts in a binding, nonviolent manner.  As first steps , we support efforts to encourage leaders increasingly to abide by and to strengthen the fragile international laws and institutions already in place, particularly the Law of the Sea Treaty, the International Court of Justice,  and the United Nations. II. Promoting A Sustainable Global Economy To improve the quality of life for the planet's rapidly expanding 4.7 billion people without courting eco-catastrophe, we call for both public and private initiatives aimed at promoting biologically sustainable economic progress, conserving resources, controlling pollution, protecting biological diversity, and curbing population growth.  These initiatives will be increasingly possible as the world redirects resources to what we really need and away from weapons we no longer can afford. First, we urge our governments to offer more assistance to the world's poor people. Savings from disarmament should be reinvested in programs promoting employment and economic progress in developing nations, particularly in rural areas.  We further endorse the recommendations of the distinguished Brandt Commission that developed nations open their markets to developing countries' producers and create mechanisms for stabilizing fluctuations in the prices of global commodities. Second, we urge all governments to adopt a policy of living within the carrying capacities of nations' ecosystems, with reasonable allowance for trade with other regions having surplus resources.  Renewable resources should be used instead of nonrenewable ones and conserved so that they are consumed at a rate no greater than they can be replenished. We strongly endorse the development of resilient, diversified, and decentralized energy supply technologies capable of tapping the renewable energy sources, including efficiency improvements, to reduce our current reliance on brittle, overcentralized, and nonrenewable sources such as uranium, coal, oil, and gas.  Where nonrenewable resources must be used, we urge their careful conservation through more vigorous programs promoting efficiency and, where possible, recycling. Third, because pollution fails to respect national borders, we recommend that global development be accompanied by vigorous programs of international pollution control. Acid rain, ozone depletion, climate shifts, pollution, radioactivity contamination, and water all of which have been caused by unwise global development, are among the areas most in need of special international attention. Moreover, nations should regulate the export of potentially hazardous substances or technologies as rigorously as they regulate them at home. Fourth, we urge a halt to the tragic destruction of our irreplaceable genetic resources.  Today, we are losing uncounted species, sacrificing agricultural sustainability, destroying potential sources of human foods, medicines, and industrial products, and irreparably tearing the entire fabric of our ecosystems.  To reverse this trend, we call upon our leaders to undertake innovative efforts to conserve Earth's biological diversity.  Particular attention should be placed on preventing further losses of tropical rainforests, which harbor an enormous share of the planet's biological wealth and the disappearance of which could well lead to cataclysmic changes in global climate. Fifth, we urge a reversal of the growth rate of the global human population, which is now expanding by more than 200,000 people per day.  If unchecked, population growth could overburden the earth with more than ten billion people by the year 2050, more than canceling any economic advancement in poor nations, spurring more disruptive global migrations, and placing unmanageable stresses on the global environment.  While the consequences of population pressures will at first be most keenly felt among the world's poor, the west will inevitably suffer the reverberations.  To avoid these consequences, we call upon developed nations to help other nations assess their resources and carrying capacities, and give international population measures the highest priority. Finally, we urge that governments, educational institutions, labor, and management develop comprehensive plans to accommodate the rechannelling of resources from military production to these development and environmental protection programs. Making A New Commitment To those who consider these proposals unrealistic, Richard Barnet's reminder is apt:  \"we now march to annihilation under the banner of realism.\"  The world has fundamentally changed, and the \"realistic\" approaches of the past no longer work.   Our task must be to expand the boundaries of what is realistic.  Many \"unrealistic\" approaches of yesterday -- energy conservation efforts, international environmental treaties, and small-scale rural development programs -- have become the success stories of today.  As the accompanying legislative agenda reflects, there is much we can do, providing we have the will.  The most unrealistic policy imaginable is to believe complacently that continued nuclear arms races can avert a nuclear winter. We call for new, enlightened leadership within all nations, but especially within the United States, which controls one-fourth of the world's wealth, an even greater amount of its scientific and technical knowledge, and half of the planet's nuclear weapons.  With a proven track record in influencing global security and environmental policies, the United States can help lead the world toward disarmament and a sustainable society, especially if it devotes more intellectual, financial, and institutional resources to these problems. Above all, survival rests in the hands of each and every one of us.  We must educate and organize our families, our friends, our neighbors, and our local leaders. From millions of individuals and groups committing themselves to finding an answer to the human predicament, new national and international institutions and policies can be shaped and implemented.  Citizens can also influence international affairs by working directly with citizens in other nations, as American and Soviet citizens have done through regular visits to one another's lands.  As the tools of global transportation and communication become more accessible, people everywhere should use them to link-up and combat the world's most pressing problems through existing and new transnational networks of nongovernmental organizations, churches, labor unions, and political parties. We are now entering the fifth decade of the nuclear age and are approaching the decisive moment in human history.  If we act decisively, we still have a chance to avert nuclear war and environmental catastrophe. But we each must make survival an uncompromised priority, for if we let life slip away, we shall never have another chance. United States Legislative Action Agenda In a democracy, one of the most powerful tools for implementing new policies is through legislation.  Consequently, to implement the goals of the Policy and Action Statement, we urge our duly elected representatives in Washington, D.C. to undertake the legislative actions described below. In making these recommendations, we do not mean to suggest that only Congress has a burden to act.  Indeed, little legislation is possible without public pressure (through letters, phone calls, and voting) or presidential leadership and support.  We instead view this agenda as a call for action to people throughout America to educate, organize, and persuade other people to make these small legislative steps possible. I.   Restoring Rationality To National Security (1) Reexamination Of U.S. Nuclear Strategy In The Face Of Nuclear Winter:  We urge the House and Senate Armed Services Committees to hold hearings on the strategic implications of the recent evidence on nuclear winter.  These hearings should critically examine the Pentagon's report on the subject, which both houses have directed it to issue by March 1, 1985. They should especially examine whether the risks of human extinction posed by a nuclear winter have now rendered nuclear war obsolete. (2) Legislate Moratoria On Destabilizing Weapons -- We endorse Congressional attempts to restrict production and recall deployment of the MX, Trident D-5, and Pershing II missiles, all of which are designed to encourage nuclear war-fighting and therefore threaten American national security at least as much as they threaten Soviet security.  We further endorse Congressional attempts to cut off the development, production, and deployment of \"Star Wars\" weapons and anti-satellite weapons. (3) Legislate A Nuclear Freeze -- We endorse recent Congressional initiatives that urge the President to enter negotiations with the Soviet Union for a bilateral, verifiable freeze on the development, testing, production, and deployment of nuclear weapons.  We also endorse the National Freeze Campaign's proposals to make defense appropriations conditional on the President faithfully pursuing a freeze. (4) Ratify Outstanding Arms Control Treaties -- We endorse Congressional resolutions (SJR 29, HJR 3) urging the President to enter negotiations for a comprehensive test ban and to submit to the Senate for ratification of the Threshold Test Ban Treaty and the Peaceful Nuclear Explosions Treaty. We further urge the Senate to ratify SALT II. (5) Conduct Hearings On Deep Cuts -- To meet the challenges posed by substantial reductions in nuclear arsenals -- including verification, the disposal of fissionable materials, and dangers posed by medium-sized nuclear powers, proliferation, and technological breakout -- we urge the Congress to undertake extensive hearings on these issues. (6) Legislate A Conversion Assistance Program -- We support efforts to create national programs aimed at easing the economic impact of defense spending cuts through adjustment assistance and retraining. (7) Strengthen The Nonproliferation Act -- To bolster the Nuclear Non-Proliferation Act, we urge Congress to prohibit domestic use of recycled plutonium and highly enriched uranium in U.S. nuclear power plants, to ban foreign reprocessing of U.S.-supplied nuclear fuel, and to halt all exports of reprocessing and uranium enrichment technology as well as separated plutonium and highly enriched uranium. (8) Create A Sunbeams For Peace Program -- To phase out nuclear power exports, we recommend that Congress discontinue subsidizing nuclear exports through the Export-Import Bank and instead begin promoting the export of nonnuclear energy sources (including conservation measures) to undercut the world's nuclear reactor vendors. (9) Strengthen The War Powers Act -- To restore the constraints on military intervention originally conceived in the Constitution, we endorse Congressional efforts (HR 6078) to strengthen the War Powers Act by requiring the President to obtain prior Congressional approval before committing U.S. troops abroad, except to rescue U.S. citizens, to repel an attack on U.S. armed forces, or to respond to an invasion of U.S. territory. (10) Pass The Common Security Resolution -- In an effort to pave the way for deep cuts in all nuclear arsenals, we support the \"Common Security Resolution\"  (SR 125, HR 123) that urges the President to reopen negotiations with the Soviet Union to implement the McCloy-Zorin principles for general and complete disarmament. (11) Ratify Outstanding International Law Treaties -- To secure the fragile instruments of international law already in place, we urge the President to submit to the Senate for immediate ratification the Convention on the Prevention and Punishment of the Crime of Genocide, the International Convention on Human Rights, and the Law of the Sea Treaty. (12) Funding For A United Nations Verification System -- To help verify global weapons control arrangements, we urge Congress to undertake hearings on the possibility of the United States offering funding and technical assistance to establish a non- partisan United Nations verification system, especially a UN satellite system -- an initiative which has already received overwhelming international support despite opposition by both superpowers. (13) Conduct Hearings On United Nations Reform -- To strengthen the only continuous forum for international discussion and cooperation the world has, we urge Congress to consider proposals for reforming the United Nations, including those aimed at giving more General Assembly votes to nations with large populations or making large contributions, directly electing General Assembly representatives, ensuring more procedural fairness for minority blocs,   abolishing the Security Council veto, and establishing a permanent peacekeeping force. II. Moving Toward Sustainable Development (1) Increase Support For Some U.S. Agency For International Development; Reevaluation Of Others -- To be added. (2) Increase Support For Multilateral Development Banks -- We urge funds disbursed to multilateral development banks (including the World Bank, the Inter-American Development Bank, and the Asian Development Bank) be more carefully tied to concrete assurances that these development programs are undertaken with careful attention to the goals of the World Conservation Strategy of the International Union for Conservation of Nature and Natural Resources (IUCN). (3) Increase Support For The United Nations Environment Program -- The United Nations Environmental Program (UNEP) is one of the most important international organizations working to promote global environmental protection by facilitating treaty negotiations on water pollution, hazardous pesticides, and ozone-protection.  Despite an authorization for ten million dollars, the Administration has requested -- and the Senate has approved -- an appropriation of only three million dollars. We urge a restoration of the prior level of funding and the gradual expansion of American contributions. (4) Strengthen Domestic Anti-Pollution Programs -- To be added. (5) Strengthen National Programs Aimed At Protective Species In The United States -- One way for the United States to protect global biological diversity is to set a good example by reauthorizing and strengthening the Endangered Species Act, inaugurating a national ecosystems protection study, and setting up a new U.S. biological survey analogous to the U.S. Geological Survey to monitor and inventory species. (6) Increase Support For National Programs Aimed At Protecting Species Abroad -- We recommend that the United States offer more financial support to the international affairs offices of both the National Fish and Wildlife Service and the National Park Service, both of which are working with their counterparts in developing countries to protect endangered species like migratory birds and to set up biological preserves.  Similarly, we support increased federal funding for the Man and the Biosphere Program, which sponsors national research on biological management techniques. (7) Increase U.S. Support For Existing International Programs Aimed At Protecting Species -- The United States can also help biological protection by contributing more money to ongoing international programs like UNEP, the United Nations Food and Agriculture Organization (FAO) and the World Heritage convention. UNEP is especially important since it provides a quarter of the budget of the IUCN, which itself carries out projects to conserve endangered species, protect national parks, and designate natural areas for inclusion in the World Heritage Convention. Finally, major funding should be provided to the World Heritage Convention, which promotes the recognition and protection of key natural and cultural areas in the world. (8) Conclude Species Protection Treaties -- We recommend that the United States sign and ratify the already completed Ramsar Wetlands Convention and the United Nations Charter for Nature. Congress should also urge the President to sponsor a treaty to protect Third World biological resources. (9) Retain And Expand Support For Existing Population Programs -- We urge Congress to oppose the Administration's attempts to cutoff $100 million to international population control programs that practice or advocate abortion. To reduce the incidence of abortion, we instead encourage increased support for programs promoting family planning methods and higher literacy among women. III. Making A New Commitment (1) Conduct Hearings On Establishing Foresight Capability -- To improve the nation's ability to evaluate population, and environmental trends effectively, resource, we recommend that Congress enact current proposals (HR 31, SR 33) to create an executive level Global Foresight Agency. (2) Hold Hearings On Means Of Expanding Public Participation -- Congress should explore means of expanding public participation in strategic planning, perhaps by opening the Arms Control and Disarmament Agency's statements on the arms control impact of each new weapons system to a more rigorous system of public scrutiny analogous to the public scrutiny that now occurs under the National Environmental Policy Act. (3) Increase Support For Global Education Programs -- We endorse legislation to increase federal funding for programs aimed at increasing students' appreciation for and comprehension of foreign languages, culture, and politics. (4) Increase Support For The Peace Corps -- We recommend increased funding for the Peace Corps, which since 1962, has sentmore than 100,000 volunteers to assist in development and environmental protection projects around the world. (5) Increase Support For Exchange Programs -- We urge greater federal support for programs like the Fulbright Fellowships that enable tens of thousands of scientists, teachers, artists and ordinary citizens to participate in international exchanges each year. (6) Create An American Peace Academy -- We endorse legislation (Amendment to HR 5167 under title IV, and SR 564, HR 1294) establishing a United States Peace Academy to research peace issues and train government leaders, military officials and civilians from all over the world in peacemaking and conflict resolution techniques.", "Brower, David ; Friends of the Earth ; Peterson, Russell W. (Russell Wilbur), 1916-2011 ; National Audubon Society", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ehzw~tur7-djbf", "00000000-0000-0000-0A47-3C99B20C5C8C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Facing the 21st Century: Threats and Promises", "101584910X311", "101584910X296", "1987", "[1987?]", "The purpose of the conference, to be held in Paris in January 1988, was to address the principle moral and political problems challenging mankind and explore solutions to them.", "Conferences, Programs (documents)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "15", "pages", "Text", "English", "Reproduced with permission of Elie Wiesel.", "Copyright may apply", null, null, "FACING THE 21st CENTURY: THREATS AND PROMISES Conference Outline The Elie Wiesel Foundation for Humanity 666 Fifth Avenue, 11th Floor New York, NY 10103 U.S.A. Telephone: (212) 399-4485 TABLE OF CONTENTS Introduction Purpose of the Conference Conference Goals and Objectives Conference Format Topics for Discussion Hate and Tolerance Political Oppression and the Rights of the Individual War and Survival _ Environmental Pollution and Ecological Equilibrium Science: Risk and Promise Program Outline (Draft) Page 10 _ FACING THE 21st CENTURY: THREATS AND PROMISES The 20th century confirms Bertrand Russell's observation that \"The human race lives in a welter of organized hatreds and threats of mutual destruction.\" Innumerable natural and human-made disasters, each one seeming to exceed in horror those it follows, dot the historical landscape. Human beings continue to suffer the ravages of famine, ecological devastation, terrorism and war. While the plea for Human Rights echoes throughout the world, the law of “might is right\" continues to prevail. Confronting complex, interconnected global problems demands technical know-how along with moral and intellectual commitment. Technology, which offers at once the greatest threat and the greatest promise for humankind, must be used to preserve our species and our environment, not to pollute the earth or destroy ourselves. The problems facing us in the 21st century must not be left to technological experts alone. Their efforts must be complemented by the efforts of scientists, humanists and political leaders, using their human ingenuity and creativity to discover, in Einstein's words, \"a substantially new manner of thinking if mankind is to survive.\" One group uniquely qualified to join together in confronting such a challenge are the Nobel Laureates. They represent different nationalities, are involved in a diversity of fields of scholarly and human endeavor, and embrace a variety of political and religious -2- orientations. Nearly 250 Nobel Laureates are living, many active scholars, others retired but still concerned world citizens. Their intellectual and ethical insights, their theoretical and practical contributions to the welfare and development of humankind are universally recognized. They are in a unique position to use their wisdom and their prestige for the benefit of humankind: to diminish human suffering and to strengthen human well being. PURPOSE OF THE CONFERENCE FACING THE 21st CENTURY: THREATS AND PROMISES is an International Conference that will be convened by the Honorable Francois Mitterrand, the President of France, and Professor Elie Wiesel, the 1986 Nobel Peace Laureate. The conference will be held in Paris, France during the third week of January, 1988. The purpose of the conference is two-fold: (1) to provide a forum for Nobel Laureates in all fields to address some of the principal moral and political problems that challenge us as we approach the next century, and (2) to enable them to discuss, explore and suggest creative solutions to those problems so that we can face the 21st century with more confidence and greater promise. CONFERENCE GOALS AND OBJECTIVES G-1 G-2 Focusing attention on the principal moral and political challenges confronting human beings now and in the 21st century. Evoking imaginative thinking about the future well-being of humankind; 0-1 To convene an international conference of all Nobel Laureates; 0-2 To invite a number of conference participants to prepare papers on issues/questions appropriate to the conference theme; 0-3 To organize seminar groups on particular topics that will meet several times during the conference; 0-4 To prepare and present a conference report intended to initiate dialogue with world leaders about selected issues of concern; 0-5 To publish the proceedings of the conference; 0-6 To produce a film related to the conference that can be used in educating the public about issues discussed during the conference; CONFERENCE FORMAT The conference format will combine plenary sessions, seminars and informal discussions. It will be designed to maximize the involvement of all the participants so that everyone will be a resource person and all will be principals in the process of reflection and discussion. There will be formal presentations of invited papers, formal discussants of each paper, seminars, and opportunities for formal and informal discussion. Skilled moderators will guide the discussions, giving all the opportunity to contribute their expertise, ingenuity and insights about issues, questions, problems and challenges’ facing humankind. During the conference, participants will sharpen their focus on the identified topics, discuss creative approaches to meeting the challenges that face us, identify the implications of such approaches, and, if possible, recommend policies, principles or strategies for meeting these challenges. Prior to the conference, papers will be distributed to all participants. Other selected readings also may be distributed to conferees in preparation for conference discussion. Because ideas and values are transmitted through the arts as well as through scholarly research and discussion, the conference will include an aesthetic component along with the intellectual. The entire conference will be audio recorded. In addition, some selected plenary sessions and seminars will be filmed and several -5- individuals will be interviewed, in order to produce a film for educational purposes. An edited book based on the _ conference proceedings also will be published. TOPICS FOR DISCUSSION The conference will focus on several areas: Hate and Tolerance; Political Oppression and the Rights of Individuals; War and Survival; Pollution and Ecological Equilibrium; and Science: Risk and Promise. HATE AND TOLERANCE: The world seems poisoned by hate -- between people and among nations, races and religions. Whether or not it erupts into cruelty and violence is almost irrelevant because hate can destroy not just its object, but the humanity of those who embrace it. Questions such as the following will be explored: What is hate and what are its forms? What are some of the ways it is manifested? Why are there certain strains of hate that seems to persist? Is it possible to eradicate hate? What are the preconditions for tolerance? How can it be fostered among people who have different ideologies, religions, or are of -different races and cultures? Is there a future for tolerance? POLITICAL OPPRESSION AND THE RIGHTS OF THE INDIVIDUAL: Since the end of World War II, political oppression, far from disappearing or declining, has increased. More governments abuse their citizens and violate their human rights than respect and defend them. In many countries, government-sponsored death squads mutilate and -6- murder political opponents, using violence as a means of controlling their own people. Periodic reports issued by Amnesty International confirm that the use of government censorship, control of the media, the imprisonment, torture and murder of political opponents, and state sponsored terrorism are widespread. Attention will be given to questions such as the following: Why do oppressive regimes flourish? What can be done to fight for the rights of individuals, to encourage and support government that serves people rather than demeans them? Which ideas and what practices encourage and support human rights and freedom? What can we do to strengthen human rights and reduce the power of oppressive governments? How can powerful governments be humanized? What are the pre-conditions for freedom and democracy? How can Memory be a shield to safeguard the dignity and human rights of people? WAR AND SURVIVAL: Nuclear weapons introduce a quantitatively and qualitatively new factor in human history. No problem is more overwhelming, no danger more unremitting than the potential for and possibility of nuclear war. It is an unimaginable, ineffable disaster which is within the realm of possibility, one which could devastate human life, destroy all human dreams and hopes, every creative work of art -- all that reflects our humanity and helps to make civilization worth saving. Scientists and humanists must find a way to stand together against death and oblivion and for a sane and humane future for all people. Questions such as the following could be discussed: What means can be developed for dealing with disparate power relationships in international relations? Why has an awareness of the threat of nuclear destruction _ hot forced us to alter our confrontational approach to international affairs? What are the checks and balances on the human control of technology? What can be done now to prevent the proliferation of nuclear weapons? Can we work cooperatively and collaboratively to apply our human genius and imagination for survival instead of destruction? Is it possible to imagine a world without nuclear, chemical or other high-tech weapons? Can our commitment to life, to a future for ourselves and our descendents, supercede our disagreements and rivalries? ENVIRONMENTAL POLLUTION AND ECOLOGICAL EQUILIBRIUM: Human beings inhabit two worlds: the natural world and the constructed world. The former is the world of plants and animals, the world of natural resources. The latter is the world of social and _ political institutions, the world human beings have built. These worlds are interrelated, although human beings are not always conscious of that interrelationship. -8- Every human decision and action has an affect on the environment and must be considered from an ethical perspective. After accidents such as those of Three Mile Island, Bhophal and Chernobyl, it seems evident that ecological issues and questions must concern not just environmentalists but everyone if Homo sapiens is to survive as a biological species on a habitable planet. Questions for discussion might include: What can be done to develop a respect for the earth as an environment suitable for human 1i fe? Are there limits to human ecological intervention that the earth can safely tolerate? Is it possible that the earth will soon be so polluted that human beings will find it difficult to inhabit the earth? Are there areas of the natural world that are seriously threatened by pollution? What must be done in the short and long-term to fight these threats? How can human beings go beyond developing mechanisms designed to prevent or minimize environmental devastation to developing new ways of relating to each other and to the environment? SCIENCE: RISK AND PROMISE: Faced with the questions and problems of their time, some scientists have always questioned whether their work served the interests of humanity. Others, interested only in their own technological competence and professional skill, irrespective of the purpose for which their research would be used, were not concerned about -9- such questions. Today, however, programs in genetic engineering, the development of sophisticated weapons and weapon systems, the complexity of new diseases, the potentiality of biotechnology are forcing many people -- not just scientists -- to ask questions about the compatability of science and human rights, and to reflect on the risks and the promises in scientific research and applied technology. Questions such as the following will be explored: What is the future of genetic engineering and its consequences on the nature of human beings? Is it ethical to alter the genetic code of animals? Is Aids a disease like any other, or is it qualitatively different from other diseases? Does the very nature of the disease demand that governments issue policies that may restrict the individual rights of people who have been infected with the Aids virus? What is the influence and impact of scientists on political debate? What should it be? In what manner must it be conducted to be effective? How can scientists and humanists mobilize the political community in their respective countries to make Aids research a priority? DAY ONE 1:00 7:30 DAY TWO 7:30 9:00 9:15 11:00 11:30 7:00 2:30 5:00 p.m. - 10:00 p.m. 8:30 a.m. 9:15 a.m. - 11:00 a.m. - 11:30 a.m. 17:00 p.m. 2:00 p.m. 4:30 p.m. -10- PROGRAM OUTLINE (Draft) Conference Registration Opening of Conference President Francois Mitterand Chamber Music Reception * * * Breakfast Welcome Keynote Address: Overall Theme Professor Elie Wiesel Two Respondents Discussion with Presenter and Respondents Coffee Break Two Respondents Discussion Lunch Simultaneous Workshops Presentation Discussion 7:00 - 8:30 p.m. DAY FOUR 7:30 - 8:30 9:00 - 10:30 10:30 - 11:00 11:00 - 1:00 7:00 - 1:15 1:15 - 2:30 2:30 - 5:00 6:30 - 7:30 8:00 p.m. DAY FIVE 7:30 - 8:30 9:00 - 10:30 a.m. a.m. a.m. p.m. p.m, p.m. p.m. p.m. a.m. a.m. >» -11- Summary of the Day's Discussion Activities Cocktails Dinner NOTE: At some appropriate moment(s) during the day, there will be a Performing Arts Interlude(s) * * Breakfast Keynote Address: Theme #3 Respondents Break Keynote Address: Theme #4 Respondents Discussion (both morning sessions) Summary of Morning Address and Discussion Lunch Working Sessions (Preparation for Conference Statement/Mani festo/Document) Dinner Concert (French Philharmonic) Reception Follows * * Breakfast Panel of Four Conference Responders 4:30 - 5:00 5:00 - 6:00 7:00 - 9:00 DAY THREE 7:30 8:30 9:00 - 10:30 10:30 - 11:00 11:00 - 12:30 12:30 - 2:00 2:00 - 3:30 3:30 - 4:00 4:00 5:30 p.m. p.m. p.m. p.m. p.m. p.m. p.m. -12- Recommendations Break Workshop Reports to Plenary Session Discussion Cocktails Dinner NOTE: At some appropriate moment(s) during the day, there will be a Performing Arts Interlude(s) * * Breakfast Simultaneous Workshops Presentation Respondents Break Continue Workshop Sessions Summary of First Session Discussion Recommendations Lunch Report from Workshop Sessions to Plenary Session Keynote Address: Theme #2 Respondents Break Discussion 10:30 - 11:00 - 1:30. - Departure 11:00 a.m. 1:00 p.m. 2:30 p.m. -13- Short Reactions/Comments Discussion Break Presentation of Conference Statement with Recommendations Discussion Summary and Final Conference Statement: Professor Elie Wiesel Closing Address: President Francois Mitterand Lunch", "Elie Wiesel Foundation for Humanity", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-c9yt~aegz.ixby", "00000000-0000-0000-8FFE-0D82E3D9DB53", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Trieste Declaration of Human Duties", "101584910X312", "101584910X301", "1993", "[1993?]", "Included in a letter from Rita Levi-Montalcini, 1968 Nobel Prize winner in Physiology or Medicine, this brochure outlines twelve principle human duties drafted by outstanding scientists and intellectuals, which include respecting human dignity and diversity, protecting nature, and promoting peace.  In the accompanying letter, Levi-Montalcini suggests that Nirenberg's approval and support would be \"of invaluable importance in the success of this project.\"", "Brochures", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "8", "pages", "Text", "English", "Reproduced with permission of Rita Levi-Montalcini.", "Copyright may apply", null, null, "International Council of Human Duties Che Crieste Declaration of Duman Duties A Code of Ethics of Shared Responsibilities University of Trieste Italy   Hntroduction rucial problems concerning humankind at the dawn of the 21st century urge the adoption of a different way of thinking and a different value system. The change must be as revolutionary as that which emerged after the Middle Ages. The new way of thinking must be centered on humans as an integral part of our planet, whose actions affect all living things. It is our plan to present and discuss these ideas through conferences, work- shops and lectures, and in particular to support women’s networks and other organizations, active in the spirit of this Declaration. Furthermore, we will seek the participation of the younger generations, which repre- sent our greatest hope for improving the quality of life on earth. Preamble ASS hereas the Declaration of Human Rights represents one of the great advances of the twentieth century, it fails to address Human Duties and Responsibilities as necessary counterparts of these Rights. Recognition of and respect for human rights demand the acceptance of specific duties, in order to assu- re an adequate quality of life for all people and the persistence of a sustainable environment for future generations. We, founding members of the International Council of Human Duties, consider a binding responsibility for ourselves, not only as human beings, but especially as scientists and educators, to carefully and explicitly carry out these duties to the best of our ability, even if their enactment may run counter to esta- blished policies generated by traditional sources of power and influence. We herewith invite all people concerned with these issues to join us in our efforts, as expressed in the following twelve points. Dre Crieste Declaration of Bauman Duties A Code of Ethics of Shared Responsibilities     IT IS THE DUTY OF EVERY HUMAN BEING TO: respect human dignity as well as ethnic, cultural and religious diversity. work against racial injustice and all discrimination of women, and the abuse and exploitation of children. work for improvement in the quality of life of aged and disabled persons. respect human life and condemn the sale of human beings or parts of the living human body. support efforts to improve the life of people suffering from hunger, misery, disease or unemployment. promote effective voluntary family planning in order to regulate world popula- tion growth. support actions for an equitable distribution of world resources. AA & avoid energy waste and work for reduction of the use of fossil fuels. Promote the use of inexhaustible energy sources, representing a minimum of environ- mental and health risks. protect nature from pollution and abuse, promote conservation of natural resources and the restoration of degraded environments. respect and preserve the genetic diversity of living organisms and promote con- stant scrutiny of the application of genetic technologies. E) gy & Promote improvement of urban and rural regions and support endeavours to eliminate the causes of environmental destruction and impoverishment which can lead to massive migrations of people and overpopulation in urban areas. SI work for maintenance of world peace, condemn war, terrorism and all other hostile activities by calling for decreased military spending in all countries and restriction of the proliferation and dissemination of arms, in particular, weapons of mass destruction. The Snternational Council of Human Duties iB p Laws I DENOMINATION AND DOMICILE The International Council of Human Duties, hereinafter called ICHD, is an intemational non-governmental and non-profit making organization. The legal domicile of [CHD is in Trieste, Italy. ICHD shall act as a World Federation of individuals and organizations which adhere to the concepts of the Trieste Declaration of Human Duties. II OBJECTIVES The objectives of ICHD shall be: To promote international understanding of the concepts of the Trieste Declaration of Human Duties and to foster throughout the world the appli- cation of these concepts, To enter into relations with the United Nations, its agencies and with national Governments for the implementation of the goals of ICHD, and to co-operate with non-governmental international, national and regional bodies whose aims are consonant with those of ICHD. To encourage the adoption by the United Nations of a “Universal Declaration of Human Duties” with comparable stature to the “Universal Declaration of Human Rights “. To promote the establishment of a “Human Duties Day”. To stimulate, design, co-ordinate and participate in the implementation of international programmes related to the concepts of the Trieste Declaration of Human Duties. To provide for and assist in the education and the dissemination of information relating to the concepts of the Trieste Declaration of Human Duties. To arrange, promote and sponsor World Congresses of the ICHD and such other meetings as may be useful or desirable for the advancement of the purposes of ICHD. : To encourage the formation of national associations for the dissemination of information of the concepts of ICHD. To actively uphold the principle of the universality of the concepts of the Trieste Declaration of Human Duties. ICHD shall not engage in any actions of political nature. TIT MEMBERSHIP ICHD shall be composed of two categories of members: Individual members International, national and regional organizations and agencies Individual Members Any individual anywhere in the world who is devoted to the concepts of the Trieste Declaration of Human Duties and supports the goals of ICHD is eligible for election as Member of ICHD. Application for membership should be submitted in writing to the Secretary General. ‘ Member Organizations International, national or regional organizations and agencies with an active interest in the objectives of the concepts of the Trieste Declaration of Human Duties are eligible for membership. Application for membership should be sent to the Secretary General and be accompanied by the sta- tutes of the organization. , IV ADMINISTRATION The work of ICHD shall be conducted by: The Executive Board The Advisory Committee The Executive Board The Executive Board shall consist of Four Officers: the President, the Vice President, the Secretary General and the Treasurer, elected by the Advisory Committee for a period of four years. They shall be eligible for re-election. , Five Ordinary Members elected by the Advisory Committee from its own membership for a period of four years. They shall not be eligible for re-election. The Executive Board shall be the governing body of ICHD and shall be empowered to take all steps necessary to fulfill its functions. The Executive Board shall actively engage in the launching of a global campaign to heighten public awareness of the concepts of the Trieste Declaration of Human Duties and shall raise funds for this purpose. The Executive Board shal] meet at least once a year. The Advisory Committee The Advisory Committee shall consist of 25 members-elected by the entire membership of ICHD, by postal ballot. The terms of the members elected shall be four years and they shall be eligible for re-election. The President and the Secretary General shall ex-officio be members of the Advisory Committee. Nominations of Committee Members may be submitted by each individual member of ICHD in writing to the Nominating Committee. The Advisory Committee shall meet at least once every second year. [t shal] outline the general policies of ICHD and co-operate with the Executive Board in the implementation of the goals of ICHD. The quorum for a meeting of the Advisory Committee shall be one-third of its mem- bers. The Advisory Committee shall elect its Chairman among its members. The Nominating Committee The Advisory Committee shall elect a Nominating Committee consisting of five members one of whom shall be designated by the Advisory Board to serve as Chairman. Members of the Committee shall serve for four years. They shall not be eligible for re-election. The Nominating Committee shall propose a slate of candidates of the Advisory Committee and shall be responsible for the nomination of candidates for the Executive Board. At least two candidates shall be nominated for each vacancy. Duties of Officers President The President shail be the Chief Executive Officer of the Council. The President shall preside at meetings of the Executive Board and shall act as spokesman of ICHD. Secretary General The Secretary General shall be responsible for managing the secretariat of the Council and for executing the policies and decisions of the Executive Board and the Advisory Committee, The Secretary General shall work in close co-operation with the President. Treasurer The Treasurer shall keep full and accurate account of receipts and disbursements of funds belonging to ICHD. The Treasurer shall render to the Executive Board and to the Council at their regular meetings an account of the financial conditions of ICHD. V FINANCES The President and the Secretary General are empowered in the name of ICHD to accept grants or donations for carrying out the activities of ICHD. The fiscal year of ICHD shall commence on the first day of January and end on the 31 of the following December. An audit of the finances shall be conducted annually at the end of the fiscal year. The Executive Board shall appoint an auditor authorized to act as a public accountant. VI - AMENDMENTS Amendments of the By-Laws may be proposed by any member of the Executive Board or Advisory Committee, The proposed amendment should be sent to all members of the Executive Board and the Advisory Committee no less than thirty days prior to the time it shall be submitted to a vote. Amendments shall go into effect following their adoption by a two-thirds majority of the Advisory Committee. FAMILY NAME... ADDRESS VOLUNTARY CONTRIBUTION I WISH TO CONTRIBUTE TQ THE PROPAGATION OF THE CONCEPTS OF THE DECLARATION ) MASTER- ) CHEQUE/EUROCHEQUE PAYABLE TO ICHD ) BANK TRANSFER TO CASSA RISPARMIO TRIESTE-ITALY c.c. NO AGENCY 23 - ViA FaBio SEvERO 152 - 34127 TRIESTE SIGNATURE TO BE SENT TO: THE SECRETARIAT OF INTERNATIONAL COUNCIL OF HUMAN DUTIES UNIVERSITA DEGLI STUDI DI TRIESTE - PIAZZALE Europa, 1 - 34127 TRIESTE - ITALY   FOUNDING MEMBERS OF THE INTERNATIONAL COUNCIL OF HUMAN DUTIES Prof. Louis Albou Dr. Enrico Alleva Prof. I Antoniou Sir Michael Atiyah Prof. André Berger Prof. Paul Blau Prof. Giacomo Borruso Dr. Noel J. Brown Prof. Luca Cavalli Sforza Prof. Jean-Pierre Changeux Prof. George Coyne Prof. Emer Colleran Prof. Benedetto de Bernard Prof. Giampaolo de Ferra Prof. Gianfranco Dioguardi * Prof. Manfred Eigen Dr. Arturo Falaschi Dr. Gabriele Gatti Prof. Frangois Gros Dr. André Hamende Prof. G. Herméren * Prof. David Hubel * Prof. Robert Huber * Prof. Frangois Jacob Prof. Joshua Jortner Prof. Sergei Kapitza Prof. Alex Keynan * Sir John Kendrew * NOBEL LAUREATE   (France} (Italy) (Belgium) (United Kingdom) (Belgium) (Austria) (Italy) (USA) (USA) (France) (USA) (Ireland) (Italy) (Italy) (Italy) (Germany) (Italy) (Rep. of San Marino) (France) (Italy) (Sweden) (USA) (Germany) (France) (Israel) (Russia) (Israel) (United Kingdom) Accademia Nazionale dei Lincei The African Academy of Sciences International Centre for Genetic Engineering and Biotechnology - ICGEB The Union of Concerned Scientists The United Nations University Prof. Iba Kone Prof. P.G. Kostyuk Prof. Ie Hyok Kwon * Prof. Rita Levi Montalcini Mr Han Been Lee Prof. Sang Soo Lee Prof. André Lichnerowicz Prof. Eleonora Masini Dame Anne McLaren Prof. Mambillikalathil G. K. Menon * Prof. Franco Modigliani Sir Gustav Nossal Prof, Michio Okamoto Prof. David Ottoson Prof. Nestor Perl * Prof. Ilya Prigogine Prof. Peter Raven Prof. Haward Ris * Prof. Carlo Rubbia Prof. Giorgio Salvini Prof. Michael Sela Prof. Eric Shooter Prof. Michel Sintzoff *Prof. Roger Sperry Prof. Hartwig Spitzer Prof. Theodore Voneida Prof. Patrick Wall Prof. Victor Weisskopf * Prof. Thomas Weller The Israe] Academy of Sciences and Humanities Third World Academy of Sciences - TWAS United Nations Environment Programme - UNEP (Kenya) (Ukraine) (Korea) (Italy) (Korea) (Korea) (France) (Italy) (United Kingdom) (India) (USA) (Australia) (Japan) (Sweden) (Argentina) (Belgium) (USA) (USA) (Italy) (Italy (Israel) (USA) (Belgium) (USA) (Germany) (USA) (United Kingdom) (USA) (USA)   LIST OF INTERNATIONAL AGENCIES SUPPORTING THE TRIESTE DECLARATION OF HUMAN DUTIES   The Suternational Council of Human Duties ICHD EXECUTIVE BOARD President Rita Levi Montalcini (Italy) Vice President David Ottoson (Sweden) Secretary General Benedetto de Bernard (Italy) Treasurer Theodore J. Voneida (USA) Noel J. Brown (USA) Jean-Pierre Changeux (France) Emer Colleran (Ireland) Michael Sela (Israel) Victor Weisskopf (USA) SECRETARIAT: Carla Savastano Universita degli Studi di Trieste Piazzale Europa 1- 34127 Trieste Italy TEL 39-40-6763002 FAX 39-40-571071 Telex 460865 UNIVTS", "International Council of Human Duties", null, null, "Università degli studi di Trieste", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-w7v3_p7bf~4qj4", "00000000-0000-0000-FA3F-36659F843A9B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics [summary of laboratory projects]", "101584910X313", "101584910X235", "1988", "September 1988", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "7", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "October 1, 1987 through September 30, 1988 Previously, many neuroblastoma and related somatic hybrid cell lines were shown to acquire voltage-sensitive ion channels and other neuronal properties when intracellular cyclic AMP levels were elevated for a number of days.  Cells with elevated cAMP acquire new proteins such as the [alpha]-subunit of voltage-sensitive calcium channels and other proteins of unknown function.   A cDNA library was constructed from poly A+ RNA prepared from NG108-15 neuroblastoma-glioma hybrid cells that had been treated for 5 days with 1 mM dibutyryl cAMP.  The library was screened and 17 cDNA clones were obtained that correspond to species of RNA that are 3 to 40 times more abundant in cells treated with dibutyryl cAMP than in cells cultured without this compound.   Each cloned cDNA was used as a probe with Northern blots to determine the number of species of poly A+ RNA responsive to dibutyryl cAMP and the chain length of each species of RNA.  The results suggest that the 17 cDNA clones correspond to species of RNA transcribed from 10 genes.  Partial nucleotide sequences of the cDNA inserts from 3 clones were obtained.  Clone NG-32 corresponds to mouse mitochondrial mRNA for ATP synthase subunit 6, a mitochondrial gene.  This protein is part of the H+ channel portion of the mitochondrial ATP synthase complex.  Treatment of NG108-15 cells with dibutyryl cAMP results in an 8-fold increase in the abundance of mRNA for this protein.  The nucleotide sequence of clone NG-10 cDNA was identified as part of the D-loop region of mouse mitochondrial DNA that contains the origin of replication for the heavy strand of DNA.  The 5'-terminal nucleotide sequence of some molecules of heavy strand mitochondrial DNA is known to consist of a short segment of RNA that is complimentary to a short light strand mitochondrial DNA sequence nearby.  Hence, NG-10 cDNA may correspond to an RNA transcript of the light strand of mitochondrial DNA that serves as a primer for the initiation of heavy strand mitochondrial DNA synthesis.   Treatment of NG108-15 cells with dibutyryl cAMP results in a 40-fold increase in this species of RNA.  These results show that treatment of NG108-15 neuroblastoma-glioma cells with dibutyryl cAMP results in marked increases in the abundance of RNA transcripts from heavy and light strands of mitochondrial DNA.  Further work is needed to determine whether cAMP regulates mitochondrial biogenesis or the ability to synthesize ATP. A [lambda]gtll cDNA library was prepared from rat brain poly A+ RNA and screened with oligodeoxynucleotide probes that correspond to the [alpha]-subunit of L-type voltage-sensitive calcium channels.   Eleven positive clones were detected that have cDNA inserts 1.6-5.5 Kb in length.  Nucleotide sequence analysis reveals strong homology as well as differences in the deduced amino acid sequences of the [alpha]-subunits of rat brain and rabbit skeletal muscle L-type voltage-sensitive calcium channels. To detect recombinant DNA clones that correspond to novel homeobox genes a Drosophila genomic DNA library was screened with multiple oligodeoxynucleotide probes, each designed to hybridize to multiple homeobox genes.  Five clones that gave positive signals with 2 or more oligodeoxynucleotide probes exhibited specificities that could not be explained on the basis of known nucleotide sequences of Drosophila homeobox genes.  Nucleotide sequence analysis of the homeobox regions of 4 clones revealed 4 new homeobox genes (NK-1, 2, 3, 4).  Two recombinant clones contained identical DNA inserts, each insert contained 2 new homeobox genes (NK-3 and NK-4).  The deduced amino acid sequence of the NK-1 homeobox exhibits the highest homology to the homeobox regions of deformed, zen-2, and zen-1 (75, 72, and 71% homology, respectively).  The relative homology of the NK-2 homeobox is as follows:   NK-4 > NK-3 > NK-1 = IAB-7.  The homology of NK-3 is: NK-2 > labial > NK-4 > NK-1; and NK-4 homology is NK-2 > zen 2 = NK-3 > labial.  Genomic DNA fragments from the 4 new homeobox genes were used to screen cDNA libraries prepared from poly A+ RNA from 0-3 hr Drosophila embryos or from 3 - 12 hr embryos.   One NK-1 cDNA clone was obtained from the 3-12 hr embryo cDNA library, but none was detected in the 0-3 hour embryo library. Comparison of the nuoleotide sequences of NK-1 cDNA and genomic DNA clones showed that the NK-1 gene has 3 exons.  One of the 2 introns detected resides within the homeobox region. Regulation of rat neuropeptide Y gene expression: Untreated PC12 rat pheochromocytoma cells and N18TG-2 mouse neuroblastoma cells possess relatively low basal levels of neuropeptide NPY (0.25 and 0.13 pg/Ug total RNA), while NG108-15 mouse neuroblastoma x rat glioma hybrid cells contain remarkably high amounts (11 pg/Ug RNA).  Untreated human neuroblastoma lines SK-BN-SH and SK-N-MC also contain relatively high amounts of NPY mRNA. During the past year we studied the regulation of NPY mRNA abundance in PC12 cells by cAMP, phorbol esters, glucocorticoids, and calcium ionophore.   The results are as follows: Cyclic AMP elevation by forskolin or 8-bromo-cAMP elicts moderate elevation (4-10-fold) over 12-48 hr of treatment and synergizes with phorbol ester. Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) that activate protein kinase C elicit little or no effect alone but synergize with cAMP to produce large elevations (20-200-fold) over 12-48 hr of treatment.  Responses to phorbol ester are enhanced by A23187, a calcium ionophore, which increases the cytoplasmic Ca ++ ion concentration. Nerve growth factor (NGF (2.5 S) strongly elevates NPY mRNA (40-l00-fold) during l-6 days of treatment. The increase is evident by as early as 3 hr of NGF exposure and is sensitive to cycloheximide, indicating a requirement for protein synthesis.  Phorbol ester synergizes with NGF to produce 300-fold elevations in NPY mRNA. Glucocorticoids such as dexamethasone (Dex) elicit 2-3-fold potentiations of the cAMP and phorbol-ester elicited elevations of NPY mRNA.  Dex biphasically modulates the stimulations by NGF, potentiating early (3-10 hr) effects of NGF but profoundly inhibiting the large stimulations at later times (l-6 days).  The latter inhibitory phase resembles previously described antagonisms by glucocorticoids of NGF inductions of specific mRNAs. We have looked for an effect of NGF treatment on the stability of NPY mRNA.  Upon addition of actinomycin D to inhibit RNA synthesis, NPY mRNA in control and NGF-treated cells decayed with half-lives of 5 and 9 hr, respectively. We conclude that part of the increase in NPY mRNA due to NGF is a consequence of increased NPY mRNA stability, but that transcriptional activation must also take place to account for the 40-100-fold elevation of NPY mRNA. Regulation of NPY mRNA levels also was observed in SK-N-MC human neuroblastoma cells, which have a high constitutive expression of the gene.  Dex, forskolin, or PMA have no effect alone, but Dex + forskolin and forskolin + PMA double the NPY mRNA level. Regulation of proenkephalin (pEnk) gene expression . Glucocorticoids and cAMP synergistically increase the abundance of pEnk mRNA in C6 rat glioma cells.  We examined the mechanism of this increase by run-on transcription experiments involving nuclei isolated from C6 cells treated with or without Dex and/or forskolin for l-24 hr. Dex alone had no effect on the pEnk transcription rate, and forskolin alone elicited a brief stimulation that reached 6-fold at 1 hr.  Dex + forskolin elicited a more sustained stimulation of 5-6 fold at 2-6 hr.  These results suggest that cAMP elevates pEnk mRNA in C6 cells by stimulating transcription and that glucocorticoids exert a permissive effect by sustaining the stimulation by cAMP. We are presently searching for a putative glucocorticoid regulatory element of the pEnk gene by transient expression assays of plasmids having portions of the pEnk gene linked to a reporter gene, chloramphenicol acetyltransferase (CAT).  Rat proenkephalin genomic clones were isolated and mapped. A fragment containing 2500 bases of the 5' upstream region and 47 bases of the first exon was ligated in both orientations in front of the CAT gene in a promoterless vector, and this construct was transfected into C6 rat glioma cells. CAT expression was found to respond as expected to forskolin but did not respond, either with or without forskolin, to Dex. Thus the putative element is not within the sequence-tested.  Since regulatory elements are sometimes found in introns, another construct that contains the above sequence plus the rest of the first exon and all of the first intron has been constructed and is being tested. We found that untreated SK-N-MC human neuroblastoma cells contain a surprisingly high abundance of pEnk mRNA.  Treatment of the cells with forskolin elevated the level 2-fold.  PMA + A23187 markedly reduced the level.  Dex did not affect basal or forskolin-stimulated levels but did increase the levels in the presence of the inhibitory combination forskolin + PMA + A23187.  Thus, cooperative regulation of the human pEnk gene by cAMP and glucocorticoids exists, as with the rat gene, but is quantitatively less significant in cells having a high constitutive expression of the gene. Cytoplasmic components of Ach receptor aggregates -- We previously showed that vinculin, [alpha]-actinin and filamin, all of which are cell adhesion associated proteins, are concentrated at the sites of newly formed nictonic acetylcholine (ACh) receptor aggregates.  However, the resolution of our techniques was not adequate to determine the localization of proteins within the aggregates.  We have devised a novel technique to overcome this problem.  Using a selective replating of myoblasts, large myotubes are grown with very few fibroblasts.  A coverslip coated with a purified polypeptide adhesive from shellfish is attached to the upper cell surface, where the ACh receptor aggregates have formed.  This coverslip is then lifted, with plasma membranes and partially disrupted myotubes attached,  allowing access of antibodies to the membrane cytoskeleton,  a high degree of spatial resolution in the plane of the membrane,  and minimal cytoplasmic background staining. With this technique, we have found that vinculin and associated actin filament bundles are adjacent to, but not superimposed upon the Ah receptor enriched domains of the aggregates, while a 43kilodalton protein (the \"43K protein\", closely associated with Ach receptors in other systems) and actin in another form are precisely colocalized with the receptors.  These results suggest the existence of at least 2 distinct membrane cytoskeleton domains within the newly formed ACh receptor aggregates. These membrane preparations also facilitated the precise localization of clathrin, the major protein of the coated vesicle \"basket\".  Clathrin is not obviously concentrated in ACh receptor aggregates and is excluded from the ACh receptor enriched domains, but most aggregates contain clathrin in the form of tiny speckles, possibly corresponding to coated pits involved in exocytosis or endocytosis. Immunogold localization of Ach receptors, the 43K protein and sodium channels.  We have succeeded in labeling 2 integral membrane proteins (ACh receptors and sodium channels) and the 43K protein at the electron microscopic level by a postembedding immunogold technique.  At both the neuromuscular junction (NMJ) and the ACh receptor aggregates formed in culture, the Ach receptors and the 43K protein are precisely colocalized.   Sodium channels are concentrated at the NMJ at a lower apparent density than ACh receptors or 43K protein.  However, unlike the ACh receptors, which are concentrated at the crests of the postsynaptic membrane folds close to the nerve ending, the sodium channels are distributed throughout the folds.  This result confirms, with higher resolution, results previously obtained with the immurioperoxidase technique. In cultured myotubes, ACh receptors and the 43K protein are also colocalized in apparently intracellular membrane compartments which remain to be characterized. Escherjcm coli the interaction of cAMP with the cAMP receptor protein (CRP) induces a conformational change in the structure of the protein thereby converting it to a form that is active in regulating gene expression.  This regulatory protein has been mutated to a form that functions in gene transcription in the absence of added cAMP.  It was the purpose of this study to isolate one of these mutant proteins, the NCR91 protein, crystallize it, and determine the X-ray crystallographic structure of the protein for comparison with that of the structure of the wild-type protein. Since the cAMP synthesizing system in Escherichia coli is regulated by the sugar transport system known as the phosphoenolpyruvate: sugar phosphotransferase system (the PTS), an interest in our laboratory has been to understand the various mechanisms by which the process of sugar transport is regulated.  One of the mechanisms that has been described in gram-positive bacteria for regulation of sugar transport is the process of inducer expulsion.  When Streptococcus pvoaenes are grown under conditions for the induction of the lactose transport system and then allowed to take up a nonmetabolizable substrate for this system (thiomethylgalactoside, TMG), the subsequent addition of glucose elicits the rapid release into the medium of the accumulated TMG.  In this organism, the uptake of TMG is via the PTS and it is presumed that the PTS is involved in the expulsion mechanism.  It was the purpose of this study to examine the mechanism of sugar transport regulation in an organism that transports sugars by a mechanism other than the PTS. A representative heterofermentative lactobacillus, Lactobacillus brevis was chosen for the study. The organisms used in the studies were Escherichia coli, Lactobacillus brevis and Lactobacillus buchneri.  Cloning the gene for the CRP protein from strain NCR91of Escherichia coli provided a basis for hyperexpression of the protein.  The hyperexpressed protein was purified by a conventional method to produce homogeneous CRP91.  Crystals of the protein were grown in the cAMP under the same conditions as for the wild-type protein (in the presence of phosphate buffer at room temperature).  Diffraction data on a single crystal were collected on a Nicolet imaging proportional counter and then processed on a VAX computer.  A difference Fourier map of the CRP91 and wild-type CRP proteins was calculated using the program PROTEIN.  Uptake of thiomethylgalactoside into Lactobacilli was measured using the radioactive sugar analog.  Pools of free or phosphorylated thiomethylgalactoside were measured in boiled extracts of cells which were then fractionated by anion exchange chromatography.  Cell-free extracts of Lactobacilli were prepared by sonication of cell suspensions.  Sugar phosphorylation in cell extracts was measured by incubating the extracts with radioactive thiomethylgalactoside or 2-deoxyglucose. The Crystal Structure of a cAMP-independent Mutatnt of the cAMP Receptor Protein: Escherichia coli NCR91 synthesizes a mutant form of the cAMP receptor protein in which alanine 144 is replaced by threonine.  This mutated form of CRP can, in the absence of adenylate cyclase, confer on cells the CRP* phenotype which is due to the ability of the mutated CRP to function as a transcription regultor in the absence of cAMP.  CRP91 has been purified and crystallized with cAMP under the same conditions as used to crystallize the wild-type CRP-cAMP complex.  X-ray diffraction data were measured to 2.4-Angstrom resolution and the CRP91 structure was determined using initial model phases from the previously determined wild-type structure.  A difference Fourier map calculated between CRP91 and the wild-type CRP showed the two alanine to threonine sequence changes in the dimer and also a change in the orientation of cysteine 178 in one of the subunits.  Refinement of the structure indicated that there were small differences in the CRP91 structure compared to that of the wild-type protein that included concerted motions in the small domains, in the hinge region between the two domains and in an adjacent loop between beta-strands 4 and 5.  These findings indicate that the mutation at residue 144 causes changes in the position of some protein atoms that are distal to the mutation site. Regulation of beta-Galactoside Transport and Accumulation in Heterofermentative Lactic Acid Bacteria. Lactobacillus brevis and Lactobacillus buchneri are examples of heterofermentative organisms.  They do not have a functional phosphoenolpyruvate: sugar phosphotransferase system and transport thiomethylgalactoside by an active transport mechanism that results in the accumulation of intracellular free thiomethylgalactoside.  When cells were preloaded with thiomethylgalactoside and then exposed to glucose, there was a rapid efflux of the intracellular galactoside.  When the glucose was depleted from the medium by uptake into the cells, then there was a restoration of the uptake and accumulation of thiomethylgalactoside.  The glucose-promoted efflux of thiomethylgalactoside required the intracellular phosphorylation of glucose.  The glucose-promoted efflux was not inhibited by iodoacetate.  These results were interpreted to indicate that a phosphorylated metabolite of glucose at or above the level of glyceraldehyde-3-phosphate was required to evoke displacement of intracellular thiomethylgalactoside from these cells.  Experiments using the counterflow technique indicated that exposure of cells to glucose converted the uptake of thiomethylgalactoside from an active uptake mechanism to a facilitated diffusion mechanism that allowed equilibration of thiomethylgalactoside between theintracellular and extracellular spaces.  Since this phenomenon in the heterofermentative Lactobacilli had similar characteristics to that of inducer expulsion that takes place in the homofermentative Streptococcus and Lactobacillus species, a possible similarity in the mechanism was explored.  The inducer expulsion mechanism in the homofermentative bacteria involves HPr, a protein component of the phosphoenolpyruvate: sugar phosphotransferase system which appears to serve also as a transport regulator.  Using complementation assays with strains of Staphylococcus that are deficient in HPr, it was established that Lactobacillus brevis extracts have HPr activity although this organism lacks a functional phosphoenolpyruuate: sugar phosphotransferase system.  This study is consistent with the idea that HPr can function as a sugar transport regulator independently of its role as a phosphocarrier in sugar transport.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3shm_8kbn-vbme", "00000000-0000-0000-F6A7-41D5443086C1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X314", "101584910X235", "1988", "September 1988", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "4", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: 01 HL 00009-14 Period Covered: October 1, 1987 - September 30, 1988 Title of Project: Cell Recognition and Synapse Formation Principal Investigator: Marshall Nirenberg, Chief, LBG, NHLBI Hemin Chin, Staff Fellow, LBG, NHLBI Li-Shan Hsieh, Visiting Fellow, LBG, NHLBI Wu-Hong Tsai, Visiting Fellow, LBG, NHLBI Maria Giovanni, Staff Fellow, LBG, NHLBI David Trisler, Guest Worker, LBG, NHLBI Dana Hilt, Staff Fellow, LBG, NHLBI Cooperating Units (if any): Bruce Schrier, LDN, NICHD Lab/Branch: Laboratory of Biochemical Genetics Section: Section of Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, Maryland 20892 Total Many Years: 10 Professional: 8 Other: 2 Summary Of Work: 1.  Treatment of NG108-15 neuroblastoma-glioma hybrid cells results in marked increases in the abundance of certain species of RNA.  Seventeen cDNA clones corresponding to these species of RNA were obtained and the nucleotide sequences of three clones were determined.  DNA clone NG-32 corresponds to mRNA for ATP synthase subunit 6, which is transcribed from the heavy chain of mitochondrial DNA and codes for a protein that is part of the H+ channel of the ATP-synthase complex.  Clone NG-10 DNA corresponds to another mitochondrial DNA of unknown function, which is transcribed from the light chain of mitochondrial DNA and may be involved in the initiation of replication of mitochondrial heavy strand DNA. 2.  Eleven cDNA clones mere obtained that correspond to mRNA for the [alpha]-subunit of the L-type voltage-sensitive calcium channel of rat brain. Analysis of the DNA sequence and the deduced amino acid sequence reveals strong homology between brain ad skeletal muscle calcium channel [alpha]-subunits.  Approximately 75% of rat brain [alpha]-subunit amino acid residues that were defined are either identical to the amino acid residues of rabbit skeletal calcium channel &subunit or are conservative amino acid replacements. 3. Four novel Drosophila homeobox genes NK-1, -2, -3, and -4 were cloned and partial nucleotide sequences were determined.  One NK-1 cDNA clone was obtained from a cDNA library prepared from poly A+ RNA from 3-12 hr Drosophila embryos, but none was detected in the 0 to 3 hr embryo library.  Six NK-3 cDNA clones were obtained from a library prepared from Drosophila poly A+ RNA from 0-3 hr embryos and 6 additional clones were obtained from a library prepared for 3-12 hr embryo poly A+ RNA.  The exon-intron structure of the NK-1 gene was determined.  One of the two introns found resides within the homeobox. Project Description Major Findings Previously, many neuroblastoma and related somatic hybrid cell lines were shown to acquire voltage-sensitive ion channels and other neuronal properties when intracellular cyclic AMP levels were elevated for a number of days.  Cells with elevated cAMP acquire new proteins such as the [alpha]-subunit of voltage-sensitive calcium channels and other proteins of unknown function.   A cDNA library was constructed from poly A+ RNA prepared from NG108-15 neuroblastoma-glioma hybrid cells that had been treated for 5 days with 1 mM dibutyryl cAMP.  The library was screened and 17 cDNA clones were obtained that correspond to species of RNA that are 3 to 40 times more abundant in cells treated with dibutyryl cAMP than in cells cultured without this compound.  Each cloned cDNA was used as a probe with Northern blots to determine the number of species of poly A+ RNA responsive to dibutyryl cAMP and the chain length of each species of RNA.  The results suggest that the 17 cDNA clones correspond to species of RNA transcribed from 10 genes.  Partial nucleotide sequences of the cDNA inserts from 3 clones were obtained.  Clone NG-32 corresponds to mouse mitochondrial mRNA for ATP synthase subunit 6, a mitochondrial gene.  This protein is part of the H+ channel portion of the mitochondrial ATP synthase complex.  Treatment of NGl08-15 cells with dibutyryl cAMP results in an 8-fold increase in the abundance of mRNA for this protein.  The nucleotide sequence of clone NG-10 cDNA was identified as part of the D-loop region of mouse mitochondrial DNA that contains the origin of replication for the heavy strand of DNA.  The 5'-terminal nucleotide sequence of some molecules of heavy strand mitochondrial DNA is known to consist of a short segment of RNA that is complimentary to a short light strand miochondrial DNA sequence nearby.  Hence, NG-10 cDNA may correspond to an RNA transcript of the light strand of mitochondrial DNA that serves as a primer for the initiation of heavy strand mitochondrial DNA synthesis.   Treatment of NG108-15 cells with dibutyryl cAMP results in a 40-fold increase in this species of RNA.  These results show that treatment of NG108-15 neuroblastoma-glioma cells with dibutyryl cAMP results in marked increases in the abundance of RNA transcripts from heavy and light strands of mitochondrial DNA.  Further work is needed to determine whether cAMP regulates mitochondrial biogenesis or the ability to synthesize ATP. A [lambda]gtll cDNA library was prepared from rat brain poly A+ RNA and screened with oligodeoxynucleotide probes that correspond to the [alpha]-subunit of L-type voltage-sensitive calcium channels.   Eleven positive clones were detected that have cDNA inserts 1.6-5.5 Kb in length.  Nucleotide sequence analysis reveals strong homology as well as differences in the deduced amino acid sequences of the [alpha]-subunits of rat brain and rabbit skeletal muscle L-type voltage-sensitive calcium channels. To detect recombinant DNA clones that correspond to novel homeobox genes a Drosophila genomic DNA library was screened with multiple oligodeoxynucleotide probes, each designed to hybridize to multiple homeobox genes.  Five clones that gave positive signals with 2 or more oligodeoxynucleotide probes exhibited specificities that could not be explained on the basis of known nucleotide sequences of Drosophila homeobox genes.  Nucleotide sequence analysis of the homeobox regions of 4 clones revealed 4 new homeobox genes (NK-1, 2, 3, 4).  Two recombinant clones contained identical DNA inserts, each insert contained 2 new homeobox genes (NK-3 and NK-4).  The deduced amino acid sequence of the NK-1 homeobox exhibits the highest homology to the homeobox regions of deformed, zen-2, and Zen-1 (75, 72, and 71% homology, respectively).  The relative homology of the NK-2 homeobox is as follows:  NK-4 > NK-3 > NK-1 = IAB-7.  The homology of NK-3 is: NK-2 > labial > NK-4 > NK-1; and NK-4 homology is NK-2 > zen 2 = NK-3 > labial.  Genomic DNA fragments from the 4 new homeobox genes were used to screen cDNA libraries prepared from poly A+ RNA from 0-3 hr Drosophila embryos or from 3-12 hr embryos.   One NK-1 cDNA clone was obtained from the 3-12 hr embryo cDNA library, but none was detected in the 0-3 hour embryo libary. Comparison of the nucleotide sequences of NK-1 cDNA and genomic DNA clones showed that the NK-1 gene has 3 exons.  One of the 2 introns detected resides within the homeobox region. Significance of the Results 1.  The demonstration of dibutyryl cAMP dependent regulation of 2 species of mitochondrial RNA raises questions that can be addressed in future studies; namely, does  cAMP regulate mitochondrial biogenesis or the ability to synthesize ATP? 2.  Voltage-sensitive calcium channels are known to play a central role in stimulus-secretion coupling and signal transmission both within cells and between cells.  The DNA clones that were obtained for the [alpha]-subunit of rat brain L-type voltage-sensitive calcium channels can be used as probes to explore the mechanisms that regulate the [alpha]-subunit gene.  The cloned DNA also can be used to direct the synthesis of the [alpha]-subunit protein of the dihydropyridine-sensitive calcium channel from brain.  Site directed mutagenesis can be used to alter the DNA and explore the relation between calcium channel structure and function. 3. Four novel Drosophila homebox homeobox genes were cloned and partially sequenced.  The homeobox family of genes code for proteins that regulate the expression of genes during development and some determine pathways of differention. These genes provide an experimental system that can be used to define the mechanisms that regulate the expression of these homeobox genes as well as regulation of gene expression by homeobox proteins. Publications 1.  Bray/P., Carter, Guo, V., Puckett, C., Kamholz, J., Spiegel, A., and Nirenberg, M.:  Human cDNA clones for an [alpha] subunit of Gi Signal-transduction protein. Proc. Natl. Acad. Sci.. USA 84, 5115-5119 (1987). 2. Fitzpatrick, L.A., Chin, H., Nirenberg, M., and Aurbach G.D.: Antibodies to an [alpha] subunit of skeletal muscle calcium channels regulate parathyroid cell secretion. Proc. Natl. Acad. Sci., USA 85, 2115-2119, (1988). 3. Carter, A., Bardin, C., Collins, R., Simons, C., Bray, P., and Spiegel, A.:  Reduced expression of multiple forms of Gs-[alpha] in pseudohypotarathyroidism type IA. Proc. Natl. Acad. Sci.. USA 84, 7266-7269 (1987). 4. Brown, D.A., and Higashida, H.:  Voltage- and calcium-activated potassium currents in mouse neuroblastoma x rat glioma hybrid cells. J of Physiology 397: 149-165 (1988). 5. Brown, D.A. and Higashida, H.:  Membrane current responses of NG108-15 mouse neuroblastoma x rat glioma hybrid cells to bradykinin. J. of Physiology 397: 167-184 (1988). 6. Brown, D.A. and Higashida, H.:  Inositol 1,4,5-trigphosphate and diacylglycerol mimic bradykinin effects on mouse neuroblastoma x rat glioma hybrid cells. J. of Physiology 397: 185-207 (1988). 7. Higashida, H.:  Acetylcholine release by bradykinin, inositol 1,4,5-trisphosphate and phorbol dibutyrate in rodent neuroblastoma cells. J. of Physiology 397: 209-222 (1988). 8. Trisler, D., and Collins, F.:  Corresponding spatial gradients of TOP molecules in the developing retina and optic tectum. Science 237: 1208-1209 (1987).", "Nirenberg, Marshall W. ; Kim, Yongsok ; Tsai, Wu-Hong ; Giovanni, Maria ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Trisler, G. David ; Hsieh, Li-Shan ; Hilt, Dana ; Chin, Hemin", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mjbr~wzr2~eqez", "00000000-0000-0000-AE7A-2C6614C32B57", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Jeffrey Denberg to Marshall W. Nirenberg", "101584910X315", "101584910X316", "1986", "2 May 1986", "Indicative of Nirenberg's prominence in the neurobiological community, Denburg asks for Nirenberg's help in filling a position in Neurobiology at the University of Iowa.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Jeffrey Denberg and the University of Iowa, Department of Biology.", "Copyright may apply", null, null, "May 2, 1986 Dear Dr. Nirenberg: The enclosed notice, which will appear in Science and Neuroscience Newsletter, describes a tenure-track position in Neurobiology.   The rank and salary for this opening are negotiable. Candidates for this position should have demonstrated expertise in cellular, molecular and developmental neurobiology.  In addition to establishing a vigorous research program, the successful applicant will be expected to develop strong research ties with the members of the Department of Biology as well as with other members of the neuroscience community.  Currently there are over 40 faculty members who work on different aspects of neuroscience in the University of Iowa.  The new faculty member will be able to draw graduate students from two sources:  the Department of Biology and the interdepartmental Neuroscience Ph.D. Program. The new appointee will have an excellent opportunity to participate in the Neurobiology Program Project which has been receiving undisrupted funding from NIH since it was first established in the Department of Biology in 1979.  The projects in the Program include:  development of identified neurons in culture; neuronal death during metamorphosis; neurosecretory control of molting hormones; genetic dissection of ionic channel functions, generation of rhythmic pattern in the CNS; development of specific connectivities and recognition molecules. We will appreciate your help in filling this position with an outstanding scientist, either by posting the enclosed announcement or by putting me in touch with an interested candidate.  Applications will be considered as they are received, until the position is filled.  However, we expect to begin the interview process by early May.  The University of Iowa is an Affirmative Action/Equal Opportunity Employer. Sincerely yours, Jeffrey Denburg", "Denberg, Jeffrey ; University of Iowa", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7dnx_pjvs_jqw2", "00000000-0000-0000-CEDD-DE8D2CFD5901", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Neurobiology Tenure Track Position", "101584910X316", "101584910X315", "1986", "2 May 1986", null, "Announcements", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Jeffrey Denberg and the University of Iowa, Department of Biology.", "Copyright may apply", null, null, "NEUROBIOLOGY-TENURE-TRACK POSITION The Department of Biology, University of Iowa, invites applications for an anticipated tenure-track faculty position, rank and salary negotiable. Any neurobiologist whose research interest is in an area of cellular, molecular or developmental neurobiology may apply.  Preference will be given to persons whose talents complement those of current faculty and whose interests will facilitate interaction among existing research programs. Postdoctoral experience is required.  In addition to establishing a strong research program, the successful applicant will be expected to participate in undergraduate and graduate teaching.  Applications will be considered as they are received until the position is filled. Send curriculum vitae, brief description of research goals, relevant publications and arrange to have three letters of recommendation sent to: Dr. C.-F. Wu, Search Committee, Department of Biology, University of Iowa, Iowa City, IA 52242. The  University of Iowa is an Affirmative Action/Equal Opportunity Employer.", "University of Iowa. Department of Biology", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9spm.3mxk-apds", "00000000-0000-0000-5F91-FD09284A93C3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Annual Report of the Laboratory of Biochemical Genetics [summary of laboratory projects]", "101584910X318", "101584910X317", "1993", "September 1993", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "October 1, 1992 through September 30, 1993 NK-2 Homeobox Gene. The first known step in the zygotic development of part of the Drosophila CNS is the expression of the NK-2 gene.  During the past year, proteins that regulate the expression of the NK-2 gene were identified by determining the expression of the NK-2 gene in embryos with mutations in various genes.   The results show that the NK-2 gene is activated in the ventral half of the embryo, presumably by dorsal protein, which is distributed in nuclei in a ventral-dorsal concentration gradient.  The NK-2 gene is-activated but not expressed in the most ventral horizontal stripe of nuclei, the mesodermal anlage, due to repression by snail, a zinc finger protein, or in the adjacent horizontal stripe of nuclei, the mesectodermal anlage, due to repression by single-minded and Enhancer of split m8, which are basic,  helix-loop-helix proteins.  However, the NK-2 gene is expressed by nuclei in the ventral half of the ventrolateral neurogenic anlage early in Prosopbila embryonic development as the nuclei undergo commitment to the neuroblast pathway of differentiation, or soon thereafter. Initially, the NK-2 gene is expressed fairly uniformly in a horizontal stripe of nuclei about 7 nuclei in width on each side that extends over 90% of the length of the embryo. During gastrulation, the horizontal stripe of cells expressing NK-2 is converted to 12 vertical stripes by repression of the NK-2 gene in some cells. Later, 26 clusters of cells that express the NK-2 gene are formed on each side, presumably by repression of the NK-2 gene in additional cells. Therefore, 2 clusters of neuroectodermal cells that synthesize NK-2 RNA are formed per hemisegment that are precursors of many neuroblasts in the ventral nerve cord. Twenty high-affinity and 13 low-affinity NK-2 binding sites were found in 2.2 kb of DNA from the 5 -upstream region of the NK-2 gene, which suggests that NK-2 protein may be required to maintain the expression of the NK-2 gene. Putative sites for other proteins that overlap or are adjacent to the NK-2 protein binding sites were found.  The conversion of neuroectodermal cells to neuroblasts is accompanied by activation of the snail gene in the neuroblasts, thereby repressing activation of the NK-2 gene by dorsal protein.  The results suggest that the NK-2 gene receives and integrates information from the ventral-dorsal and anterior-posterior gradients of gene regulators that is needed to generate a pattern of clusters of neuroectodermal cells that synthesize NK-2 RNA that are precursors of different types of neuroblasts. One of the major goals in neurobiology is to understand how the nervous system is assembled. Studies on the NK-2 homeobox gene led to some novel ideas and to a hypothesis which predicts the overall strategy of the gene program (that is the rules) for the early development of part of the CNS of Drosophila. Every aspect of the hypothesis can be tested experimentally using the NK-2 gene. With a slight modification the hypothesis also applies to the assembly of part of the mammalian CNS. Circular dichroism measurements and 1D NMR spectra showed that the tm for denaturation of the NK-2 homeodomain, NK-2H, is approximately 25 C at pH 4.4 and that denaturation is fully reversible.  NK-2H was found to have relatively little [alpha]-helical content.   No dramatic change in the CD spectra was observed on addition of an oligodeoxynucleotide with a high-affinity NK-2 binding site.   NK-2H uniformly enriched with 15N was examined by 2D and 3D NMH.   The results suggest that NK-2H has a novel homeodomain secondary structure. Genes Expressed In The Developing Nervous System.  Transposition of a P-element that contains the [beta]-galactosidase gene from 1 site in the Drosophila genome to another yielded many transgenic fly lines that express [beta]-galactosidase only in the nervous system during embryonic development. The developmental time and location of [beta]-galactosidase expression then is determined by regulatory signals of the genes that contain the inserted [beta]-element DNA. DNA flanking the P-element insertion sites were cloned from 15 of the most interesting transgenic fly lines and corresponding cDNA clones were obtained and were sequenced partially. Clone 393C-2 was shown to encode Drosophila high-mobility-group protein D (HMG-D), a DNA binding protein.  A homologous mammalian protein, HMG-1, recognizes DNA conformation rather than nucleotide sequence; HMG-1 binds to cruciform DNA and to DNA with axial distortion due to cisplatin. The functions of HMG-1 and HMG-D proteins have not been identified; however, the proteins are thought to play a role in chromatin structure. Also the HMG domain has been found in many DNA binding proteins that regulate transcription.  We find that the HMG-D gene is expressed ubiquitously during early embryonic development but later in development is expressed exclusively in the nervous system. The homozygous P-element insertion is a lethal mutation and is accompanied by striking morphologic defects in the central nervous system. Clone 367C-3 DNA corresponds to a gene that encodes a novel zinc finger protein that is expressed in the CNS and anterior sensory organs.  The homozygous P-element insertion is a lethal mutation that results in extraordinary morphologic defects in the ventral nerve cord of developing embryos.  Clone 7D3C-1 corresponds to a novel Drosophila gene that encodes a member of the kinesin heavy chain gene family.  Kinesin functions as a molecular motor for axonal fast transport of organelles or cell membranes on microtubule tracks from soma of neurons towards axon tips. Clone 314-4C-2 encodes a protein that is similar to the human QM protein, an apparent suppressor of Wilm's tumor, a pediatric nephroblastoma.  Sequence analysis of cDNAs from other transgenic fly lines suggest that the cDNAs correspond to novel genes expressed in the nervous system. Mammalian Homeobox and POU-Domain Genes.  Approximately 8 kb of a novel mouse homeobox gene, NKx-1, a homolog of the Drosophila NK-1 homeobox gene, was sequenced.  The amino acid sequences of the NKx-1 and NK-1 homeodomains differ by only 3 of the 60 amino acid residues.  Both proteins also contain an acidic domain. However, most of the other regions of the protein that have been defined differ markedly. NKx-1 poly A+ RNA was found to be most abundant in 10-day mouse embryos; the abundance progressively decreases thereafter. Northern analysis of poly A+ RNA from adults revealed 1 major band of NKx-1 poly A+ RNA in brain and trace bands in RNA from testes or spleen. The NK-1 gene is expressed in discrete regions of 14-day old mouse embryo mesencephalon and myelencephalon and also in spinal cord, vertebrae, and ribs. A mouse genomic DNA library was screened with oligodeoxynucleotide probes for novel homeobox genes.  Seventy-two positive recombinants were cloned. Thus far five novel homeobox genes have been found.  Restriction site analysis of the 72 clones revealed additional classes of clones that have not yet been sequenced. In addition, novel POU-domain genes related to Brain-3 POU-domain cDNA were cloned from mouse and human genomic DNA and were sequenced. Two additional, novel human POU-domain genes related to Ott-3 were cloned and the POU-domain regions were sequenced. Sites of expression of Brain-l, Brain-2, Brain-4, and SKIP POU-domain genes in the mouse nervous system were determined by in situ hybridization as a function of mouse embryo developmental age and also were defined in the adult mouse. Hox 4.1 cDNA and genomic DNA were cloned and the complete Hox 4.1 open RNA reading frame was sequenced. Regulation of a Calcium Channel [alpha]-1 Subunit Gene.   The [alpha]-l subunit of a voltage-sensitive calcium channel previously was shown to be inducible in NG108-15 cells and the expression of the gene was shown to control the ability of the cells to form synapses with striated muscle cells. The 5 -upstream regulatory region of the calcium channel gene was cloned and sequenced. A nucleotide sequence was found that is a powerful activator or an enhancerless chloramphenicol acetyltransferase reporter gene. A protein was found in NG108-15 nuclei that specifically binds to the activating sequence. A cDNA expression library in [lambda]gtll was screened for recombinants that direct the synthesis of proteins that bind to the nucleotide sequence and 35 positive clones were obtained. Seven kinds of clones were found that encode proteins that bind to oligonucleotides with appropriate sequence specificity but differ in specificity for double-stranded DNA, or (+) or (-) single-stranded DNA. Further work is needed to determine whether one or more of these proteins regulate the expression of the Ca2+ gene channel. Enhancer and Promotor Selection.  During the past year further work has been done on the selective amplification of DNA clones that contain enhancer or promoter nucleotide sequences that activate gene expression.  The method is based on the observation that the synthesis of polyoma virus DNA in mouse cells requires viral enhancer sequences that also are required for the synthesis of mRNA from polyoma genes. Mouse genomic DNA fragments were ligated to polyoma DNA that lack the enhancer region of the virus. The E. coli origin of replication and [beta]-lactamase gene also were inserted in the polyoma coat protein gene.   Promoters or enhancers in the mouse genomic DNA inserts that activate plasmid DNA synthesis in mouse cells are able to replicate and hence are selectively amplified; whereas, plasmids that lack functional enhancer sequences do not replicate.  Plasmid DNA was harvested from mouse cells that had been transfected and incubated for several days.   Recovered DNA then was amplified in E. coli. The selection method is highly effective; some clones were shown to increase in abundance more than 100,000-fold.  Fragments of the recovered DNA inserts were shown to bind proteins from nuclei and to activate the expression of an enhancerless chloramphenicol acetyltransferase reporter gene.  Previously, cDNA clones were obtained that encode proteins that specifically bind to oligonucleotide sequences that were identified by the oligonucleotide selection method.  Partial sequences of some of the cDNA clones were obtained. Differentiation of Excitable Membranes and Myofibrils of Striated Muscle. Ventral horn neurons from fetal rats or mice were cocultured with rat striated myotubes.  Neurites induce accumulation of acetylcholine receptors on the myotube surface where neuritis contact the muscle cells and also reduce the concentration of acetylcholine receptors where neurites contact a pre-existing patch with a high receptor density. Steps in the assembly of functional triads in cultured skeletal myotubes were studied by calcium imaging, immunocytochemistry, and electron microscopy. Developing triads and punctate structures containing ryanodine receptors (calcium release channels) and dihydropyridine receptors were found in 3-day old myotubes. Excitation-contraction coupling was found in cells with only a few of these structures. Changes in the maximal calcium release and uptake rates were observed during the course of myotube development that were correlated with the elaboration of the sacroplasmic reticulum around the myofibrils. Polyclonal antibodies were obtained directed against a protein from fetal pig brain that induces the formation of acetylcholine receptor aggregates on skeletal muscle cells in culture. The antibodies immunoprecipitate all acetylcholine receptor aggregating activity in crude fractions of brain and spinal cord extracts as well as in purified preparations and recognize a protein with a molecular weight of 120,000. Genes Associated with Programmed Cell Death.  During embryonic development, normal cell turnover, and other physiological and pathological processes, cells die by the activation of a mechanism of self-destruction, termed apoptosis or programmed cell death. The available information shows that apoptosis often requires gene activation and the synthesis of proteins presumably needed for cell death. Radiation and chemotherapeutic agents also elicit cell death by apoptosis.  Since little is known about the genes or biochemical mechanisms involved in apoptosis, a study was initiated to identify genes involved in apoptosis in radiation-sensitive fetal rat brain neuroblasts following radiation treatment.  It is anticipated that among the genes activated by irradiation will be genes required for neuroblast apoptosis, either for the triggering or for the execution of this process. Fetal 17 day-old rats were irradiated (or sham-irradiated) with sublethal  neutron/gamma irradiation in utero, and RNA isolated from brains 5 and 24 hours later was used to construct a subtractive cDNA library enriched in sequences of transcripts increased by irradiation.  Clones from this library were screened and analyzed by differential colony and Northern blot hybridization. At least 76 out of 682 analyzed clones were found to represent transcripts increased in abundance by irradiation, generally by a factor of 2-3 but much greater for at least one transcript.  Sequencing of the inserts of these clones is in progress.  Some clones were found to have previously unreported insert DNA sequences, while others represent ubiquitin, ferritin light chain, 12.3 protein (related to G-[beta] proteins) and subunits of mitochondrial NADH dehydrogenase. These results indicate that a variety of transcripts are increased moderately during radiation-induced neuroblast cell death. Additional more strongly activated genes, which are expected in the triggering of cell death, are being sought currently. Sequence and Characterization of the Hox A7 Gene.  The Hox A7 gene is a mouse gene characterized by the presence of a homeobox region typical of the Antennapedia class of proteins found in Drosophila.  Starting with a clone derived from mouse genomic DNA, the complete sequence of the structural gene, as well as upstream and downstream regulatory regions, were determined. A variety of studies have led to the assignment in this sequence of the probable transcription start site as well as likely regions for the interaction with proteins that regulate transcription. Site-specific Mutagenesis of Presumptive ATP Binding Sites in Escherichia coli Adenylyl Cylcase.  The region of the enzyme bounded by Alanine 190 and Arginine 197 was studied as a probable region for substrate binding. Site-directed mutagenesis was carried out on Lysine 196 by replacing this residue with a variety of other amino acids. It was found that replacement with other basic amino acids led to retention of some activity, while replacement with other classes of amino acids generally led to abolition of activity. The conclusion from these studies is that the charge in the vicinity of Lysine 196 is crucial for enzymatic activity. Sequence and Organization of a Monocistronic ptsH Operon in Mycoplasma. A sequencing project, designed to isolate genomic clones for the enzymes of the transport system known as the phosphoenolpyruvate: sugar phosphotransferase system (PTS), was undertaken.  Several clones were isolated that allowed the determination of the sequence of the gene encoding the PTS phosphocarrier protein known as HPr as well as of flanking regions upstream and that the gene encoding HPr (the ptsH gene) was located in an operon that is monocistronic.  This is in marked contrast to the organization of all previously sequenced ptsH genes in other organisms where the ptsH gene is found in a polycistronic operon.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7pt7_26eq_y7s6", "00000000-0000-0000-AAF0-FFB7EB206BFA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Cell Recognition and Synapse Formation\"", "101584910X319", "101584910X317", "1993", "September 1993", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 00009-19 LBG Period Covered: October 1, 1992 - September 30, 1993 Title of Project: Cell Recognition and Synapse Formation Principal Investigator: P.I.: Marshall Nirenberg, Chief, LBG, NHLBI Others: Lan-Hsiang Wang, Staff Fellow LBG, NHLBI Dervla Mellerick-Dressler, Staff Fellow, LBG, NHLBI Hsui-Ming Saunders, Prat Staff Fellow, LBG, NHLBI Hsi-Ping Li, Visiting Associate, LBG, NHLBI Zheng-Mei Liu, Visiting Associate, LBG, NHLBI Rebecca Chmelik, Biologist, LBG, NHLBI Norma Heaton, Chemist, LBG, NHLBI Cooperating Units (if any): D.H.H. Tsao, LBC, NHLBI J. Gruschus, LBC, NHLBI J.A. Ferretti, LBC, NHLBI Lab/Branch: Laboratory of Biochemical Genetics Section: Section on Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, MD Total Man Years: 6.5 Professional: 5.5 Other: 1.0 Summary of Work: A. NK-2 Homeobox Gene. The NK-2 homeobox gene is expressed in nuclei in the ventral half of the ventrolateral neurogenic anlage very early in the development of part of the CNS of Drosophila. The distribution of NK-2 RNA in various mutants was determined to identify genes that regulate NK-2 expression. Four genes were found that encode DNA binding proteins that regulate NK-2 gene expression. In addition, the 5-flanking region of the NK-2 gene was shown to contain many binding sites for NK-2 protein, which suggests that NK-2 protein may be required to maintain NK-2 gene expression. These results suggest that the NK-2 gene receives and integrates information from the ventral-dorsal and anterior-posterior gradients of gene regulators to generate a pattern of clusters of neuroectodermal cells that synthesize NK-2 RNA and are precursors of different types of neuroblasts. The NK-2 homeodomain was shown by NMR to have a novel secondary structure. B. Gene Expression in the Developing Nervous System. A Drosophila gene was found that encodes a novel zinc finger protein that is restricted to the CNS. Homozygous P-element insertions are lethal and are accompanied by massive morphological defects in the ventral nerve cord. Another cDNA clone was identified as a DNA binding protein which is widely distributed during early embryonic development, but is expressed exclusively in the nervous system during later embryonic development. Another cDNA clone was found that corresponds to a Drosophila gene that encodes a novel member of the kinesin heavy chain gene family. Other cDNAs were found that correspond to genes that encode novel proteins that are specifically expressed in the developing nervous system. Cell Recognition And Synapse Formation Project Description Major Findings NK-2 Homeobox Gene. The first known step in the zygotic development of a considerable portion of the Drosophila CNS is the expression of the NK-2 gene. During the past year, proteins that regulate the expression of the NK-2 gene were identified by determining the expression of the NK-2 gene in embryos with mutations in various genes.  The results show that the NK-2 gene is activated in the ventral half of the embryo, presumably by dorsal protein, which is distributed in nuclei in a ventral-dorsal concentration gradient.  The NK-2 gene is activated but not expressed in the most ventral horizontal stripe of nuclei, the mesodermal anlage, due to repression by snail, a zinc finger protein, or in the adjacent horizontal stripe of nuclei, the mesectodermal anlage, due to repression by single-minded and Enhancer of split m8, which are basic, helix-loop-helix proteins.  However, the NK-2 gene is expressed by nuclei in the ventral half of the ventrolateral neurogenic anlage early in Drosophila embryonic development as the nuclei undergo commitment to the neuroblast pathway of differentiation, or soon thereafter. Initially, the NK-2 gene is expressed fairly uniformly in a horizontal stripe of nuclei about 7 nuclei in width on each side that extends over 90% of the length of the embryo. During gastrulation, the horizontal stripe of cells expressing NK-2 is converted to 12 vertical stripes by repression of the NK-2 gene in some cells. Later, 26 clusters of cells that express the NK-2 gene are formed on each side, presumably by repression of the NK-2 gene in additional cells. Therefore, 2 clusters of neuroectodermal cells that synthesize NK-2 RNA are formed per hemisegment that are the precursors of many neuroblasts in the ventral nerve cord. Twenty high-affinity and 13 low-affinity NK-2 binding sites were found in 2.2 kb of DNA from the 5'-upstream region of the NK-2 gene, which suggests that NK-2 protein may be required to maintain the expression of the NK-2 gene. Other putative sites for proteins that overlap or are adjacent to the NK-2 protein binding sites were found.  The conversion of neuroectodermal cells to neuroblasts is accompanied by activation of the snail gene in the neuroblasts, thereby repressing activation of the NK-2 gene by dorsal protein. The results suggest that the NK-2 gene receives and integrates information from the ventral-dorsal and anterior-posterior gradients of gene regulators, which is needed to generate a pattern of clusters of neuroectodermal cells that synthesize NK-2 RNA that are precursors of different types of neuroblasts. One of the major goals in neurobiology is to understand how the nervous system is assembled. Studies on the NK-2 homeobox gene led to some novel ideas and to a hypothesis which predicts the overall strategy of the gene program (that is the rules) for the early development of part of the CNS of Drosophila.  Every aspect of the hypothesis can be tested experimentally using the NK-2 gene. With a slight modification the hypothesis also applies to the assembly of part of the mammalian CNS. Circular dichroism measurements and 1D NMR spectra showed that the tm for denaturation of the NK-2 homeodomain, NK-2H, is approximately 25 C at pH 4.4 and that denaturation is fully reversible. NK-2H was shown to have relatively little [alpha]-helical content. No dramatic change in the CD spectra was observed on addition of an oligodeoxynucleotide with a high-affinity NK-2 binding site.  The results show that NK-2H has an unusual homeodomain secondary structure. Genes Expressed In The Developing Nervous System.  Transposition of a P-element that contains the [beta]-galactosidase gene from 1 site in the Drosophila genome to another yielded many transgenic fly lines that express [beta]-galactosidase only in the nervous system during embryonic development. The developmental time and location of [beta]-galactosidase expression then is determined by regulatory signals of the genes that contain the inserted P-element DNA. DNA flanking the P-element insertion sites were cloned from 15 of the most interesting transgenic fly lines and corresponding cDNA clones were obtained and were sequenced partially. Clone 393C-2 was shown to encode Drosophila high-mobility-group protein D (HMG-D), a DNA binding protein. A homologous mammalian protein, HMG-1, recognizes DNA conformation rather than nucleotide sequence; HMG-1 binds to cruciform DNA and to DNA with axial distortion due to cisplatin. The functions of HMG-1 and HMG-D proteins have not been identified; however, the proteins are thought to play a role in chromatin structure. Also the HMG domain has been found in many DNA binding proteins that regulate transcription. We find that the HMG-D gene is expressed ubiquitously during early embryonic development but later in development is expressed exclusively in the nervous system. The homozygous P-element insertion is a lethal mutation and is accompanied by striking morphologic defects in the central nervous system. Clone 367C-3 DNA corresponds to a novel gene that encodes a zinc finger protein that is expressed in the CNS and anterior sensory organs.  The homozygous P-element insertion is a lethal mutation that results in extraordinary morphologic defects in the ventral nerve cord of developing embryos.  Clone 7D3C-1 corresponds to a novel Drosophila gene that encodes a member of the kinesin heavy chain gene family.  Kinesin functions as a molecular motor for axonal fast transport of organelles or cell membranes on microtubule tracks from soma of neurons towards axon tips.  Clone 314-4C-2 encodes a protein that is similar to the human QM protein, an apparent suppressor of Wilm's tumor, a pediatric nephroblastoma.  Sequence analysis of other cDNA clones suggest that the cDNAs correspond to novel genes that are expressed in the nervous system.", "Nirenberg, Marshall W. ; National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Wang, Lan-Hsiang ; Mellerick-Dressler, Dervla ; Heaton, Norma ; Li, Hsi-Ping ; Chmelik, Rebecca ; Liu, Zheng-Mei ; Saunders, Hsui-Ming", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-76be.gjp2-5558", "00000000-0000-0000-2A03-0F4E21C1D243", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Laboratory project: \"Regulation of Gene Expression\"", "101584910X320", "101584910X317", "1993", "September 1993", null, "Reports, Excerpts", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Project Number: Z01 HL 0155-01 LBG Period Covered: October 1, 1992 - September 30, 1993 Title of Project: Regulation of Gene Expression Principal Investigator: P.I.: Marshall Nirenberg, Chief, LBG, NHLBI Others: Sadamitsu Asoh, Visiting Associate, LBG, NHLBI Bruce Bethke, IRTA, LBG, NHLBI Yoshinobu Hara, Visiting Associate, LBG, NHLBI Adil Nazarali, Visiting Associate, LBG, NHLBI Alessandra Rovescalli, Visiting Fellow, LBG, NHLBI Dong-Ping Tan, Visiting Associate, LBG, NHLBI Wu-Hong Tsai, Visiting Fellow, LBG, NHLBI Vicki Guo, Chemist, LBG, NHLBI Cooperating Units (if any): Scott Young, LCB, NIMH Adam Hartman, LCB, NIMH Yongsok Kim, LMC, NHLBI Lab/Branch: Laboratory of Biochemical Genetics Section: Section of Molecular Biology Institute and Location: NHLBI, NIH, Bethesda, MD Total Man-Years: 8.5 Professional: 7.5 Other: 1.0 Summary of Work: A. Mouse Homebox And POU-Domain Genes.  A novel mouse homebox gene NKx-1 was cloned an approximately 8 kb was sequenced.  The deduced amino acid sequence of the NKx-1 homeodomain differs from Drosophila NK-1 homeodomain by only 3 of 60 amino acid residues.  Both NKx-1 and NK-1 proteins contain an acididc region before the homeodomain.  Northern analysis revealed one major band of NKx-1 poly A+ RNA in brain and trace bands in testes and spleen.  The abundance of NKx-1 poly A+ RNA is highest in 10 day mouse embryos and progressively decreases thereafter.  In situ hybridization with sections of 14-day mouse embryos revealed NKx-1 RNA in the mesencephalon, myelencephalon, spinal cord, vertebrae, and ribs.  Five additional novel mouse homebox genes were cloned and partially sequenced.  A novel mouse POU-domain gene related to Brain-3 POU-domain cDNA was cloned and about 3 kb was sequenced.  The expression of Brain-1, Brain-2, Brain-4 and Skip POU-domain genes in the mouse nervous system was determined by in situ hybridization as a function of developmental age. B. Regulation of a Calcium Channel a-1 Subunit Gene.  A nucleotide sequence was found in the 5-upstream regulatory region of a voltage-sensitive calcium channel [alpha]-1 subunit gene that is a powerful activator of an enhancerless chloramphenicol acetyltransferase reporter gene.  A cDNA expression library was screened for recombinants that direct the synthesis of proteins that bind this nucleotide sequence.  Seven kinds of clones were found that encode proteins that bind to oligonucleotides with appropriate sequence specificity. C. Promotor And Enhancer Selection. cDNA clones were obtained that encode proteins that specifically bind to oligonucleotide sequences by a method that selects for gene regulatory sequences.  Partial sequences of the cDNA clones were obtained. Regulation Of Gene Expression Project Description Major Findings Mouse Homeobox And POU-Domain Genes.  Approximately 8 kb of a novel mouse homeobox gene, NKx-1, a homolog of the Drosophila NK-1 homeobox gene, was sequenced. The amino acid sequences of the NKx-1 and NK-1 homeodomains differ by only 3 of the 60 amino acid residues present. Both proteins also contain an acidic domain. However, most of the other regions of the protein that have been defined differ markedly. NKx-1 poly A+ RNA was found to be most abundant in 10-day mouse embryos; the abundance progressively decreases thereafter. Northern analysis of poly A+ RNA from adults revealed 1 major band of NKx-1 poly A+ RNA in brain and trace bands in RNA from testes or spleen. The NKx-1 gene is expressed in discrete regions of the 14-day old mouse embryo mesencephalon and myelencephalon and also in spinal cord, vertebrae, and ribs. A mouse genomic DNA library was screened with oligodeoxynucleotide probes for novel homeobox genes.  Seventy-two positive recombinants were cloned. Thus far five novel homeobox genes have been found.  Restriction site analysis of the 72 clones revealed additional classes of clones that have not yet been identified. In addition, a novel mouse POU-domain gene related to Brain-3 POU-domain cDNA was cloned and 3 kb was sequenced.  Sites of expression of Brain-l, Brain-2, Brain-4, and SKIP POU-domain genes in the mouse nervous system were determined by in situ hybridization as a function of mouse embryo developmental age and also were defined in the adult mouse.  Hox 4.1 cDNA and genomic DNA were cloned and the complete Hox 4.1 open reading frame was sequenced. Regulation of a Calcium Channel [alpha]-l Subunit Gene.  The [alpha]-1 subunit of a voltage-sensitive calcium channel previously was shown to be inducible in NG108-15 cells and the expression of the gene was shown to control the ability of the cells to form synapses with striated muscle cells. The 5'-upstream regulatory region of the calcium channel gene was cloned and sequenced. A nucleotide sequence was found that activates an enhancerless chloramphenicol acetyltransferase reporter gene.  A protein was found in NG108-15 nuclei that specifically binds to this sequence. A cDNA expression library in [lambda]gtll was screened for recombinants that direct the synthesis of proteins that bind to the nucleotide sequence and 35 positive clones were obtained.  Seven kinds of clones were found that encode proteins that bind to oligonucleotides with appropriate sequence specificity but differ in specificity for double-stranded DNA, or (+) or (-) single-stranded DNA. Further work is needed to determine whether one or more of the proteins regulate the expression of the Ca2+ gene channel. Enhancer and Promotor Selection.  During the past year further work has been done on the selective amplification of DNA clones that contain enhancer or promoter nucleotide sequences that activate gene expression.  The method is based on the observation that the synthesis of polyoma virus DNA in mouse cells requires viral enhancer sequences that also are required for the synthesis of mRNA from polyoma genes. Mouse genomic DNA fragments were ligated to polyoma DNA that lack the enhancer region of the virus. The E. coli origin of replication and [beta]-lactamase gene also were inserted in the polyoma coat protein gene.  Promoters or enhancers in the mouse genomic DNA inserts that activate plasmid DNA synthesis in mouse cells are able to replicate and hence are selectively amplified; whereas, plasmids that lack functional enhancer sequences do not replicate.  Plasmid DNA was harvested from mouse cells that had been transfected and incubated for several days.   Recovered DNA then was amplified in E. coli. The selection method is highly effective; some clones were shown to increase in abundance more than 100,000 fold.  Fragments of the recovered DNA inserts were shown to bind nuclear protein and activate the expression of an enhancerless chloramphenicol acetyltransferase reporter gene.  Previously, cDNA clones were obtained that code for proteins that specifically bind to oligonucleotide sequences that were identified by the oligonucleotide selection method.  Partial sequences of some of the cDNA clones were obtained.", "Nirenberg, Marshall W. ; Bethke, Bruce,Tsai, Wu-Hong,Rovescalli, Alessandra,Tan, Dong-Ping,National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics,Hara, Yoshinobu,Nazarali, Adil J. (Adil Jafferali), 1954-2017,Asoh, Sadamitsu,Guo, Vicky", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cghm_8azb-r43a", "00000000-0000-0000-61D9-C1035F0559FA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Summary of Dr. Nirenberg's Research", "101584910X322", null, "1991", "21 November 1991", "This narrative covers the main areas of research in Nirenberg's career for a thirty year period from about 1960 through 1991.  Major areas of study mentioned include the early study of DNA, RNA, proteins, and the genetic code, work in the field of developmental biology with the establishment and study of cell lines, and his early work with Drosophila genes.", "Narratives (document genres)", null, "Biographical Information", "2", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "NG ence Weare SUMMARY OF DR. NIRENBERG'S RESEARCH Dr. Nirenberg and his coworkers studied the steps that relate DNA, RNA, and protein. They showed that messenger RNA is required for protein synthesis and that synthetic oligo - and polyribo nucleotides have messenger activity. Over a five year period they deciphered the genetic code. Dr. Nirenberg then began to work in the field of developmental neurobiology. He and his colleagues established many clonal lines of mouse neuroblastoma cells and somatic hybrid cells that express neural properties in culture, and cell lines were found that form synapses in culture with striated muscle cells. The cell lines were given to many investigators and now are widely used as model systems for studies in neurobiology and related fields. Nirenberg and his coworkers found that synaptogenesis and other neural properties are regulated by cAMP. Prolonged elevation of cellular cAMP levels shifts the cells to a more differentiated state that enables the cells to form synapses. cAMP was found to activate the expression of a gene for an L-type voltage-sensitive calcium channel a-1 subunit that is required for stimulus-secretion coupling at synapses. The calcium channel gene was cloned and sequenced and factors that regulate the expression of this calcium channel gene, which have long term effects on the efficiency of transynaptic communication, are being studied currently. In addition, Dr. Nirenberg and his coworkers recently have cloned and sequenced Drosophila or mouse genomic DNA or cDNA for 10 novel homeobox genes or Pou box-homeobox genes. These genes code for proteins that bind to DNA and thereby regulate gene expression. Current studies center on defining the functions of these genes during the development of the nervous system. Dr. Nirenberg has received many honors and awards including the Nobel Prize in Medicine.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-s5hu-n7dt.eiiy", "00000000-0000-0000-973B-C0F9E7030A31", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Molecules Localized at the Neuromuscular Synapse", "101584910X323", null, "1983", "18 May 1983", "These notes are from the period when Nirenberg studied the development of synapses between neurons and muscles.  A list of eleven molecules including acetylcholine and various antibodies are listed and some characterized.", "Laboratory notes", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "SHO | ho Re OTE: eee 1 RAR tu ROS WS, |e 5 a3 [erotonek SEH | a part oan sped od 4 _ : ! Y \\\\ Ap +o. Say A f- t eH | & pe ) , 58 BH = haha ys G MASS AW Datayoun * 10PLV     i. G ho | . \\\\ ~ \\\\ <-. _ a . \\\\ i \". * . ; A - 2 — 4 . : « “ \" - é K, ‘ D", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bb32.89t7-2ug3", "00000000-0000-0000-F6A7-5EFCB0D91074", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Takehiko Amano to Marshall W. Nirenberg", "101584910X325", null, "1987", "8 January 1987", "Amano provides an update on his latest research and informs Nirenberg that he is planning on returning to the United States to attend courses at the Cold Spring Harbor Laboratory. He also requests another position in Nirenberg's lab.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Isamu Sojyo and the Mitsubishi Chemical Corporation.", "Copyright may apply", null, null, "January 8, 1987 Dear Dr. Nirenberg: It was a great pleasure to talk with you at Bethesda Last Fall.  I am now planning to change my research directions with molecular biological techniques and also with postnatal neurons in tissue culture.  Somehow we managed to present the first paper at the last neuroscience meeting entitled \"beta-NCF gene expression in glial cells\". This year I am planning to take the neurobiology courses at the Cold Spring Harbor Lab. this summer.   After the courses, I am looking for the laboratory in which I can learn some things and also have possible collaboration with molecular biology people. I wonder whether you could accept me in your laboratory for several months.  I can pay most of the expenses for my stay at N.I.H. I would greatly appreciate if you consider the matter seriously. With best regards Takehiko Amano", "Amano, Takehiko", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-pxr9.abmu~a3py", "00000000-0000-0000-2595-9AEE60764328", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Joe Marwah to Marshall W. Nirenberg", "101584910X326", null, "1987", "9 January 1987", "Marwah invites Nirenberg to speak at the National Science Foundation sponsored joint Indo-USA meeting on \"Neuroreceptor Plasticity and Brain Function\" to be held in New Delhi.  The letter includes a short handwritten postscript.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Joe Marwah.", "Copyright may apply", null, null, "January 9, 1987 Dear Dr. Nirenberg: I am writing to let you know that the National Science Foundation has partly sponsored a joint Indo-USA meeting entitled \"Neuroreceptor Plasticity and Brain Function\" to be held in New Delhi, India, April 6-10, 1987.  This is an official invitation to you to be a speaker at this meeting.  Your round trip airfare (economy and by American carriers only) between your point of origin and New Delhi will be covered by the NSF.  The All-India Institute of Medical Sciences, through Dr. Sharma and the Indian government, will cover your hotel, food, and transportation expenses in New Delhi. If you are willing to accept this invitation, please immediately send me a confirmation, together with a title for your talk (45 minutes to 1 hour).  The proceedings of this meeting will most likely be published as a monograph.  At this time, please note that all American Nationals travelling to India are required to have a valid U.S. passport, as well as a visa, from the Indian High Commission or Consulate (New York/Washington, D.C., etc.). I look forward to hearing from you. Best wishes, Joe Marwah, Ph.D. Associate Professor [Handwritten postscript:] P.S. Dr Sharma wrote me, saying that you may be available.  Please confirm.", "Marwah, Joe", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-gvdi~tgt6.z7wg", "00000000-0000-0000-F00B-C135A6753CEA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from David Schiff to Marshall W. Nirenberg", "101584910X327", null, "1986", "January 1986", "Schiff, Director of Newborn Nurseries and Professor of Pediatrics, Obstetrics, and Gynecology at the University of Alberta Hospitals, thanks Nirenberg for providing him with cell lines and supporting his research.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of David Schiff.", "Copyright may apply", null, null, "Dear Dr. Nirenberg: Thank you very much for the supply of N-115 cells that you sent to us.  They arrived a little over 48 hours after you sent them and are thriving extremely well.  I really appreciate the support that you have given us in this endeavor. I did not receive any invoice with respect to the cost of shipping the cells.  I would appreciate it if you would forward such an invoice so that we can reimburse you for this cost. With very best wishes for the new year. Yours sincerely, D. Schiff, M.D., Ph.D., FRCPC Director Newborn Nurseries Professor of Pediatrics, Obstetrics & Gynecology University of Alberta Hospitals", "Schiff, David", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8i9k-nm9f~ssz7", "00000000-0000-0000-A0A5-9CA90F04D169", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Stephen Thesleff to Marshall W. Nirenberg", "101584910X328", null, "1986", "17 December 1986", "Thesleff invites Nirenberg to participate in a Swedish symposium on \"The Neuromuscular Junction,\" designed to gather leading scientists in a number of fields for a \"coordinated discussion\" of the different aspects of neuromuscular function.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Stephen Thesleff.", "Copyright may apply", null, null, "December 17, 1986 Dear Dr. Nirenberg, On behalf of the Organizers of the Eric K. Fernstroms Symposium on The Neuromuscular Junction, I hereby cordially invite you to participate in this symposium.  The preliminary program and list of invited speakers are enclosed. Recent years have brought about an abundance of information about processes such as transmitter release, the acetylcholine receptor, acetylcholine-receptor interactions, excitation-contraction coupling and the genetic regulation of synapse formation and maintenance and degenerative changes resulting from neuromuscular disorders.  Even if many of these subjects have recently been discussed at various meetings it seems appropriate to gather leading scientists in those fields for a coordinated discussion of all these aspects of neuromuscular function. They Symposium will take place during the Midsummer Season at Orenas Castle (35 km from Lund) beautifully situated by the Sound (Oresund), and overlooking the coast of nearby Denmark (see enclosed brochure).  It will be organized as an official satellite symposium preceding the European Society for Neurochemistry Seventh General Meeting in Goteborg June 12-17 1988.  In addition, the XIXth Nordic Conference of Physiology and Pharmacology will be held in Oslo, Norway, June 13-15 1988.  Information regarding this latter congress can be obtained from Professor Harald Aars, University of Oslo, Box 1052, Blindern, 0316 Oslo 3, Norway. We can guarantee to cover the costs of your stay at Orenas and are applying for grants to cover your travel expenses as well (air fare, economy class).  Information about your travel support will be given not later than July 1987.  No registration fee is requested and all participants will be accommodated in modern hotel rooms at the Castle.  Each speaker will be asked to submit an Abstract before January 1, 1988 (Abstract forms will be sent separately).  Your contribution will be published as a full length paper in the Fernstrom Symposium series by Elsevier Biochemical Press of which you will receive a free copy. We are very much looking forward to receiving your, hopefully positive, answer as soon as possible. Sincerely yours, Stephen Thesleff", "Thesleff, Stephen,Eric K. Fernström Symposium", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-eiib-8bqb~d62j", "00000000-0000-0000-2EF2-AFCD3EDD6C1E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Shiro Takashima to Marshall W. Nirenberg", "101584910X329", null, "1988", "2 December 1988", "Takashima discusses his study of neuroblastoma, and following the advice of Dr. Mary Glick of the Children's Hospital, requests some clonal cell lines for use in research.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Shiro Takashima.", "Copyright may apply", null, null, "December 2, 1988 Dear Dr. Nirenberg, We are interested in the study of excitability of neuroblastoma, particularly in Na and Ca fluxes using the patch clamp method and Ca-fluorescence microscopy.  I consulted Dr. Mary Glick of the Children's Hospital with regard to this problem and she suggested me to contact you either by telephone or in writing. To my opinion, the clones N1E115 and NG 108-15 may be best suited for the purpose of our experiments.  Dr. Glick told me that although she has both of them, perhaps, the best thing to do is to obtain these cells from you directly. If you are kind enough to give these cells to us, we will be most grateful.  Since Philadelphia is not far from Bethesda, we are able to come to your lab on a specified day and bring them back to our laboratory.  I am looking forward to hearing from you.   In the mean time, Sincerely yours, Shiro Takashima Professor", "Takashima, Shiro", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ycsc.26y3_vtgw", "00000000-0000-0000-44F0-0ECD133CD9B6", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Dominick Purpura to Marshall W. Nirenberg", "101584910X330", null, "1985", "31 October 1985", "In this letter Purpura suggests, \"Directing your study to the general neuroscience community that reads Brain Research [a publication devoted to molecular studies] and its various sections should greatly enhance the awareness of investigators interested in brain mechanisms to the important new field of molecular neurobiology.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Dominick Purpura.", "Copyright may apply", null, null, "October 31, 1985 Dear Dr. Nirenberg: I was quite interested in the study you presented at the recent Society for Neuroscience meeting in Dallas.  As you know Brain Research will initiate a new section, Molecular Brain Research, with a distinguished Editorial Advisory Board.  The first issue of this new journal is scheduled for early 1986.  It is my impression that the subject of your study would be suitable for publication in Molecular Brain Research.  I hope that when you have prepared the study for publication you will submit it for review by our Editorial Board for consideration of publication in Molecular Brain Research.  Directing your study to the general neuroscience community that reads Brain Research and its various sections should greatly enhance the awareness of investigators interested in brain mechanisms to the important new field of molecular neurobiology. Sincerely, Dominick P. Purpura Editor", "Purpura, Dominick", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wcaf.fzja~73xq", "00000000-0000-0000-8B81-168D1F1D583F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Paul Greengard to Marshall W. Nirenberg", "101584910X332", null, "1979", "10 July 1979", "Greedgard invites Nirenberg to participate in the Fourth International Congress on Cyclic Nucleotides in Brussels, where over one thousand scientists were expected to convene.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Paul Greengard.", "Copyright may apply", null, null, "July 10, 1979 Dear Marshall: The 4th International Congress on Cyclic Nucleotides is to be held in Brussels from July 22-26, 1980.  At a recent meeting of the Organizing Committee, a tentative program was set up for the Congress.  I proposed your name as one of the speakers and it was enthusiastically and unanimously accepted by all the members of the Organizing Committee.  You should be receiving a letter from Professor Jacques Dumont of the Free University of Brussels formally inviting you to participate.  However, I am writing this informal letter of invitation on behalf of and at the request of the Organizing Committee to indicate how pleased we would be if you were to accept this invitation. It is anticipated that more than one thousand scientists will attend this meeting.  The proposed program for the neurobiology symposium includes Drs. Bockaert, Benzer, Hamprecht, you and myself.  I know that a presentation of your beautiful work, of the sort you gave at the Gordon Conference, would have a most exciting impact on the Congress. I greatly enjoyed our discussions at the Gordon Conference and should be in touch with you within a few weeks concerning the collaborative program. I do hope that within a short period of time we will have greatly improved the methodology for quantitative analysis of Protein I. Thanks again for your excellent contribution to the Gordon Conference. With best wishes, Yours sincerely, Paul Greengard Professor of Pharmacology", "Greengard, Paul, 1925-", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-f8yp.432p.kk5h", "00000000-0000-0000-EEE4-A99AF5DAFC18", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Bimal K. Bachhawat to Marshall W. Nirenberg", "101584910X333", null, "1984", "18 September 1984", "Bachhawat asks Nirenberg if he can provide an opportunity for P.K. Sarkar, of the Indian Institute of Chemical Biology, to do some collaborative work in Nirenberg's lab as a visiting scientist.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Sept 18, 1984 Dear Marshall, It was indeed a pleasure to meet you during my last visit to USA.  This letter is to request you, if possible, to provide an opportunity to Dr P.K. Sarkar, one of the senior scientists of the Institute, to do some collaborative work in your laboratory as a Visiting Scientist. The main result of the work from Dr Sarkar's laboratory here established that in developing rats and chicks, thyroid hormones promote brain maturation by eliciting an induction of tubulin.  They now have probes (cDNAS for alpha- and beta-tubulin which are being employed to test the effects of the hormone on transcription of tubulin mRNA.  Dr Sarkar and his colleagues developed a new procedure for the isolation of protoplasmic astrocytes and are currently using monoclonal antibodies to cell surface proteins to fractionate various subclasses of neurons. Based on the effect of thyroid hormones on fractionated neuronal and glial cells, they have shown in manuscript just communicated that contrary to the expectation, the glial cells are the target cells for the hormone and the neurons are virtually insensitive to induction of tubulin by thyroid hormones.  Most interestingly, chase experiments show that under the influence of the hormone, there is a translocation of tubulin from glial to neuronal cells at the time of brain maturation presumably to satisfy the large need of this protein for neuronal differentiation and synaptogenesis. Dr Sarkar, whose CV and list of publications are enclosed, desires to pursue further research on the hormone induced translocation of tubulin using cultures and cocultures of neuronal and glial cells.  He is also interested to work on mechanisms of regulation of synaptogenesis. In view of your interest in the area of brain maturation during development, I do believe that such collaboration would be of great help to Dr Sarkar and his efforts to develop the Neurobiology unit here.  Dr Sarkar wishes to spend about a year at NIH beginning April, 1985.  I would appreciate if you please let me know your opinion in the matter including the possibility of financial support for Dr Sarkar. With kind regards, Sincerely yours, Bimal K. Bachhawat", "Bachhawat, Bimal K.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2hrf.gb58~2d9a", "00000000-0000-0000-0F43-17F92E22A8DF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Uriel Littauer to Marshall W. Nirenberg", "101584910X334", "101584910X335", "1978", "4 October 1978", "Littauer invites Nirenberg to the Satellite Symposium on Biochemical Development of Nervous Tissue in Culture at the Weizmann Institute of Science in Israel.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Uriel Littauer.", "Copyright may apply", null, null, "October 4, 1978 Dear Marshall, Just a short note to tell you how delighted we will be to see you again in Israel.  I believe that the Satellite Symposium on Biochemical Development of Nervous Tissue in Culture, as well as the following congress will be of great interest and I do hope you will find the time and energy to come. Please also note that we do have plans to ask you to participate in the Symposium on Developmental Neurobiology which will take place in Jerusalem within the framework of the 13th FEBS Meeting, 1980. These are all the invitations in stall and I wish you and Perla a Happy New Year. Best wishes Sincerely yours, Uriel Littauer", "Littauer, Uriel ; The Weizmann Institute of Science", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3ef9.pvae.wd7h", "00000000-0000-0000-906D-CC311D157B72", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from the local organizing committee of the Bat-Sheva Symposium to Marshall W. Nirenberg", "101584910X335", "101584910X334", "1978", "4 October 1978", "Littauer invites Nirenberg to the Satellite Symposium on Biochemical Development of Nervous Tissue in Culture at the Weizmann Institute of Science in Israel.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Reproduced with permission of Abraham Shahar.", "Copyright may apply", null, null, "BAT-SHEVA SYMPOSIUM ON BIOCHEMICAL DEVELOPMENT OF NERVOUS TISSUE IN CULTURE AUGUST 26-31, 1979 Dear Dr. Nirenberg, We are organizing a Satellite Symposium to the VII International Meeting of the International Society for Neurochemistry which is being held in Jerusalem, Israel, September 2-7, 1979. The Symposium will be held at the Weizmann Institute of Science, Rehovot, Israel, from August 26 to August 31, 1979. The first three days (August 26-28) of the symposium will consist of a workshop on methodology of nerve and muscle cultivation, to be attended by 20 students.  The remaining three days (August 29-31) will be devoted to lectures by invited speakers and poster sessions.  The symposium will focus on (a) neuronal development and synapse formation in culture (plasticity and cell recognition, neurotransmitter mechanisms, synaptogenesis, epigenetic factors during development; (b) neuronal-glial interactions (primary glial cultures, hybrids, neural tumor cell lines; (c) Myelin formation and demyelination in vitro (biochemistry of myelin formation, pathology and immunobiology of demyelination). Because of your significant contributions, we would feel honored to have you participate as an invited speaker to deliver a lecture on the subject of biochemical development of nervous tissue in culture. The local organizing committee will cover expenses for the period of the meeting.  Although we are at this moment unable to promise any additional financial assistance, we are exploring the possibility of allocating a part of our budget for partial reimbursement of travel expenses. The proceedings of the symposium will be published as one volume of a series entitled \"Advances in Developmental Neuroscience\", edited by Ezio Giacobini and Antonia Vernadakis, and published by S. Karger A.G., Basel. We would be honored if you would accept our invitation and would be thankful if you could let us have your response by the end of October, 1978. Yours sincerely, Dr. Ezio Giacobini Dr. Abraham Shahar Dr. Antonia Vernadakis", "Giacobini, Ezio ; Shahar, Abraham ; Vernadakis, Antonia", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fhf8_a9wn-jk5r", "00000000-0000-0000-8338-580F38DD8A25", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from University of Colorado's Special Events Committee to Marshall W. Nirenberg", "101584910X336", null, "1969", "14 May 1969", "Coffin invites Nirenberg to speak at the University of Colorado's Summer Lecture Series while he attends the Neuroscience Conference.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of the University of Colorado at Boulder.", "Copyright may apply", null, null, "May 14, 1969 Dear Dr. Nirenberg: I have just been talking with Miss Kathryn Cusick at the Neuro-Science Center in Brookline, Massachusetts and she tells me that you will be on campus during the Neuro-Science Conference, July 20 to August 8, 1969. On behalf of Dean J.R. Little, Associate Dean of the Faculties, Secretary of the University and of the Board of Regents, and the Special Events Committee of which I am the Administrative Assistant, we would like to extend an invitation to you to speak on our Summer Lecture Series. Each summer we invite persons of eminence in various academic and professional fields to deliver scholarly but non-technical lectures to a general audience of University faculty, students, and citizens of the Boulder-Denver area.  The lecture topic should be of your own choosing relating to some phase of your work in genetics, biochemistry, etc.  In addition to giving such a lecture, and attending the Conference, if you should find time to also speak informally to one or more of our graduate seminars, our students and faculty would welcome such an opportunity. We would be pleased to have you accept an honorarium of $750 for this lecture, and we would pay your round trip air transportation from Bethesda to Denver.  We would be happy to house you as a guest of the University for one or two nights, either on campus in one of our new residence halls, or at the Harvest House. We have an open date of July 23 in the 1969 Summer Lecture Series if this would fit into your plans.  Also, we appreciate having our lecturers furnish us with a manuscript for publication in our non-profit University journal, The Colorado Quarterly, which is sent to the libraries of all the high schools in Colorado, as well as to the junior colleges, community colleges, senior colleges, and other universities in the state.  This further provides a lasting value for the individual lectures and we do keep a file in the Archives of this University. I am enclosing a copy of last summer's Lecture Series together with a statement of University policy concerning public lectures, and a tape release form which we hope you will sign and permit the University to use for non-commercial and classroom purposes. I hope you will not object to my calling you on Monday, May 19 (301-496-5208) to see what your reaction is to our invitation.  If you prefer, please call me collect (303-443-2211 X7425).  Thank you for your consideration of our proposal. Sincerely yours, (Mrs.) Mildred W. Coffin, Adm. Ass't., Special Events Committee", "Coffin, Mildred W. ; University of Colorado. Special Events Committee", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ercu-mrm9_q34z", "00000000-0000-0000-52DF-E87A0C7F8B5A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from George F. Wilgram to Marshall W. Nirenberg", "101584910X337", null, "1969", "9 April 1969", "Wilgram invites Nirenberg to be the main speaker at the genetics symposium to be held at the American Academy of Dermatology.  He suggests that Nirenberg would \"do genetics, dermatology, and dermatologic genetics a great service\" if he accepted.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of George F. Wilgram.", "Copyright may apply", null, null, "April 9, 1969 Dear Doctor Nirenberg: This note is written in hopes that you would be willing to accept an invitation to be the main speaker at the genetics symposium to be held at the forthcoming meeting of the American Academy of Dermatology on Saturday, December 6th in Miami, Florida. The American Academy of Dermatology is the largest national organization in this specialty.  I have been asked to organize the yearly symposium on genetics in dermatology, and I am making strong efforts not only to keep the field alive, but also to improve its research and teaching performance. You would do genetics, dermatology, and dermatologic genetics a great service if you were in a position to accept our invitation. The Academy would be happy to defray all your travel expenses and to offer an honorarium of $200.00. The topic of your talk would be left up to you, but your work and views on the genetic code would be just fine. Hoping that you might accept this invitation, I remain Sincerely yours, George F. Wilgram, M.D., Ph.D. P.S.  The first letter sent to you on March 24 apparently went astray in the mail.", "Wilgram, George F. ; New England Medical Center Hospital", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jrv5.scj4-2vmw", "00000000-0000-0000-D2AD-D5F7B5436686", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Biographical Sketch", "101584910X338", null, "1993", "22 August 1993", "This biographical sketch includes handwritten corrections and additions in covering five areas of Nirenberg's work.  1. Maintaining and managing creative environments in the Biochemical Genetics laboratory; 2. Pursuing and directing a vigorous research program; 3. Maintaining an active role in the profession; 4. Serving on committees at NIH and elsewhere; and 5. Supporting NIH equal employment opportunity programs.", "Resumes (personnel records)", null, "Biographical Information", "9", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "ONE-PAGE BIOGRAPHICAL SKETCH FOR PERMANENT NIH INTRAMURAL SCIENTIST               NAME TITLE BIRTHDATE & PLACE CITIZENSHIP Marshall Nirenberg Chief, Lab. of Biochemical Genetick 4-10-27 New York USA institute /Division /Laboratory OFFICE (Bidg./Room No.) LABORATORY (Bidg./Room No.) NHLBI/Lab. of Biochemical GeneticB 36/1C-27 4C-20   EDUCATION and TRAINING: - Years institution Degree Disciptine   1952-57 Univ. of Mich.Ann Arbor Ph.D. Biochemistry 1950-52 Univ. of Florida M.S. Biology 1945-48 Univ. of Florida B.S. Biology and Chemistry       CHRONOLOGY OF EMPLOYMENT (Limited to space provided) 1966.Now | Chief, Laboratory of Biochemical Genetics, NHLBI 1962-66 Chief, Section on Biochemical Genetics, NHLBI 1960-62 Research Biochemist, Section of Metabolic Enzymes, NIAMD 1957-60 Postdoctoral Fellow with Dewitt Stetten, Jr. (2 yrs.), Gordon Tomkins (3rd year)     RESEARCH INTERESTS AND ACCOMPLISHMENTS (Limited to space provided) Interest. Regulation of gene expression. Neurobiology, Genetics, Drosophila,Neuroblastoma. Accomplishments. Deciphered.the genetic code. Showed that RNA is a template for protein Syiithesis. Established clonal lines of neuroblastoma and somatic hybrid cells that form Synapses in vitro as model systems for neurobiology. Discovered 10 novel homeobox genes and 2 novel Pou-domain genes.   IMPORTANT AND RECENT PUBLICATIONS (Limited to 5) Total Number of Publications: 159 1. Hara, Y., Rovescalli, A., Kim, Y., and Nirenberg, M.: Structure and Evolution of Four (i992) Genes Expressed in Mouse Brain. Proc. Natl. Acad. Sci., USA 89: 3280-3284 1992). 2. Nazarali, A., Kim, Y., and Nirenberg, M.: Hox 1.11 and Hox-4.9 Homeobox Genes. Proc. Natl. Acad. Sci., USA 89: 2883-2887 (1992). 3. Kim, Y. and Nirenberg, M.: Drosophila NK-homeobox Genes. Proc. Natl. Acad.Sci. 7716-7720 (1989). 4. Nirenberg, M. W. and Leder, P.: RNA codewords and protein synthesis. I. The effect of 5 yinu leotides upg the binding OF BRN to yibosomes. P-free’ pede eaneee (1964). Ee colt Gon natura ty oceuzring or synthetic polyribonuc ESEigegtepe synphesis in, Sci. USA, 47: 1588-1602 ———         AWARDS, HONORS, EDITORSHIPS, AND COMMITTEES (Examples limited to space provided) Nobel Prize in Medicine or Physiology (1968). Shared with H.G. Khorana and R.W.Holley. Member National Academy of Science. Member National Academy of Medicine. Editoria] Advisory Board - Molecular Neurobiology. Eqitoria Advisory Board —- Cotecyaa and Mobecul ay Neurobiology Editorial Board - Molecular Neurobiology               | DATE FORM COMPLETED: August }2, 1993 SIGNATURE Watt bb. Trendrer~ L/L ft? = Dr. Marshall Nirenberg (1992-1993) CRITICAL ELEMENT fi: Maintains and Manages a creative environment in the Laboratory of Biochemical Genetics. Encouragement of Seminars and Journal Clubs. Laboratory seminars are held Monday, Tuesday, and Wednesday. The most recent interesting papers that have been published are presented at the Monday noon seminar. The Tuesday 4:00 PM seminar is for Dr. Nirenberg’s group. This is a highly informal seminar with no scheduled speakers. Each person discusses the experimental results that he or she obtained during the previous week, technical problems encountered, methods, and so forth. Ideas also are discussed. The Wednesday noon seminar series focuses on the research of the investigators in the laboratory. Each person gives a one hour talk on the research that he or she has done over the past 4-6 months, answers questions, and considers critical comments. n j j ff Progress and Encourage Collaborations where Adviseable. Usually, Dr. Nirenberg discusses results of the current or last experiment with each postdoctoral fellow each day and also discusses plans for the next experiment or set of experiments. However, the frequency of discussion often is tailored to the needs of the postdoctoral fellow and the experimental difficulties that are encountered. Large projects usually are designed as collaborative projects; i.e., different postdoctoral fellows work on separate but related parts of the same problem. In addition, A projects are being studied in collaboration with investigators in other laboratories at the NIH. Additional discussions also are held with the other permanent, independent investigators in the laboratory on the results of their research and their plans for future research. Effective Performance of Support Staff. The performance of support staff is monitored closely and suggestions are made as needed. Problems are discussed with Dr. Nirenberg and are monitored until they are solved. Dr. Nirenberg’s technicians ¢+—full-tiis—2ohe- ere chbicioms) are effective and productive. A medical problem has impaired the effectiveness of Dr. Daniel’s technician, but this problem should be resolved soon,   diener in the lab, Derek Tang, from Viet Nam, Nirenberg vooe fe oie with £.E.0. and affirmative action objectives. es de ot AE we he ba eer taught many technigal procedures used in molecular biology, and now is doing      research for of each day, which should enable him to obtain a promotion. Afro-Americans in the laboratory also have been taught many technical procedures.   . Dr. Nirenberg makes sure that all members of the laboratory conserve resources in a responsible manner. Years ago, to save money some policies were instituted in this laboratory which are still being followed; ca. because of the expense no one in this laboratory uses NIH sterile glassware or thessefithses—cfi-the NIH media room. The NIH Art Department is used very infrequently; figures are drawn on the computer by members of the laboratory. C   howevre-—Monte Sa bare re comoute acj o5-are—tsedrather than the NIH—> IY Ap as because ees A actin TEESE ae ovcelient. ANI : © orders are approved by Dr. Nirenberg or Dr. Peterkofsky prior to placement. Computer printouts of ee aed are examined each month by Dr. Nirenberg and appropriate actions are taken needed, Dr. Marshall Nirenberg (1992-1993) CRITICAL ELEMENT 2: PURSUES AND DIRECTS A VIGOROUS RESEARCH PROGRAM. Publications. Three manuscripts were written that were supposed to be in press by now but have been delayed a few months because several months ago Dr. Nirenberg thought it was more important for him to-focus intensively on some research concerning the pattern of expression of the NK-2 homecbox gene during the development of Drosophila embryos. This analysis led to predictions,-—~ whteh—have_abigh—prebabitityeftbetrg-eorkect. of the rules for assembling part of the central nervous system of Drosophila and explains how positional information that d ES the structure of the nervous system is—enceded in _ DNA and_retrieved! he eas will change the direction of Dr. Nirenberg’s esearch for the n 3 or 4 years. prediction can be teste experimentally us as the experimental system.4\\\\These ideas represent a major advance in Dr. renbdeérg s undeé anding of how the nervous system is assembled. With a slight modification the hypothesis also applies to the assembly of part of the mammalian CNS.                                           Dr. Nirenberg was a coauthor of 7 abstracts that were presented as posters or talks at the following meetings: Society for Cell Biology, Society for Neuroscience meeting, Neurobiology of Drosophila meeting at Cold Spring Harbor, Biophysical Society meeting, Eastern Analytical Symposium, and the Third Forum on Peptides and Proteins, Biarritz, France. Dr. Nirenberg also presented a symposium talk at the International Meeting on the Biochemistry of Cell Membranes, sponsored by the International Union of Biochemistry and Molecular Biology in Bari, Italy. Peer Recognition. Was invited to select the speakers and cochairperson for a symposium on Drosophila Neurobiology and to be the Chairman of the symposium at the International Congress of Biochemistry in New Delhi in 1994. Was invited to be a plenary speaker at the European Pharmacology Society Meeting in Berlin in 1994. Was a member of the Organizing Committee and an invited lecturer for the Elba International Neuroscience Meeting in Marina di Campo, Isola d'Elba, Italy for 1993. Was invited to be a symposium speaker at a meeting “DNA: The Double Helix, Forty Years: Perspective and Prospective” sponsored by the New York Academy of Science. Invited lecturer at the Department of Biochemistry, College of Medicine, University of Illinois at Chicago, Illinois and Georgetown University. Many other invitations to give talks were not accepted. Member of the Editorial Advisory Board, Member of the Editorial Advisory Board of Cellular and Molecular Neurobiology, Member of the Editorial Board, Journal of Neurogenetics. Adjunct Professor, Department of Biochemistry, George Washington Medical School, Washington, D.C. Member of an American College of Neurology Committee to choose the recipient of a major award for research. influence on Research Projects. Essentially all of the research objectives of Dr. Nirenberg’s group, past and present, originated from Dr. Nirenberg. The ideas for 2 of the 3 collaborative projects that Dr. Nirenberg currently is working on were originated by Dr. Nirenberg. The idea for the third collaborative project in collaboration with a former postdoctoral fellow of Or. Nirenberg’s was equally obvious to Dr. Nirenberg and the other investigator. SPAR HP Depa Oe, EN. MOGs As |) pede oy v L r) iw Ay KLf “<i, 4 2 a fj ; < <a % ALL ee Dr.. Marshall Nirenberg (1992-1993) CRITICAL ELEMENT (#2: PURSUES AND DIRECTS A VIGOROUS RESEARCH PROGRAM.               Wier 2 oe int on i IteATO ON han BU riled ye?) th + tN 2 a — = 4 2) /, >i = cub tsa ak eas ae TTS “s os = | een ob e. ee wr maha aed 4 0) tb I FOMETF IF) S OPA I WAAL) $ A tos 4.7       Dr. Nirenberg was a coauthor of 7, posters ( or talks thetowett—efescrted at the following meetings: Society for Cell Biology, Society for Neuroscience meeting, Neurobiology of Dresephila meeting at Cold Spring Harbor, Biophysjcah Society meet , Eastern Analytica] Symposium, and the g jo« é oY y 7 . E ~rre€ . , Foe On - Or. Ntrenbétg~also resen symposium talk at anes the International Meeting on the Biochemistry of Cell Membr , sponsored by the iology in Bari, Italy. To Peer Recognition. Was invited to select the speakers| for a symposium on Drosophila Neurobiology and to be the Chairman of the symposium at the International Congress of Biochemistry in New Delhi in 1994. Was invited to be a International Union of Biochemistry and M oH Nes          plenary speaker at the European Pharmacology Society Meeting in Berlin in 1994. Was a member of the Organizing Committee and an invited lecturer for the Elba International Neuroscience Meeting in Marina di Campo, Isola d’Elba, Italy for 1993, Was invited to be a symposium speaker at a meeting “DNA: The Double Helix, Forty Years: Perspective and Prospective” sponsored by the New York Academy of Science. Invited lecturer at the Department of Biochemistry, College of Medi , University of Tilinois at Chicago, Illinois and Georgetown University. han N tartone to give talks were not accepted. Member of the Editorial Advisory Board, Molecular Neurobiology. Member of the Editorial Advisory Board of Cellular and Molecular Neurobiology, Member of the Editorial Board, Journal of Neurogenetics. Adjunct Professor, Department of Biochemistry, George Washington Medical School, Washington, D.C. Member of an American College of Neurology Committee to choose the recipient of a Se award for research. N aq Influence on Research Projects. Essentially all of the research objectives of Dr. Nirenberg’s group, past and present, originated from Dr. Nirenberg. The ideas ror ae 3 collabprative projects that Dr. Nirenberg GsfourrenttyV py 1 On Apes iri iad Se ui Ns paterercal aes thir, ad \"A collaborati rome) VS y obvicug to Dr. Nirenberg and the er investigator.            Dr. Marshall Nirenberg (1992-1993) CRITICAL ELEMENT f: MAINTAINS AN ACTIVE ROLE IN THE PROFESSION AS INDICATED BY; Reviews Manuscripts for Scientific Journals. Dr. Nirenberg is a member of the Editorial ards of 3 scientific journals and reviews manuscripts for these Journals also serves as an ad hoc reviewer of many manuscripts for other scientific journals. Active Participation in Scientific Societies. Dr. Nirenberg is an active member of the following societies: National Academy of Sciences, U.S.A. National Academy of Medicine, U.S.A. American Academy of Arts and Sciences American Society of Biological Biological Chemistry and Molecular Biology American Chemical Society American Neurochemistry Society Biophysical Society American Association for the Advancement of Science Society for Neuroscience The Society for Developmental an American seqhetyterNeurologtZaX Dae European Academy of Arts and Sciences Pontifical Academy of Sciences, the Vatican Federation of American Scientists International Society for Neuroimmunomodulation Dr. Nirenberg is a Sponsor of The Federation of American Sewientists and > Maver of the Advisory Board of the The International Society for Neuroimmunomodulation. During the past year, Dr renberg signed about 10 letters from different scientific SDE TETTES or W an eee cri mm      We 7 \\\\ . Marshall Nirenberg (1992-1993). NONCRITICAL ELEMENT fe: SERVES ON COMMITTEES WITHIN NIH AND ELSEWHERE AS REQUESTED. As discussed on @ previous Pe Nirenberg is a member of the Editorial Advisory Boards of 3 Scientific journals, eS a member of the Advisory Board of the Wess member ae aye Neuroimmunomodulation,and was a member,of a cqmmittée to select the recipients ie major’ award presented by the American Neurological Association. e is a member of the Advisory Board of the Beckman Institute of the University of Illinois at Ubana-Champaign, IL. He also was a member of the doctoral committee of Mrs. Wha Kwon and directed the research of Mrs. Kwon, who received a Ph.D. degree several months ago from the Department of Biology, University of MD. Dr. Marshall Nirenberg (1990-1995) CRITICAL ELEMENT fs: SUPPORTS THE NIH EQUAL EMPLOYMENT OPPORTUNITY PROGRAMS BY ACTIONS THAT INSURE EQUAL TREATMENT OF EMPLOYEES. Dr. Nirenberg always has supported the National Institutes of Health and Public Health Service Equal Employment Opportunity programs and has a long, well established record of treating all employees equally and of recruiting, developing, and advancing minorities, women, and persons with disabilities. About half of the investigators in the Laboratory af Biochemical Genetics are women ane ie embers of minorities. Only one individual with a neant ith applied rie lal crippled byt polio in ch Idhood S$ a postdoctoral are              Ttion, The individualW who was             Was         a woman, and a member of a minority group   fellow.   accepted As discussed on an ea er page, a young man with little or no background in science, a member of a minority croup, mda was recruited to maintain liquid nitrogen rreezersf and other laboratory \\\\ equipment@ and to take care of the laboratory stock room, was taught many technical procedures during the past year that are required for recombinant DNA research and now spends most of the day doing research. Other individuals who are members of minorities also were taught various technical procedures during the past year. Dr. Korn came to the Laboratory of Biochemical Genetics and gave a talk which was attended by all members of the laboratory on issues related to sexual harassment", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-57zd~sumj-2vmj", "00000000-0000-0000-4E5E-1DBA3F75DE86", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Bradykinin- Activated Transmembrane Signals are Coupled via No or Ni to Production of Inositol 1,4,5- Trisphosphate, a Second Messenger in NG108-15 Neuroblastoma-Glioma Hybrid Cells", "101584910X321", null, "1986", "February 1986", "The article explores the interaction of bradykinin (a hormone which causes muscle contractions, hypertension, pain generation, and blood coagulation) with other hormones and enzymes.", "Articles", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 83, pp. 942-946, February 1986 Biochemistry Bradykinin-activated transmembrane signals are coupled via N, or N; to production of inositol 1,4,5-trisphosphate, a second messenger in NG108-15 neuroblastoma—glioma hybrid cells (GTP-binding proteins / pertussis toxin /adenylate cyclase/ion channels /phosphatidylinositol turnover) HARUHIRO HIGASHIDA*, RICHARD A. STREATY!, WERNER KLEE‘, AND MARSHALL NIRENBERG* *Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute and +Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 Contributed by Marshall Nirenberg, October 7, 1985 ABSTRACT The addition of bradykinin to NG108-15 cells results in a transient hyperpolarization followed by prolonged cell depolarization. Injection of inositol 1,4,5-trisphosphate or Ca?* into the cytoplasm of NG108-15 cells also elicits cell hyperpolarization followed by depolarization. Tetraethylam- monium ions inhibit the hyperpolarizing response of cells to bradykinin or inositol 1,4,5-trisphosphate. Thus, the hyper- polarizing phase of the cell response may be due to inositol 1,4,5-trisphosphate-dependent release of stored Ca** into the cytoplasm, which activates Ca’*+-dependent K* channels. The depolarizing phase of the cell response to bradykinin is due largely to inhibition of M channels, thereby decreasing the rate of K* efflux from cells and, to a lesser extent, to activation of Ca”*-dependent ion channels and Ca?* channels. In contrast, injection of inositol 1,4,5-trisphosphate or Ca”* into the cytosol did not alter M channel activity. Incubation of NG108-15 cells with pertussis toxin inhibits bradykinin-dependent cell hyperpolarization and depolarization. Bradykinin stimulates low K,, GTPase activity and inhibits adenylate cyclase in NG108-15 membrane preparations but not in membranes prepared from cells treated with pertussis toxin. Reconstitution of NG108-15 membranes from cells treated with pertussis toxin with nanomolar concentrations of a mixture of highly purified N, and N; [guanine nucleotide-binding proteins that have no known function (N,) or inhibit adenylate cyclase (N,)] restores bradykinin-dependent activation of GTPase and inhibition of adenylate cyclase. These results show that [bradykinin-recep- tor] complexes interact with N, or Nj, and suggest that N, and/or N; mediate the transduction of signals from bradykinin receptors to phospholipase C and adenylate cyclase.   Bradykinin (BK) is a nonapeptide derived by proteolysis of higher molecular weight precursor proteins synthesized in the liver and hypothalamus (1, 2). The pharmacological actions of BK or precursor proteins include muscle contrac- tion, hypertension, pain generation, increase in vascular permeability, and blood coagulation 3-5). However, rela- tively little is known about the function of BK in the nervous system. NG108-15 neuroblastoma—glioma hybrid cells have BK receptors (6, 7) and respond to BK by a transient cell hyperpolarization followed by a long-lived depolarization (6, 8) accompanied by an increase in secretion of acetylcholine from NG108-15 cells at synapses with myotubes (8). BK also stimulates phosphatidylinositol turnover in NG108-15 cells (8) and increases cellular levels of inositol 1,4,5-trisphosphate (InsP;) (9) and the release of Ca2* from intracellular stores into the cytoplasm (10).   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 942 In this report we show that pertussis toxin partially inhibits the effects of BK on NG108-15 cells and that activation of BK receptors by BK results in activation of GTPase of Nj (11) or N, (12) [guanine nucleotide-binding proteins that have no known function (N,) or inhibit adenylate cyclase (Nj)], which are substrates for pertussis toxin (13, 14). The results suggest that N; or N, are involved in signal transduction initiated by [BK-receptor] complexes that result in activation of phos- pholipase C and inhibition of adenylate cyclase. METHODS AND MATERIALS BK was obtained from Sigma or Calbiochem; InsP; and inositol 2-phosphate were obtained from Sigma; [p- Ala”,Met*]enkephalinamide was from Calbiochem. Pertussis toxin was a gift from R. Sekura (National Institute of Child Health). “CaCl, was obtained from Amersham, and [y- 32P|GTP was from New England Nuclear. NG108-15 cells used for electrophysiological studies were cultured in polyornithine-coated 35-mm Petri dishes and were treated with 10 uM prostaglandin E, and 1 mM theophylline for 1-3 weeks before use (15). RESULTS Cell Responses to BK, InsP3, or Ca?*+. Addition of BK by iontophoresis to the external surface of an NG108-15 cell resulted in cell membrane hyperpolarization followed by depolarization (Fig. 1A) (8). Injection of InsP; (Fig. 1B) or Ca** (Fig. 1D) into the cytoplasm of a cell also resulted in cell hyperpolarization followed by depolarization. Injection of inositol 2-phosphate into the cytoplasm had little or no effect on the cell membrane potential (Fig. 1C). These results suggest that the responses of NG108-15 cells to BK are dependent on an increase in cellular levels of InsP3, presum- ably due to activation of the phospholipase C that catalyzes the formation of InsP; and diacylglycerol from phosphati- dylinositol 4,5-bisphosphate followed by an InsP3-dependent release of stored Ca”+ into the cytoplasm. The relation between the amount of iontophoretic current used for intracellular InsP, injection and the magnitude of the hyperpolarizing and depolarizing cell responses is shown in Fig. 2A. Both responses of cells increased as the quantity of InsP; iontophoretic currents (A:sec) was increased up to about 65 nC. With some cells, injection of InsP; with relatively high amounts of iontophoretic current, such as —220 nC, elicited a depolarizing cell response but little or no hyperpolarizing response (Fig. 2 B and C). The mean hyper-   Abbreviations: BK, bradykinin; InsP3, inositol 1,4,5-trisphosphate; Et,NCI, tetraethylammonium chloride; N, or N;, guanine nucleotide- binding proteins that couple some species of activated receptors with adenylate cyclase stimulation or inhibition, respectively; N,, an N protein of unknown function. Biochemistry: Higashida e¢ al.           Membrane potential, mV         l i ] l j 20 40 60 80 =: 100 —20   Time, sec Fic. 1. Typical membrane potential changes of NG108-15 cells evoked by extracellular BK or intracellular injection of InsP; or Ca?*. (A) Cell hyperpolarization followed by depolarization elicited by extracellular application of BK. The potential changes were recorded with an intracellular microelectrode filled with 3 M KCI. A second micropipette filled with 0.1 mM BK dissolved in 0.1 mM HCl] was located extracellularly close to the cell surface. BK was applied at zero time by iontophoresis (50 nA for 1 sec). (B) Cell hyperpolar- izing and depolarizing responses induced by intracellular injection of InsP;. The recording electrode (3 M KCl) and a micropipette filled with 1 mM InsP, dissolved in H,O were inserted into an NG108-15 cell. At zero time —100 nA was passed through the InsP; pipette for 0.5 sec. (C) One mM inositol 2-phosphate dissolved in H,O was injected intracellularly (—100 nA for 1 sec). (D) Ca?* was injected into the cytoplasm of an NG108-15 cell from a micropipette filled with a solution containing 0.5 M CaCl, by iontophoresis with 100 nA for 1 sec at zero time. The upward and/or downward deflections of the traces between 0 and 2 sec are due to the iontophoretic current rather than the compound applied. polarizing and depolarizing responses of many cells to injected InsP; are shown in Fig. 2C as a function of the amount of iontophoretic current applied. Tetraethylammonium ions (Et,N*) completely blocked BK- or InsP3-dependent cell hyperpolarization but had little or no effect on cell depolarization (Fig. 3A and C). EtyN* is known to inhibit some Ca?*-dependent K* channels (16), which suggests that BK- or InsP3-dependent hyperpolariza- tion may be due to activation of K* channels by Ca**, thereby increasing the rate of K* efflux from the cells. In contrast, Co*t had little effect on the hyperpolarizing re- sponse of NG108-15 cells to BK or InsP3 but inhibited most of the BK- or InsP3-dependent cell depolarization (Figs. 3 B and D, respectively). When NG108-15 cells were incubated without Ca** and with or without 0.1 mM EGTA, the amplitudes of BK-dependent hyperpolarizations and depo- larizations were reduced greatly (not shown). Incubation of cells in the absence of Na* or in the presence of 1 uM tetrodotoxin had little or no effect on cell responses to BK. The Ca?* channel blocker nifedipine (0.1 pM) had no effect on BK- or InsP3-dependent hyperpolarizing or depolarizing responses of NG108-15 cells (not shown). The mean input membrane resistance, determined from the amplitudes of hyperpolarizations induced by repetitive or constant currents applied through the intracellular recording electrode, was reduced to 42 + 9% (+ SEM, n = 5) of the mean control value (12.9 + 1.1 MQ, n = 8) during the hyperpolarizing phase of the responses of NG108-15 cells to BK (Fig. 4A). The membrane resistance increased to 131 + 10% (16.8 MQ) of the control value during the BK-dependent depolarization phase. BK-dependent increases in membrane resistance during the depolarizing phase were found over a Proc. Natl. Acad. Sci. USA 83 (1986) 943   T T T T T T T TT\"         $e tf ao) )     ~ —100     —200 Membrane potential, mV         Time, sec Fic.2, The relation between the amount of iontophoretic current used for intracellular InsP; injection and the magnitudes of the hyperpolarizing and depolarizing cell responses. (A) Typical exam- ples of hyperpolarizing and depolarizing responses of NG108-15 cells evoked by intracellular injection of InsP; for 2 sec; the amount of current in nC is indicated in each panel. (B) An example of InsP; intracellular injection by iontophoresis using a large amount of current (—220 nC), which elicited primarily cell depolarization. The discontinuities in the trace at approximately 10~15 sec suggest some cell hyperpolarization. At 30-60 sec passive hyperpolarizing cur- rents were injected to test input membrane resistance. (C) The mean changes in membrane potential during the hyperpolarizing and depolarizing phases of NG108-15 cell responses to intracellular injection of InsP; with different amounts of iontophoretic current (nC). Each point represents the mean of values obtained from 5-19 cells; each cell was used for only one determination. The hyper- polarization and depolarization values shown are the maximum membrane potential deflections at 5-20 and 30—45 sec, respectively, after injection of InsP3. wide range of membrane potentials (—80 to —10 mV) (not shown). These results suggest that the addition of BK to NG108-15 cells results in the closure of some ion channels that are open in the absence of BK. In contrast, the average membrane resistance values during the hyperpolarizing and depolarizing phases of the cell response to injection of InsP; decreased to 40 + 6% (n = 6) and 58 + 7% (n = 18), respectively, of the control value (27.1 + 2.5 MQ, n = 14) (Fig. 4 B and C). The reversal potential for hyperpolarization induced by extracellular application of BK or intracellular injection of Ca2+ was —70 to —80 mV, which is close to the K* equilibrium potential and suggests that the hyperpolar- izing phase is due to the activation of Ca?*-dependent K* channels, thereby increasing the rate of K* efflux from cells. BK elicited cell hyperpolarization after a delay of 1-2 sec. The delay in cell response was shorter when InsP; was injected into the cytosol, and no delay in response was detected when Ca?* was injected. The decreasing delay in cell responses to BK, InsP3, or Ca2* suggests the following sequential reactions; BK receptor occupancy results in activation of phospholipase C; phosphatidylinositol 4,5- bisphosphate is converted to InsP; and diacylglycerol; InsP; induces release of stored Ca?* into the cytosol; Ca?*-depen- dent K* channels are opened; and K* efflux hyperpolarizes the cell. Addition of 10-1,000 nM phorbol 12-tetradecanoate 13- acetate or 1-oleoyl-2-acetylglycerol had no effect on the resting membrane potential or on cell responses to BK 0-60 min after addition (not shown). Also, 10 4M A23187 did not mimic the effects of BK on cell membrane potential in control or phorbol! ester-treated cells (not shown). 944 Biochemistry: Higashida et al.                                                             TAT ELN\" 7“ Te LAAT. rc tt bit do ry PD oe T it L cot J 59     —20 O 20 40 60 80 100 120 Time, sec Fic, 3. Effects of Et,N* (A and C) or Co?* (B and D) on the responses of NG108-15 cells to extracellular BK (A and B) or intracellular InsP; (C and D). (A) NG108-15 cells were incubated for 20-30 min in a 35-mm Petri dish containing 2 ml of Dulbecco’s modified Eagle’s medium supplemented with 5 mM Et,N*. At zero time 2 ul of a solution containing 10 »M BK dissolved in 150 mM NaCl was added to the surface of the medium close to the cell being tested. Action potentials were evoked by passing depolarizing pulses of current (0.2 nA for 60 msec) through the intracellular recording microelectrode. (B) NG108-15 cells were incubated in 2 mM Co?* /20 mM Tris-HCl, pH 7.2/150 mM NaCl/5.4 mM KC1/0.8 mM Mg- Cl,/1.8 mM CaCl,/20 mM glucose for approximately 10-20 min. Hyperpolarizing currents (0.5 nA for 60 msec) were passed through the intracellular recording microelectrode but failed to evoke off- spikes. BK was applied at zero time in a 2-yl drop as described above. (C) NG108-15 cells were incubated in the presence of 5 mM Et,Nt as described above. InsP; (0.1 mM) was injected into the cytoplasm by iontophoresis (—100 nA for 1 sec). (D) Cells were incubated in the presence of 2 mM Co** in the medium as described above for 10-20 min. InsP; was injected intracellularly at zero time (—200 nA for 1 sec). BK-Dependent Efflux of “Ca?+. As reported (8), BK stimulates “Ca?+ influx into NG108-15 cells. The effect of BK on “Ca?* efflux from NG108-15 cells is shown in Fig. 5. Cells were incubated in the presence of 1.8 mM “CaCl, for 8 min to promote *Ca?* uptake and washed for 3 min, and then dishes were perfused with control medium or medium with BK. BK stimulated the rate of ®Ca?*+ efflux approxi- mately 3-fold during the first 20 sec, but by 80 sec the rate of 45Ca?* efflux had returned to the control value. Effects of Pertussis Toxin on Cell Responses to BK. Both the hyperpolarizing and depolarizing responses of NG108-15 cells to BK were inhibited by treatment of the cells with pertussis toxin (200 ng per ml, 15 hr) (Fig. 6). The maximum inhibition by pertussis toxin was obtained with 1 ~M BK; higher concentrations of BK resulted in less inhibition by pertussis toxin. The maximum inhibition of hyperpolariza- tion (72%) was obtained with 1000 ng of pertussis toxin per ml, the highest concentration tested; 50% of the maximum observed inhibition was obtained with 35-100 ng of pertussis toxin per ml of medium. These results suggest that GTP- binding regulatory proteins such as Nj; (13, 14) or N, (12) mediate the hyperpolarizing cell response to BK. The results also are consistent with the suggestion that pertussis toxin acts primarily to decrease receptor affinity for ligands (17). Because all known GTP-binding regulatory proteins also display hormone-stimulated GTPase activities, we measured the effect of BK on GTPase activity of NG108-15 cell membranes. The data shown in Table 1 demonstrate that BK or the opioid peptide [D-Ala?,Met*]enkephalinamide stimu- lated the activity of a low K,, GTPase in NG108-15 mem- branes and that the stimulatory effects of BK or [p- Proc. Natl. Acad. Sci. USA 83 (1986)   TI00 nAT T T T T A 5 sec     Membrane potential, mV | |         Time, sec Fic. 4. Changes in membrane resistance elicited by BK iontophoresis (A), InsP; injection (B), or Ca** injection (C). (A) Membrane resistance was monitored by passing repetitive hyperpo- larizing pulses of current (0.5 nA for 400 msec) through the intra- cellular recording microelectrode. BK was applied extracellularly by iontophoresis (100 nA for 5 sec). The amplitudes of the hyperpolar- izing pulses are measures of membrane resistance. (B) InsP; was injected intracellularly by iontophoresis (—100 nA for 400 msec). Membrane resistance was measured by passing hyperpolarizing pulses (0.2 nA for 200 msec) through the intracellular recording microelectrode. (C) Ca?* was injected intracellularly by iontopho- resis (100 nA for 120 msec). Repetitive hyperpolarizing pulses of current (0.2 nA for 300 msec) were passed through the recording intracellular microelectrode. Ala’,Met*}Jenkephalinamide were inhibited by treating the cells with pertussis toxin before the membranes were pre- pared. BK-dependent stimulation of low K,, GTPase activity was restored to membranes of pertussis toxin-treated   160-77 ToT 0 140 4 120;- 4 e 8 Qo oS T T 1 1 Ca** released, nmol per dish na Ss T 1     40   | I I ! 1 1 I I | -60 —-40-20 0 20 40 60 80 100 120 140 Time, sec Fic. $. BK-stimulated “Ca?* efflux from NG108-15 cells. The cells were incubated in the medium described in the legend to Fig. 3B without Co?* for 2 min and then for 8 min at room temperature in medium supplemented with 4 «Ci (1 Ci = 37 GBq) of *CaCl,, washed three times with medium without *°Ca?*, and then perfused for 2.9 min. At zero time cells were perfused with medium that contained 1 uM BK (e) or medium without BK (0). Fractions (1 ml) were collected at 20-sec intervals. Each point represents the mean of 3-4 values + SEM. Each Petri dish contained approximately 5 x 10° cells. Biochemistry: Higashida et al.   10 5 o an. -10 Membrane potential, mV I a -15         2 i i ! i 5 7 6 5 4 3 Bradykinin, — log M Fic. 6. The effects of pertussis toxin on the hyperpolarizing (@, ©) and depolarizing (@, 0) responses of NG108-15 cells grown in dishes that were not coated with polyornithine. Droplets (2 1 each) of a solution containing the indicated concentration of BK dissolved in 150 mM NaCl were applied to the surface of the medium near the cell that was being tested. Each point represents the mean value of responses of nine cells. Cells were incubated for 15 hr with 0.34 mM ammonium sulfate (control cells) or with 200 ng of pertussis toxin/ml of medium and 0.34 mM ammonium sulfate before being tested. o, Hyperpolarizing responses of control cells to BK; e, BK-dependent hyperpolarizing responses of NG108-15 cells that had been treated with pertussis toxin; 0, depolarizing responses of control cells to BK; mg, BK-dependent depolarizing responses of cells that had been treated with pertussis toxin. NG108-15 cells by the addition of a mixture of bovine brain N, and N; (approximately 70% N, and 30% Nj) estimated to be >95% pure (19) (Fig. 7A); half-maximal stimulation of GTPase by BK was obtained with <1 nM N,/Nj. As reported earlier (19) and as shown in Fig. 7A, the N,/N; mixture also reconstituted a [D-Ala?,Met°]enkephalinamide-dependent, opiate receptor-mediated stimulation of GTPase. Interesting- ly, BK-stimulated GTPase activity was reconstituted at appreciably lower concentrations of the N,/Nj mixture than those needed for opiate-stimulated GTPase. We found also that BK stimulation of GTPase activity was diminished only slightly in the presence of a saturating concentration (10 4M) of [p-Ala?,Met*]enkephalinamide (Fig. 7B). Such additivity of effects suggests that BK receptors may interact preferen- tially with Ny, and opiate receptors may interact preferen- tially with N;. Additivity of the effects of BK and opiates is confirmed by the observation that BK-dependent inhibition of adenylate cyclase still was observed in the presence of saturating concentrations of opiates (Fig. 7C). Ligands for other species of NG108-15 receptors that mediate inhibition of adenylate cyclase, such as norepinephrine, which acti- vates a-adrenergic receptors (21), or somatostatin (22), no Table 1. Attenuation of peptide-stimulated GTPase by pertussis toxin   P; formed, pmol/min per mg   of protein Pertussis Control toxin-treated Addition cells cells Water 26.0 + 1.5 19.9 + 1.6 BK (10 4M) 32.6 + 1.8 18.9 + 1.8 [p-Ala?,Met*]- enkephalinamide (10 4M) 38.0 + 2.3 15.7 + 1.6   Attenuation of peptide-stimulated low K,, GTPase activity by pertussis toxin. Membranes prepared from NG108-15 cells were incubated with or without 20 ng of pertussis toxin per ml of medium for 22 hr. Low K,, GTPase activity was assayed as described (18). Proc. Natl. Acad. Sci. USA 83 (1986) 945           < 36 B TT 6 C ne 86 3 34F PS) 4782.5 oS fo \\\\ E E 3210 0 {Bs a i) af oo go 30 7 44 ES 5 ‘eo a = 285 ON 9.06/70 se Bu . 2o oy 26 < 0 966 2 as 24 Pix OW 162 & = E a OO A 8 > 2r Of 453 = > z 20k , . S Cad 54 1 1 4 4 1 1 J 1 i i % 2.5 5 7.5 1012.5 By -7 -6 -5 -4 -7 -6 -5 =4° N protein, nM BK, log M Fic. 7. (A) Reconstitution of BK-stimulated (@) or opiate- stimulated (A) low K,, GTPase activities of membranes prepared from pertussis toxin-treated NG108-15 cells. Membranes were incu- bated with the indicated concentrations of a mixture of bovine brain N; and N, estimated to be >95% pure for 15 min at 30°C in the standard assay mixture (18) without radioactive GTP and then were incubated for an additional 15 min in the presence of approximately 60,000 cpm (1 4M) [y-*P]GTP. *2P; released in the absence of BK or [p-Ala?,Met*}Jenkephalinamide has been subtracted. Mean values (three experiments) are shown. (B) Low K,, GTPase activity of membranes prepared from untreated NG108-15 cells is shown as a function of BK concentration. o, BK with 10 wM [p-Ala?,Met*]en- kephalinamide; @, BK without [D-Ala?,Met*]enkephalinamide. The ordinate scale is pmols of P; released per min/mg of protein. (C) Adenylate cyclase activity of membranes prepared from untreated NG108-15 cells, assayed as described (20), is shown as a function of BK concentration in the absence (@) or presence (©) of 10 uM [p-Ala?,Met*]Jenkephalinamide. longer inhibit adenylate cyclase or stimulate low K,, GTPase in the presence of saturating concentrations of opiates. Thus, the mechanism of coupling BK receptors to inhibition of adenylate cyclase differs from the mechanism coupling a2- receptors or somatostatin receptors to inhibition of adenylate cyclase. DISCUSSION Exposure of NG108-15 cells to BK elicits increases in InsP; and diacylglycerol levels (8); in turn, InsP3 stimulates the release of stored Ca?‘ into the cytoplasm (10), thereby activating Ca*+-dependent K* channels and increasing the rate of K* efflux. Cell hyperpolarization results from the increase in K* efflux. The observations that support these conclusions are: (i) injection of InsP; or Ca?* into the cytoplasm of NG108-15 cells results in cell hyperpolarization followed by depolar- ization; (ii) hyperpolarization of cells elicited by BK, InsP3, or Ca?* is accompanied by a decrease in membrane resist- ance—i.e., an increase in cell permeability to ions; (iii), the reversal potential for BK-dependent hyperpolarization is approximately —80 mV, which is close to the equilibrium potential for K*; (iv) the amplitudes of the hyperpolarizing responses of 108CC-25 (23) and NG108-15 cells (unpublished results) to BK are dependent on the concentration of extra- cellular K*; and (v) BK-dependent hyperpolarization is blocked by EtsN*, which is known to inhibit some Ca?*- dependent K* channels (16). The long-lasting depolarization evoked by BK is associated with an increase in membrane resistance—i.e., cell perme- ability to ions decreases. Other species of receptors have been shown to mediate cell depolarization by decreasing cell permeability, such as muscarinic acetylcholine receptors (24) and receptors for luteinizing hormone-releasing hormone (25—27), substance P (28, 29), thyrotropin-releasing hor- mone (30, 31), neurotensin (32), somatostatin (33), angioten- sin (34), prostaglandin D2 (35), and the small cardioactive 946 Biochemistry: Higashida et al. peptide (36). Cell depolarization mediated by muscarinic acetylcholine receptors has been attributed to inhibition of M channels for K*, which decreases K* efflux from cells (24). Recent results obtained with voltage-clamp conditions show that NG108-15 cells have M channels that are inhibited by BK (Higashida and Brown, unpublished data). In contrast, injection of InsP; or Ca?* into the cytoplasm of NG108-15 cells results in cell hyperpolarization followed by a long-lasting depolarization that is accompanied by a decrease in membrane resistance—i.e., an increase in cell permeability to ions. Injection of InsP; or Ca2* into the cytoplasm did not affect the activity of M channels; therefore, elevation of cytosolic levels of InsP; or Ca2* elicited most, but not all, of the cell responses to extracellular BK. Because BK stimulates the formation of InsP3 in NG108-15 cells, part of the BK-induced depolarization probably also is due to an increase in cell permeability to ions. The most likely candi- dates for the ion channels that are activated during the depolarizing phase are the Ca?*-dependent cation channel (37) and a Ca?* channel. We find that BK-dependent cell hyperpolarization and depolarization are inhibited by pertussis toxin, which sug- gests that a GTP-binding regulatory protein such as N, or N; plays a role in the signal transduction process. BK was shown to stimulate a low K,, GTPase and to inhibit adenylate cyclase in NG108-15 membrane preparations, and these effects of BK were blocked by pertussis toxin. Reconstitution of mem- branes from pertussis toxin-treated NG108-15 cells with nanomolar concentrations of a mixture of highly purified bovine brain N, and Nj; restored BK-dependent activation of low K,, GTPase and inhibition of adenylate cyclase. BK- and [D-Ala*, Met>Jenkephalinamide-dependent inhibitions of ade- nylate cyclase were additive, whereas norepinephrine- and [D-Ala”,Met*]enkephalinamide-dependent inhibitions were not additive, which suggest that BK and opiates inhibit adenylate cyclase by different mechanisms. Because the [By] subunits of the N proteins are functionally interchangeable (38), activation of any N protein, with dissociation of [a-B-y] complexes to a and [6-y] will result in inhibition of adenylate cyclase by mass action, since the released [f-y] subunits will combine with the free a subunit of activated N,, the N protein that stimulates adenylate cyclase. In addition, our results suggest that N, or Nj are involved in the transduction of signals from BK receptors to Ca?* mobilization, presumably via activation of phospholipase C, as has been suggested for several other species of receptors (39-42). 1. Takagaki, Y., Kitamura, N. & Nakanishi, S. (1985) J. Biol. Chem. 260, 8601-8609. 2. Perry, D.C. & Snyder, S.H. (1984) J. Neurochem. 43, 1072-1080. Regoli, D. & Barabe, J. (1980) Pharmacol. Rev. 32, 1-46. Correa, F. M. A. & Graeff, F. G. (1975) J. Pharmacol. Exp. Ther. 192, 670-676. 5. Rocha e Silva, M. (1976) in Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease, Publ. No. 76-791, Ed. Pisano, J.J. & Austen, K. F, (Department of Health, Education and Welfare, Washington, DC), pp. 7-35. 6. Higashida, H., Wilson, S. P., Adler, M. & Nirenberg, M. (1978) Soc. Neurosci. Abstr. 4, 591, AY 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22, 23. 24. 25. 26. 27. 28. 29, 30. 31. 32. 33. 34, 35. 36. 37. 38. 39, 4l. 42. Proc. Natl. Acad. Sci. USA 83 (1986) Reiser, G., Walter, U. & Hamprecht, B. (1984) Brain Res. 290, 367-371. Yano, K., Higashida, H., Inoue, R. & Nozawa, Y. (1984) J. Biol. Chem. 259, 10201-10207. Yano, K., Higashida, H., Hattori, H. & Nozawa, Y. (1985) FEBS Lett. 181, 403-406. Ohsugi, T., Uchida, S. & Yoshida, H. (1985) Folia Pharmacol. Jpn., in press. Katada, T. & Ui, M. (1982) Proc. Natl. Acad. Sci. USA 79, 3129-3133. Sternweis, P. C. & Robinshaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813. Burns, D. L., Hewlett, E. L., Moss, J. & Vaughan, M. (1983) J. Biol. Chem. 258, 1435-1438. Kurose, H., Katada, T., Amano, T. & Ui, M. (1983) J. Biol. Chem. 258, 4870-4875. Nirenberg, M., Wilson, S., Higashida, H., Rotter, A., Krueger, K., Busis, N., Ray, R., Kenimer, J. G. & Adler, M. (1983) Science 222, 794-799. Findlay, I., Dunne, M. J., Ullrich, S., Wollheim, C. B. & Petersen, O. H. (1985) FEBS Lett. 185, 4-8. Hsia, J. A., Moss, J., Hewlett, E. L. & Vaughan, M. (1984) J. Biol. Chem. 259, 1086-1090. Koski, G. & Klee, W. A. (1981) Proc. Natl. Acad. Sci. USA 78, 4185-4189. Milligan, G. & Klee, W. (1985) J. Biol. Chem. 260, 2057-2063. Sharma, S., Nirenberg, M. & Klee, W. A. (1975) Proc. Natl. Acad. Sci. USA 72, 590-594. Sabol, S. L. & Nirenberg, M. (1979) J. Biol. Chem. 254, 1913-1920. Traber, J., Glaser, T., Brandt, M., Klebensberger, W. & Hamprecht, B. (1977) FEBS Lett. 81, 351-354. Reiser, G. & Hamprecht, B. (1982) Brain Res. 239, 191-199, Adams, P. R., Brown, D. A. & Constanti, A. (1982) J. Physiol. (London) 332, 223-262. Jan, L. Y. & Jan, Y.N. (1982) J. Physiol. (London) 327, 219-246. Katayama, Y. & Nishi, S. (1982) J. Physiol. (London) 333, 305-313. Kuffler, S. W. & Sejnowski, T. J. (1983) 7. Physiol. (London) 341, 257-278. Murase, K. & Randic, M. (1984) J. Physiol. (London) 346, 203-217. Surprenant, A. (1984) J. Physiol. (London) 351, 343-364. Ozawa, S. (1981) Brain Res. 209, 240-244. Dubinsky, J. M. & Oxford, G. S. (1985) Proc. Natl. Acad. Sci. USA 82, 4282-4286. Nakagawa, Y., Higashida, H. & Miki, N. (1984) J. Neurosci. 4, 1653-1661. Delfs, J. R. & Dichter, M. A. (1983) J. Neurosci. 3, 1176-1188. Brown, D. A., Constanti, A. & Marsh, S. (1980) Brain Res. 193, 614-619. Higashida, H., Nakagawa, Y. & Miki, N. (1984) Brain Res. 295, 113-119. Abrams, T. W., Castellucci, V. F., Camardo, J. S., Kandel, E. R. & Lloyd, P. E. (1984) Proc. Natl. Acad. Sci. USA 81, 7956-7960. Yellen, G. (1982) Nature (London) 296, 357-359. Gilman, A. G. (1984) Cell 36, 577-579, Berridge, M. J. & Irvine, R. F. (1984) Nature (London) 312, 315-321. Okajima, F. & Ui, M. (1984) J. Biol. Chem. 259, 13863-13871. Bokoch, G. M. & Gilman, A. G. (1984) Cell 39, 301-308. Cockcroft, S. & Gomperts, B. D. (1985) Nature (London) 314, 534-536.", "Nirenberg, Marshall W. ; Higashida, Haruhiro ; Streaty, Richard A. ; Klee, Werner A.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6jux_bnup-p3g7", "00000000-0000-0000-D033-C6329F18C544", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Carl W. Cotman and Earl Usdin to Marshall W. Nirenberg", "101584910X339", null, "1983", "25 August 1983", "Usdin thanks Nirenberg for agreeing to give one of the Distinguished Lecture Series in Basic and Medical Neuroscience at the University of California, Irvine, and provides guidance on addressing the audience.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "2", "pages", "Text", "English", "Reproduced with permission of Carl W. Cotman.", "Copyright may apply", null, null, "August 25, 1983 Dear Marshall: On behalf of the Department of Psychiatry and the Neuroscience Association of the University of California, Irvine, we should like to thank you for agreeing to give one of the Distinguished Lecture Series in Basic and Medical Neuroscience.  These Lectures are given on Wednesday evenings in the Science Lecture Hall of the UCI campus from 7:00 to 8:30 p.m. and attract a good-sized cap and gown audience.  Some are quite knowledgeable in the neurosciences; quite a few are psychiatrists; almost all are at least at the \"Scientific American level\".  In general, we ask our speakers to include some material for this later group, but also to include considerable material at the forefront of the field to whet the appetite of the experts present. A typical Distinguished Lecturer travel program has included arriving here on Tuesday.  The local airport is named Orange County or Santa Ana or John Wayne - depending on where you look. If the Lecturer wishes to see some of the really spectacular local scenery (e.g. Laguna Beach), he/she arrives early Tuesday afternoon; if not, Tuesday evening. If the Lecturer is willing, we arrange a luncheon-rap session with graduate students and psychiatry residents on Wednesday.  Often the Lecturer wishes to meet on a one-to-one basis with Neuroscience and Psychiatry faculty in the afternoon: just let us know who you would like to see.  Dinner with a few distinguished members of the community is shortly after the end of the Lecture.  Just before the Lecture, there is an informal wine-and-cheese on campus. In addition to your travel expenses (air coach) and local expenses, you will receive an honorarium of $700.00.  Please let us know your travel schedule so that we can make local arrangements.  Also let us know if you would prefer to stay in a hotel or if you would like to stay with Dr. Usdin and his wife.  Please send Dr. Usdin a copy of your Curriculum Vitae (primarily so that you may be given a proper introduction), your social security number (so that you may receive a check), and your home address (same reason). If you have any questions, please do not hesitate to call, write or ESP.  Also let us know if you need any audio-visual materials besides a 35 mm slide projector and a chalk board. Sincerely, Carl W. Cotman, Ph.D. Professor of Psychobiology Earl Usdin, Ph.D. Professor of Psychiatry P.S.:  We have listed your February 1, 1984 talk as \"Synapse Formation by Neuroblastoma Cells\".  It would be most helpful if you would send some words on your words, for us to use in preparing promotional announcements.", "University of California, Irvine ; Cotman, Carl W. ; Usdin, Earl", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rd25-se4x-g9y7", "00000000-0000-0000-535A-B3734B3E0CE6", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Nicholas W. Seeds, University of Colorado Health Sciences Center to Marshall W. Nirenberg", "101584910X340", null, "1982", "25 August 1982", "Seeds invites Nirenberg to participate in the second Gordon Research Conference on the \"Central Nervous System,\" focusing on the molecular and cellular factors influencing neural development.  Seeds suggests that Nirenberg's participation in the Synaptogenesis section \"will be a special asset to the success of the conference.\"", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of Nicholas W. Seeds.", "Copyright may apply", null, null, "August 25, 1982 Dear Marshall: We are organizing the second Gordon Research Conference on the \"Central Nervous System\", which will focus on the molecular and cellular factors influencing neural development, and will be held June 12-17, 1983, at the Tilton School in Tilton, NH. We would be very honored if you would be able to participate in this conference: Session 6; Topic - Synaptogenesis. We will be able to reimburse you for part of your expenses with funds made available from the Gordon Research Conference.  Although additional funding is being sought, we will not know of its availability until next spring; therefore, we suggest that you seek travel funds from other sources if possible. We would be most grateful if you would let us know of your decision at your earliest convenience (and a tentative title for your presentation). A tentative program is enclosed for your information. Additional participants will be invited to present poster sessions. As you probably know, the aim of the Gordon Research Conference is non-formal with total participation in discussion between speakers, discussion leaders and audience. Your participation will be a special asset to the success of the Conference, and we are looking forward to your acceptance. Best Regards, Hope you will still be able to attend. Sincerely, Nicholas W. Seeds, Ph.D, Professor Dept. Biochem/Biophys/Gen. on behalf of P.S. Timiras, M.D., Ph.D. Professor and Chairman Dept. Physiology - Anatomy University of California Berkeley", "Seeds, Nicholas W. ; University of Colorado Health Sciences Center", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kutu_i3hx.urcy", "00000000-0000-0000-4C16-C238580C6DBB", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Flier for the Annual Ewing Halsell Lecture, delivered by Marshall Nirenberg", "101584910X341", null, "1982", "1982", "This flier describes Nirenberg's lecture on Synapse Plasticity.  He was to deliver this lecture to the University of Texas Health Science Center in San Antonio.  The flier is annotated by an unknown person.", "Fliers (printed matter)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of the University of Texas Health Sciences Center.", "Copyright may apply", null, null, "Manckx0f \\\\ {haar 2 gee panes Leta 2 ~ - \\\\ ct Lee - 4 LA A ( co > tee Cpe pews scat Asa tess fo on The University of Texas Health Science Center at San Antonio invites you to . Cen Arcteunco The Annual Ewing Halsell Lecture sey ec.~ Monday, December 6, 1982 YPC ove, 4 p.m., room 3.104A (new lecture hall building) wag */ oor, rk Synapse Plasticity ©   delivered by Marshall W. Nirenberg, Ph.D. 1968 Nobel Laureate in Physiology or Medicine Chief, Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 4 i o Hi F Dr. Nirenberg, along with Drs. Robert W. Holley and Har Gobind Khorana, was awarded the 1968 Nobel Prize in Physiology or Medicine for their interpretation of the genetic , code and its function in protein synthesis. Dr. Nirenberg in 1961, using a simple nucleic acid composed of a chain of repeating uridylic acid molecules was able in vitro to produce a protein which contained only the amino acid phenylalanine. This experiment deciphered the first element in the code, and in less than five years through his efforts and the efforts of Dr. Khorana, all the details of the genetic code were established. This work without question will remain one of the greatest landmarks in the history of biology.   The Ewing Halsell Annual Lectureship is made possible by a gift to The University of Texas Health Science Center at San Antonio by the Ewing Halsell Foundation.", "University of Texas. Health Science Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-b6q3.by6y~cvmt", "00000000-0000-0000-AE10-CCD2D317271E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Andrew Wolfe to Marshall W. Nirenberg", "101584910X342", null, "1995", "1 December 1995", "Wolfe, from the Division of Endocrinology at the Children's Hospital of Harvard Medical School requests Brn-2 cDNA from Nirenberg's laboratory for use in examining the role of Brn-2 in the regulation of gene expression.  A hand note at the top of the letter from Nirenberg approves the request.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of the Harvard University Medical School Children's Hospital.", "Copyright may apply", null, null, "[Handwritten note: 12-1-95 Yoshi -- would you send Brn-2 genomic DNA clone to Wolfe. Marshall] Dear Dr. Nirenberg, We are interested in obtaining the Brn-2 cDNA from your laboratory for use in examining the role of Brn-2 in the regulation of gonadotropin-releasing hormone (GnRH) gene expression. Evidence obtained from our NLT and Gn11 cell lines, both of which are neural GnRH expressing cell lines (Radovick et al. PNAS (1991) 88:3402-3406), suggests that Brn-2 might play an important role in this process. We would, of course, acknowledge your contribution in any publications that resulted from the use of this cDNA, and we would not give this cDNA to anyone else without your permission. If you have any questions feel free to call (617-355-6957) or fax (617-730-0741) us. Thank you very much. Sincerely, Andrew Wolfe, Ph.D.", "Harvard Medical School. Children's Hospital ; Wolfe, Andrew", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e3xz-xx8q-pfcz", "00000000-0000-0000-1B55-9705F9B45619", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Daniel J. Minnema to Marshall W. Nirenberg", "101584910X343", null, "1990", "18 June 1990", "Minnema, from the Department of Environmental Health at the University of Cincinnati Medical Center, asks Nirenberg for access to cell lines for examining the effects of chronic toxicant exposure on nerve cells.", "Letters (correspondence)", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Text", "English", "Reproduced with permission of Daniel J. Minnema.", "Copyright may apply", null, null, "June 18, 1990 Dear Dr. Monsma, I recently read an article in Brain Research by Dr. Monsma et al. in which they characterize D1 and D2 dopamine receptors in neuroblastoma cell lines. One important use of this cell line is for the in vitro testing of neurotoxicants that potentially affect dopamine receptors.  Dr. Monsma has referred me to you as the supplier of the NS20Y cells.  I would kindly appreciate obtaining some of these NS20Y cells as a generous gift to initiate a colony here at the University of Cincinnati, Department of Environmental Health, specifically for examining the effects of \"chronic\" toxicant exposure on such nerve cells. In addition I would need the appropriate information as to any special requirements necessary to maintain these cells in culture.  We will not pass the cell line on to anyone, and would make you aware of any results obtained with the cell line.  Of course, I am more than willing to pay the necessary expenses for transportation and handling. Thank you in advance. Sincerely, Daniel J. Minnema, Ph.D.", "University of Cincinnati. Medical Center ; Minnema, Daniel J.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3miw_ms4i-4n2r", "00000000-0000-0000-8071-462C96DD1239", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Centre National De La Recherche Scientifique to Marshall W. Nirenberg", "101584910X344", null, "1980", "[1980?]", "Nirenberg began his research in developmental neurobiology when the field was still rather young and underdeveloped.  This letter from the Centre National De La Recherche Scientifique in Strasbourg informs Nirenberg that the first meeting of the International Society for Developmental Neuroscience will be held in June 1980.  A list of potential Symposia is included.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Reproduced with permission of the Centre National de la Recherche Scientifique (France).", "Copyright may apply", null, null, "Dear ISDN Member, The 1st Meeting of the International Society for Developmental Neuroscience will be held in Strasbourg on June 30th - July 4th, 1980.  The programme includes the following Symposia (given by invited speakers): - Early stages of nervous system development - Factors of growth and differentiation - Synaptogenesis in the peripheral nervous system - Synaptogenesis in the central nervous system - Neuronal-glial interactions - Genetic and environmental factors affecting nervous system development - Behavioural and clinical aspects of nervous system development. Contributions are invited for poster presentations, and Round Tables discussions, based on these, will be organized. Those interested in attending should contact the organizers, Prof. G. Vincendon and Dr. G. Gombos (Centre de Neurochimie du CNRS, 11 rue Humann, 67085 Strasbourg Cedex, France).  Registration fee $75 (for members of the Society) and $100 (for non members).  Abstracts, Registration and Hotel accommodation forms should be sent to the organizers by April 1st, 1980. Abstracts: No special form for Abstracts will be sent to authors. Draw in pencil a 15 cm x 24 cm frame on heavy white typing paper.  Type (new ribbon, preferably electric typewriter) within this frame.  Spacing can be at your convenience, but remember that in the Abstract book, the abstracts will be reduced to about 2/3rd the actual size. Erase the pencil frame.  Mail abstracts between two pieces of cardboard. N.B.: To write on the envelope \"Do not bend\" without including cardboards inside the envelope is useless.  Apparently mailmen take perverse pleasure in crumpling envelopes thus labelled. Registration and hotel accommodation: It is not necessary to ask for additional forms.  Photocopies of them may be used if needed. Authors from overseas: Please don't forget to check that your secretary sends Abstracts and Applications \"Air Mail\".  According to our experience, not everybody is aware that about two months are needed for the surface mail from U.S.A. or India to reach Strasbourg. Italian participants: All mail to and from Italy should be \"express\" in order to arrive in time. G. Vincendon, G. Gombos", "Centre National de la Recherche Scientifique France ; Vincendon, G. ; Gombos, G.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ik9d_rrvd.ke2n", "00000000-0000-0000-A035-92939ADE59A2", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Robert H. Broyles to Marshall W. Nirenberg", "101584910X345", null, "1995", "29 June 1995", "Broyles, from the University of Oklahoma Health Sciences Center, thanks Nirenberg for participating in the Distinguished Lecturer Series and graduate course \"Advanced Topics in Human Molecular Genetics.\" Nirenberg delivered a videotaped lecture on NK-2 homeobox genes in Drosophila.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "3", "pages", "Text", "English", "Reproduced with permission of Robert H. Broyles.", "Copyright may apply", null, null, "June 29, 1995 Dear Dr. Nirenberg: Thank you again, very much, for participating in our Distinguished Lecturer Series and graduate course \"Advanced Topics in Human Molecular Genetics.\"    Your talk was truly outstanding, and I have received many laudatory comments from both students and faculty. As you may recall from your visit, all the talks were recorded on videotape; and I probably mentioned that these tapes are kept as a lending library for educational purposes.   I may also have mentioned that if the tape were to be used for any other purpose, I would first ask your permission.   I am doing that now.   Since the lectures this year were all of very high quality and since the recordings were also good, we have decided to sell the videotapes to generate income for helping to support the course in subsequent years.  I have enclosed a release form for that purpose, which I hope that you will sign and mail back to me.  Also, please Fax (405/271-3092) a copy of the form to me before you mail it, as my TV producer is eager to get going on this project and would like to know that the form is on its way.   If you need to discuss this first, or if there is a problem or objection that we have not previously discussed, please call me at (405)271-1644. Thanks again for the outstanding lecture.  The students were very impressed and greatly stimulated by your visit. You helped make this our best of the three years the course has been conducted. I appreciate it very much. Sincerely, Robert H. Broyles, Ph.D. Professor, Course Director Enclosures. [Handwritten note: Marshall, If this is O.K., leave the original with Shirley and I will stop by the office on July 7th, when I will be at NIH. Thanks.] \"Advanced Topics In Human Molecular Genetics\" Biochemistry 6502 - A seminar/discussion course Sponsored by The Oklahoma Center for Molecular Medicine Taught by OUHSC Faculty and Invited, Internationally-known Lecturers Lecture is at 4:00 p.m. in BSEB-320 except as noted.* Topics and Distinguished Guest Faculty for the Spring Semester. 1995: * Jan 19: Arthur Kornberg, Stanford*# \"Inorganic Polyphosphate: A Molecular Fossil Come to Life\" West Lecture Hall, BSEB Jan 26: Griffin Rodgers, NIH, NIDDK \"Reversing Ontogeny: Therapeutic Trials to Modulate Human Globin Expression\" Feb. 2: Stuart Orkin, Harvard(H)* \"Genetic Control of Hematopoietic Development\" Feb. 9: Anita Roberts, NIH, NCI \"New Insights From the TGF-[beta]1 Knockout Mouse\" Feb. 16: Larry Simpson, UCLA (H) \"RNA Editing in Trypanosome Mitochondria\" Feb. 23 Alan Wolffe, NIH, NICHD \"Nucleic Acid Presentation: Mundane Packaging Proteins Regulate Gene Expression\" Mar. 2: All students to attend Presbyterian Health Foundation 10th Anniversary Symposium: \"A Salute to Molecular Biology\". Speakers: James D. Watson#, James Wyngaarden*, Elizabeth Zimmer, W. French Anderson, James W. Gusella, Mark H. Skolnick, Ganesh M. Kishore, and David Suzuki. Mar. 3: Elizabeth Zimmer, Smithsonian Meeting with students in BMSB-833 Mar. 9: Carol Clayberger, Stanford Scanlan Lecture - \"Induction of Immune Tolerance by HLA Peptides\" Mar. 29: Jean-Marc Egly, CNRS/INSERM** \"The Basic Transcription Factor BTF2/TFIIH\" Wileman Auditorium, OMRF Mar. 30: Curtis Harris, NIH, NCI* \"p53, Cell Cycle Control, and Apoptosis\" (Clinical Implications of Tumor Suppressors) Apr. 4: Joseph Gall, Carnegie* \"Spliceosomes, Snurposomes, and Coiled Bodies: Keys to RNA Processing\" Apr. 27: Elizabeth Hay, Harvard* \"Genes Controlling Tissue Transformations in the Embryo\" *May 4: Marshall Nirenberg, NIH, NHLBI*# \"The NK2 Homeobox Gene and Early Development of the Central Nervous System\" West Lecture Hall, BSEB May 16: Elizabeth Neufeld, UCLA* \"Molecular Approaches to Lysosomal Storage Diseases\" (H) Howard Hughes Medical Institute * Member, National Academy of Sciences ** Member, EMBO (European Molec. Biol. Organiz.) # Nobel Laureate Particulars: Meets in BSEB-320 3-3 1/2 hrs. per week (each Tues. and Thurs., 4-5pm., plus extra time with Guest Faculty), 2 credits, letter-graded.  Each student presents one 20-minute oral analysis of a research paper as part of class discussion.  Registration: By permission (see Dr. Broyles). Students and Fellows: The main purpose of this course is for students to have personal contact with internationally-recognized scientists (several are Nobel Laureates and many are members of the National Academy of Sciences).  Most of the lectures will be heavily attended; only registered students will be guaranteed seats and will meet with the guest speakers.  You are encouraged to register for the course and take advantage of this opportunity. Consent For Use Of Picture And Voice Name: Marshall W. Nirenberg, Ph.D. Address: National Institutes of Health Date: The undersigned hereby authorizes or ratifies the taking of videotapes, motion pictures, photographs, and/or voice recordings by the University of Oklahoma subject to the following conditions: 1. That said videotapes, motion pictures, photographs, and/or voice recordings, the publication, showing, or other use thereof will be for the purpose of educational use only. 2. Further, I relinquish and give to the University of Oklahoma all right, title and interest I may have in the finished and/or reproduced videotapes, motion pictures, photographs, and/or voice recordings. 3. That the nature, purpose and proposed use of said videotapes, motion pictures, photographs, and/or voice recordings has been fully explained to and is understood to me.  I acknowledge that no guarantee has been made as to the results that may be obtained. 4. That no royalty, fee or other compensation of any character shall become payable by reason of the taking or use of such videotapes, motion pictures, photographs, and/or voice recordings. Signature", "University of Oklahoma. Health Sciences Center ; Broyles, Robert H.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ddzh-rxjp~psz2", "00000000-0000-0000-68ED-ACB82B281AA9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Harvard University,The Biological Laboratories to Marshall W. Nirenberg", "101584910X346", null, "1979", "9 April 1979", "In this letter, an unidentified person from Harvard University's Biological Laboratories attests to the importance of Nirenberg's work with neuroblastoma, repeating an earlier statement that \"the use of cell lines has make neurobiology accessible to biochemists.\"  He thanks Nirenberg for opening the door and emphasizes that many will follow in his footsteps in the years ahead.", "Letters (correspondence)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "[stamped, APR 16 1917] April 9, 1979. Dear Marshall, Just a note to say thank you for that superb speech and your coming to Dallas in the first place. I meant what I said in the introduction that I believe the use of cell lines has made neurobiology accessible to biochemists. You opened the door and I believe many will follow in your footsteps in the years ahead. The new results are particularly exciting. Many thanks Cordially, Dan.", "Harvard University. The Biological Laboratories", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e7bu~q9ay-k83k", "00000000-0000-0000-5866-3CB135697D89", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Gaylord P. Harnwell to Marshall W. Nirenberg", "101584910X348", "101584910X349", "1969", "19 February 1969", "Harnwell, President of the University of Pennsylvania, invites Nirenberg to receive an honorary degree and participate in commencement ceremonies at the University.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of the University of Pennsylvania.", "Copyright may apply", null, null, "February 19, 1969 Dear Dr. Nirenberg: On behalf of the Trustees of the University of Pennsylvania it gives me great pleasure to invite you to be present at our 213th Annual Public Commencement on Monday, May 19, 1969, and receive at that time an honorary degree from the University. The Trustees have acted following the recommendation of a Committee of the Faculty, endorsed by the Administration and the Trustees' Honorary Degree Committee. You will wish to know that the ceremonies will be held in The Municipal Auditorium of The Philadelphia Civic Center, and those who are to participate will be requested to assemble in the ballroom of the Civic Center prior to ten o'clock on the morning of Commencement Day. The program will begin precisely at ten-thirty and will conclude at approximately twelve o'clock. I hope very much to hear that it will be possible for you to be with us and, in the event you accept, we shall be in touch with you concerning detailed arrangements for the Commencement weekend. Very sincerely, Gaylord P. Harnwell", "Harnwell, Gaylord P. ; University of Pennsylvania", null, null, null, "National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-v7f7-yajp.ih6p", "00000000-0000-0000-5C47-4A1E48977DAC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to Gaylord P. Harnwell", "101584910X349", "101584910X348", "1969", "11 March 1969", "Nirenberg accepts Harnwell's invitation to attend the Commencement at the University of Pennsylvania and accept an honorary degree.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "March 11, 1969 Dear Dr. Harnwell: Thank you for your invitation to attend your Commencement and to accept an honorary degree from the University of Pennsylvania. I am deeply honored and will be delighted to accept. I would like to convey my sincere thanks to you, the faculty, and the trustees, and I am looking forward with pleasure to the occasion. Sincerely, Marshall Nirenberg Laboratory of Biochemical Genetics National Heart Institute", "Nirenberg, Marshall W.", null, null, null, "Harnwell, Gaylord P. ; University of Pennsylvania", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-m9ay-x4v5-8xep", "00000000-0000-0000-3B57-051EFAD093DB", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Hugh Rawson to Marshall W. Nirenberg", "101584910X350", "101584910X121", "1967", "17 August 1967", "Rawson, editor for Thomas Y. Crowell Company book publishers, asks Nirenberg to consider the possibility of writing a book describing advances in genetics and assessing their probable social consequences.  He mentions Nirenberg's Science editorial \"Will Society Be Prepared?\" as an example of demonstrating the need for \"greater understanding of recent advances.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Hugh Rawson.", "Copyright may apply", null, null, "August 17, 1967 Dear Mr. Nirenberg: The editorial in the August 11 issue of Science, adapted  from remarks you made when accepting the Research Corporation's 1966 award, points out very clearly the need for greater public understanding of recent advances in genetics. Would you consider the possibility of writing a book that would describe these advances and assess their probable social consequences?  If such a project appeals to you, I would be very interested in getting together with you to discuss the idea in more detail. Sincerely yours, Hugh Rawson Editor", "Rawson, Hugh ; Thomas Y. Crowell Company", null, null, null, "National Heart Institute (U.S.) ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-x69u.f7d8_beyh", "00000000-0000-0000-13D7-15CA2305C677", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Frank J. Ayd, Jr. to Marshall W. Nirenberg", "101584910X351", null, "1967", "3 November 1967", "Ayd requests Nirenberg's views on the topic of when life begins.  He suggests that doctors would be interested in Nirenberg's ideas and that Nirenberg's response would help to make his intended article on the topic more authoritative.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Frank J. Ayd Jr.", "Copyright may apply", null, null, "November 3, 1967 Dear Doctor Nirenberg: I am planning to write an article on \"When Does Human Life Begin?\" in which I would include the views of world renowned authorities on this subject.  As you well know, there are physicians who hold that human life begins at the moment of conception and others who say human life does not begin until implantation.  What would be your answer to this?  I would be indebted to you, if you would send me your answer with the reasons for it, along with permission to include it as part of my article.  Your reply could be as long as you wish to make it. I solicit your cooperation because I am convinced that doctors would be interested in what you think is the proper answer to this timely question. It is my desire to make this article as authoritative as possible.  Enclosed is a partial list of those to whom copies of this letter is being sent.  Since you undoubtedly know other authorities in this field, I appeal to your generosity by asking you to send me the names and addresses of others whose opinion I should solicit.  I am particularly anxious to obtain the names of authorities outside the United States. I truly would appreciate your suggestions and your answer to the question, \"When Does Human Life Begin?\". Hoping that some day I will have the privilege of meeting you, I am Sincerely yours, Frank J. Ayd, Jr., M.D.", "Ayd, Frank J., Jr", null, null, null, "National Heart Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hewu.w68a~d4qf", "00000000-0000-0000-580D-DF635B825CEF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Israel Halperin to Marshall W. Nirenberg", "101584910X352", "101584910X353", "1989", "17 February 1989", "Halperin, Director of the International Campaigns for Human Rights, provides Nirenberg with a bulletin detailing the rampant torture and abduction in Chile under the regime of Augusto Pinochet.  Halperin asks Nirenberg to add his name to a message speaking out against these abuses that will be sent to every member of the Human Rights Commission of the United Nations.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "February 17, 1989 Dear Dr. Nirenberg, Nobel Laureate From the enclosed Bulletin you will see that an extraordinary community of persons and groups in all walks of life have agreed to speak out with determination and persistence to stop all torture and abduction in Chile. This community now includes many, many thousands of persons, among them 125 Nobel Laureates, eminent writers, artists, scientists, clergy, trade unionists, National Academies of several countries, Presidents of many leading universities in many countries. Chile is just the focus of the present campaign. When it ends successfully, we will campaign to end apartheid in South Africa.  By focusing on one issue at a time, we intend to build an ever-increasing community to ensure that every government lives up to the Universal Declaration of Human Rights. These Campaigns are the donation of time and energy by some hundreds of busy scientists, writers, and others.  We have no salaried employees and our expenses are just the unavoidable ones (but high): printing, postage, telephone. We separate these Campaigns from all political movements and we are neither controlled, nor financed, by any group of any kind.  If we could raise sufficient funds, we could use a part-time secretary, and greatly expand the reach of our Campaigns. Would you be willing to have your name appear in our Bulletins as supporting the Chile Campaign? the Apartheid Campaign, when it begins? There would be no other obligation involved, although any other help you could give the Campaigns: financial, publicity, would be welcome. Israel Halperin Director of the International Campaigns For Human Rights", "Halperin, Israel ; International Campaigns for Human Rights", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wsyc.vjwt.cxbv", "00000000-0000-0000-A9D9-CF21162834CE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Campaigns for Human Rights", "101584910X353", "101584910X352", "1989", "[February-March 1989]", null, "Manifestoes", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "8", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Da. Martner, Mag met odd stour mama Ae prey 3 ¢ CAMPAIGNS FOR HUMAN RIGHTS i INTERNATIONAL CAMPAIGN - PROFESSOR J.L. MASSERA (URUGUAY) INTERNATIONAL CAMPAIGN - ORLOV AND SHCHARANSKY (USSR) INTERNATIONAL CAMPAIGN - ORLOV (USSR) INTERNATIONAL CAMPAIGN - ALL VICTIMS (CHILE) c/o Department of Mathematics, University of Toronto Toronto, Ontario, Canada, M5S 1A1 Telephone (416) 978-4156 BULLETIN of February-March 1989 1981-1984 1984-1986 1986-1986 1986-   This Chile Campaign is directed by Professor Israel Halperin (Ph.D. Princeton, 1936), Fellow and Gold Medalist of the Royal Society of Canada. It will escalate until world-wide opinion, without violence, brings an end to all abduction, torture, and murder by agents and unofficial agents of the Pinochet regime. This Campaign has the support of: Sir Yehudi Menuhin Claudio Arrau Leonard Bernstein Sir Colin Davis Sir Georg Solti Charles Dutoit § Samuel Beckett Arthur Miller Yves Montand Margaret Atwood Robertson Davies Northrop Frye Maureen Forrester Saul Bellow Patrick White Odysseus Elytis William Golding  Czeslaw Milosz ~ Elie Wiesel Sir George Porter (President, The Royal Society) Jo Benkow (Speaker of the Norwegian Parliament) The Accademia Nazionale dei Lincei (National Academy, Italy) | Lord Brian Flowers (Rector, University of London) Dr. Paulo Renato Costa Jonga (Rector, Unicamp, Brazil) Dr. Georges Verhaegen (Rector, Free University of Brussels) Dr. Klaus Ring (Rector, Univ. J.W. Goethe, Frankfurt). Dr. O. Bastiansen (Pres., Norwegian Academy of Science & Letters) Ephriam Katzir (former President of Israel) Jack Lemmon (Star of the movie ‘*Missing’’) Lord Alexander Todd, Linus Pauling, and §23other Nobel Laureates and thousands and thousands of others, in all walks of life, in many countries. Bulletins are distributed in English, French, Spanish, Portuguese, Japanese and German, three times a year.   Famous or not, you can raise your voice. Send a letter to the President or Prime Minister of your country; urge that all finan- cial or economic aid given to Chile should be conditioned on the elimination of torture; then send a copy of that letter to: Ambassador, Embassy of Chile, Capital City of your country (a more precise address is not necessary).   Pinochet has announced measures which promise a decrease in abuse of human rights. But abduction and torture continue. The promises are not actually carried out. Msgr. Cristian Precht, Vicar General of Santiago, August, 1987: “*The time has come to stop arguing about whether or not there is torture; we all know that there is torture.”’ In August, 1987, the Chilean Bishops’ Conference denounced the continued use of torture.   Typical cases, reported to the Chilean Commission on Human Rights: Pedro Marin Hernandez, 31 year old doctor, beaten by the Chilean Security Forces (CNN), electric current applied to his geni- tals, then raped by a male CNI agent. Leopoldo Francisco Orrego Saez, 28 years old, electric- current applied to temple, hands, ears, legs: warned he would pay dearly if he reported the torture. Raquel Marcela Espinoza Orellana, 24 year old woman, raped. Claudio Patricio Pino Cortes, beaten, died from injuries.   The Chilean Commission on Human Rights, 1987: The number of cases of kidnapings by paramilitary groups during the first six months of 1987 was more than double the number for'1986. By the end of September, 108 kidnapings had been reported. Not one agent has ever been punished. Amnesty International, May, 1988: Cases in Chile of nearly 700 ‘‘disappeared”’ persons have been sufficiently documented to have been presented to the courts, but judicial investigations were blocked by higher courts or by the security forces.   The Canadian Inter-Church Commitiee on Human Rights, September 8, 1988: Abuses documented during the past 12 months include: systematic torture, imprisonment of journalists, attacks on church and human rights organizations, abductions and death threats by paramilitary groups clearly linked to the official security forces.   Actions of Foreign Governments: 15 years ago, in a shockingly bloody coup, Pinochet’s armed thugs murdered the legally elected President, abolished the Par- liament, replaced all mayors by Pinochet appointees, subjected each university to the control of a military boss, smashed the unions. Within two weeks of this coup, thirty foreign countries gave Pinochet formal recognition. Today, an international storm of condemnation of Pinochet has led many governments to express disapproval of abuse of human rights in Chile. But their agencies finance an enormous flow of investment and military supplies to Chile without which Pinochet could hardly continue; and this support makes no reference to a required elimination of torture.   Letter of December 22, 1987, from the Government of Canada: .-.. Canadian representatives in November, 1987 voted for the Mexican sponsored resolution in the United Nation’s Third Committee condemning the human rights situation in Chile. But the Chilean Ambassador in Ottawa wrote on September 12, 1988: The Canadian representatives, when voting for the Mexican resolution, have consitently denounced the bias against Chile in this matter of human rights. And Canada Member of Parliament Dan Heap, at a public meeting in Toronto on September 11, 1988 stated: When I visited Chile in 1985, the Canadian Ambassador said to me, ‘‘Chile, under Pinochet, was right to reduce the buying power of the people so as to compete better in the international market; Canada has the same problem and must use the same remedy.’’.   From a letter sent to 316 Mayors in Chile, July 1, 1988: ...Canadians are disturbed by the reports of abduction, torture, and even murder, of persons not in favour with the present government...many Canadians believe that, behind closed walls, the grave abuse of human life continues.... we urge you to use your influence to bring about a state of affairs in which all persons in Chile enjoy the protection of their civil rights. (signed) Arthur Eggleton, Mayor of Toronto Sylvia Sutherland, Mayor of Peterborough David Burr, Mayor of Windsor Ron Wallace, Mayor of Halifax John Counsell, Mayor of Guelph Peter Wong, Mayor of Sudbury Angus Read, Mayor of Cobourg Brian Lynch, Mayor of Comwail Brad Woodside, Mayor of Fredericton Dominic Cardillo, Mayor of Kitchener Manning MacDonald, Mayor of Sydney Larry Schneider, Mayor of Regina Robert Morrow, Mayor of Hamilton Joseph McCaffery, Mayor of St. Catherines Gordon Campbell, Mayor of Vancouver   Message to every member nation of the Human Rights Commission of the United Nations: The inhuman practice of abduction, torture and oppression in Chile has been condemned by world-wide opinion. The Human Rights Commission has rebuked the Government of Chile but has failed to take effective measures to stop the practice. This failure undermines the credibility of the Commission and weakens the United Nations. The under- signed Nobei Laureates call on the Commission to take action that will be effective in stopping torture and oppression in Chile. (This message is co-signed by the Nobel Laureates listed with a star on the next page.) -3- (i) Among the supporters of this Campaign are also the following y?¢ Nobel Laureates “** (a1 havelco-signed the mes- sage to the Human Rights Commission of the United Nations printed on the previous page). Philip Anderson (USA)* Julius Axelrod (USA)* Sir Derek Barton (USA)* Baruj Benacerraf (USA)* Gerd Binnig (Germany)* Daniel Bovet (Italy)* S. Chandrasekhar (USA)* A.M. Cormack (USA)* James Cronin (USA) < Renato Dulbecco (USA)*. Adolfo Perez Esquivel (Angentina)® Murray Gell-Mann (USA) * William Golding (U.K.)* Herbert Hauptman (USA)* Antony Hewish (U.K.)* Robert Hofstadter (USA)* Francois Jacob (France)* Sir Bernard Katz (U.K.)* Arthur Kornberg (USA)* Jean-Marie Lehn (France)* Andre Lwoff (France)* AJ.P. Martin (U.K.)* Simon van der Meer (Switzerland)* Peter Mitchell (U.K.)* Daniel Nathans (USA)* Linus Pauling (USA)* John Polanyi (Canada)* Burton Richter (USA)* Emst Ruska (Germany)* (since . Frederick Sanger (U.K.)* Glenn Seaborg (USA)* Herbert Simon (USA)* Wole Soyinka (Nigeria)* Howard Temin (USA)* James Tobin (USA)* Desmond Tutu (South Africa)* ~ Patrick White (Australia)* Robert Wilson et Salvador Luria(USA)* Jacob Goldstein(USA)* Michael Brown(USA)* Chien Shuing Wu(USA)* Christian Anfinsen (USA)* David Baltimore (USA)* Samuel Beckett (Ireland)* Paul Berg (USA)* Konrad Bloch (USA)* Willy Brandt (Germany Stanley Cohen (USA)* Sir John Cornforth (U.K.)* Jean Dausset (France)* __ Manfred Eigen (Germany)* E.O. Fischer (Germany)* Donald Glaser (USA)* Ragnar Granit (Sweden)* Dudley Herschbach (USA)* Sir Alan Hodgkin (U.K.)* Robert Holley (USA)* B.D. Josephson (U.K.)* Lawrence Klein (USA)* Sir Polycarp Kusch (USA)* Rita Levi-Montalcini (Italy)* Sean MacBride (Ireland)* JE. Meade (U.K.)* Czeslaw Milosz (USA)* Francisco Modigliani (USA)* Louis Neel (France)* Aro Penzias (USA)* Sir George Porter (U.K.)* Alfonso Garcia Robles (Mexico)* Abdus Salam (Italy)* Arthur Schawlow (USA)* Robert Schrieffer (USA)* George Snell (USA)* Roger Sperry (USA)* Jan Tinbergen (Netherlands)* Lord Todd (U.K.)* George Wald (USA)* Elie Wiesel (USA)* Sir James Black(U.K.)* Leon Ledemman(usa)* Leo Esaki(USA)* Isaac B. Singer(USA)* Kenneth Arrow (USA)* John Bardeen (USA)* Saul Bellow (USA)* Hans Bethe (USA)* Baruch Blumberg (USA)* Adolph Butenandt (Germany)* Leon Cooper (USA)* Andre Cournand (USA)* Gerard Debreu (USA)* _ Odysseus Elytis (Greece)* William Fowler (USA)* Sheldon Glashow (USA)* Roger Guillemin (USA)* Gerhard Herzberg (Canada)* Dorothy Hodgkin (U.K.)* David Hubel (USA)* Jerome Karle (USA)* Aaron Klug (U.K.)* Yuan T. Lee (USA)* William Lipscomb (USA)* Mairead (Corrigan) Maguire (Ireland)* Sir Peter Medawar (U.K.)* (since deceased) Cesar Milstein (U.K.)* Sir Nevill Mott (U.K.)* George Palade (USA)* Max Perutz (U.K.)* Tadeus Reichstein (Switzerland)* Carlo Rubbia (USA)* Paul Samuelson (USA)* Julian Schwinger (USA)* Kai Siegbahn (Sweden)* Robert Solow (USA)* Henry Taube (USA)* Nicolaas Tinbergen (U.K.)* (since deceased) Charles Townes (USA)* Lech Walesa (Poland)* Torsten Wiesel (USA)* Jack Steinberger(Switzerland )* Georg Bednorz(Switzerland)* George Hitchings(USA)* Susumu Tonegawa(USA)* Heinrich Rohrer(Switzerland )* -4- (ii) Among the supporters of this Campaign are also Academicians in many countries. As examples, we list those in Aus- tralia, Norway, and the U.K. , Australia: The Australian Academy of Science: H.G. Andrewartha C.A. Appleby R.J. Baxter, FRS Sir N. Bayliss A.L.J. Beckwith, FRS MR. Bennett L.C. Birch — P.O. Bishop, FRS A. Boden RP. Brent K.S. Campbell D.G. Catcheside, FRS J.P. Chalmers WN. Christiansen L.M. Clarebrough H.C. Coombs DR. Curtis, FRS (President) FJ. Fenner, FRS Sir O. Frankel, FRS J. Gani F.W. Gibson, FRS MF. Glaessner D.H. Green H.S. Green $.D. Hamann EJ. Hannan W. Hayes, FRS T.W. Healy B.W. Holloway G.A. Horridge, FRS C.A. Hurst B.G. Hyde L.A. Johnson GM. Kelly A. Kerr, FRS KH. Key PL Komer K. Lambeck J.W. Lance KJ. Le Couteur A.W. Linnane, FRS K.G. McCracken A.D. McEwan B.H. McKellar JJ. Mahony LN. Mander A.McL. Mathieson J.H. Michael J.F.A. Miller, FRS P.A.P. Moran, FRS J.0O. Newton K. Norrish Sir G. Nossal, FRS AG. Ogston, FRS Sir M. Oliphant, FRS M.S. Paterson J.D. Pettigrew, FRS JR. Philip, FRS R.B. Potts Sir J.R. Price SJ. Redman AE. Ringwood, FRS Sir R. Robertson, FRS D.W. Robinson GE. Rogers WPP. Rogers A.M. Sargeson, FRS G.B. Sharman C.W. Shoppee, FRS W.J. Simmonds JF. Sprent F.D. Stacey J, Stone R, Street J.M. Swan G. Szekeres SR. Taylor J.S. Turner, FRS C.H. Tyndale-Biscoe R.G. Wake E. Weigold DE. Weiss G.K. White Norway: The Royal Norwegian Society of Science and Letters, and The Norwegian Academy of Science and Letters S. Aanderaa J. Alstad J. Andenaes P. Andersen L Asheim O. Bastiansen F, Benestad E. Beyer N. Bjorgo K. Bjorlykke P. Borgen O. Bratteli J. Bremer J. Brockmann J. Brogger N. Dahl OJ. Dahl R. Djupedal T. Eckhoff K. Eimhjelien L. Eitinger K. Elgjo K. Elgmork A. Eliassen P. Enoer Y. Espmark J. Faarlund O. Faltinsen H. Fehn O. Gjaerevoll L. Gjerlow L. Gjessing J. Gjonnes J. Goksoyr H. Granum P. Grotvedt B. Gullvag A. Hannay J. Hareide O.M. Heide M. Helvig P. Hemmer S.D. Henriksen F. Hodbebo M. Hoffmann H. Holgersen A.E. Holme H. Holtedahl A. Holter E. Husebye G. Hygen J. Jansen I. Johansen A. Johnson A. Kjonstad P, Kvaerne O. Laerum S. Langholm H. Larsen B, Lassen O. Ledang R. Leivestad G. Lorentzen E. Lundeby S. Lunden L. Maehle H. Mageroy A. Michaelsen J. Moe P. Moe K. Mork P. Munthe B. Myhre H. Myklebost O. Myklebust A. Naess J. Natvig O. Nijastad H. Ofstad J. Ofstad A. Ofsti H. Olsen T. Opsahi T. Ostvold H. Oye O. Oyslebo J. Pedersen O. Reiersol R. Rommetveit T. Rosenquist E. Samuelsen ALL. Seip H. Seip P. Seyersted T. Sikkeland E. Sivertsen S. Skard L. Skattebel A. Skuiberg H. Skullerud K, Smidt H. Sorum E. Spjotvoll E, Steen J. Steen E. Steinnes N. Stenseth B. Stigum O. Storstein E. Somme A. Stromnes — E. Sverdrup T. Thonstad E. Torgersen K. Tranoy W. Velle FE. Vinje F.M. Vokes B. Waaler J. Weber E. Wyller V. Ystad T.W.Blackstad R. Rokseth U.K.: The Royal Society (names of Nobel Laureates and names of Fellows of the Australian Academy of Science will not be repeated here) JF, Adams AR. Battersby M.V. Berry Sir Herman Bondi Sir Malcoim Brown P. Chadwick - W. Cochran D.H. Copp Gordon Cox AL. Cullen K.G. Denbigh J.H. Edwards _ D.S. Falconer — AR. Fersht Sir Otto Frankel R.M. Gaze C.S. Hanes R. Hide R.W.K. Honeycombe J.T. Houghton K.L. Johnson J.S. Kennedy Peter A. Lawrence HS. Lipson NJ. Mackintosh A.L. McLaren P.D. Mitchell Joseph Needham JF. Nye W.D. Ollis R.M. Perham T.G. Pickavance J.C. Polkinghorne M.C. Raff LM. Roitt ELC. Slater G.H. Stafford J.E. Sulston S.A.S. Tait Grenville Turner J.C. Waterlow Sir James Baddiley Sir Robert Boyd B.Crossland _ B.E.Johnson J Mandelstam Sir Philip Randle JR.L. Allen G.H. Beale J. Bingham A.D. Bradshaw B.D. Bums J. Chatt P. Cohen VE. Cossiett HLS.M. Coxeter A.W. Cuthbert Richard Doll M.A. Epstein G.BR. Feilden JR.S. Fincham F.G. Friedlander R.G.H. Gell J.L. Harper Sir Austin Bradford Hill H.H. Hopkins N.C. Hughes Jones B.D. Josephson T.W.B. Kibble J.D. Lawson JF. Loutit P.M. Maitlis DJ. McLaren J.M. Mitchison J.A. Nelder _ Sir Charles W. Oatley William Parry J.D. Pettigrew L.M. Pickford G. Pontecorvo R.C, Rainey G.B. Segal M. Smith Sir Frederick Stewart 1.C. Swallow Sir George Taylor Sir Michael Atiyah J.S. Bell P.O. Bishop M.S. Bretscher I, Butterworth P.H. Clarke D. Colquhoun T.G. Cowling DJ. Crisp Lord Dainton J.D. Dowell L. Essen WS. Feldberg David J. Finney G. Fryer Sir John Gray B.S. Hartley R.A. Hinde Gabriel Horn H.E. Huppert N. Kemmer Francis King CJ. Leaver G. Lowe E.H. Mansfield L. Mestel H.K. Moffatt S.R. Nockolds P.H. Nye RS. Pease C.G. Phillips N.W. Pirie AF. Posnette D.A. Ramsay N. Sheppard LM. Sneddon WJ. Strang David Tabor J.C. Taylor Marthe L. Vogt J.Z. Young T.B.Benjamin D.P.Burkitt G.K. Batchelor RE. Bell Sir Walter Bodmer D.M. Brown F.W. Campbell J.H. Coates R.C. Cookson Sir David Cox M.J. Crumpton P. de Mayo J.D. Dunitz N.L. Falcon M.A. Ferguson-Smith CE. Ford S.D. Garrett R.W. Guillery O.V.S. Heath E.D. Hondros M.R. Home A.J. Jeffreys David G. Kendall H.G. Kuhn AJ. Leggett F.C. Macintosh R.EF. Matthews Sir Ashley Miles A.E. Mourant D.G. Northcott A.G. Ogston Charles Pereira Sir David Phillips R.V. Pitt-Rivers J.R. Postgate J.A. Ramsay L. Siminovitch Sir Richard Southwood J.T. Stuart JF. Tait G.FJ. Temple C.T.C. Wall John G. Thompson P.M.Biggs B.A.Cross Sir Francis Graham-Smith A.E.Green R.Loudon S.E.Moorbath L.Weiskrantz I.G.Macdonald 0.Penrose D.H.Williams -6- (iii)Among the supporters of this Campaign are also present and former Rectors, Vice-Chancellors, and Presidents of Univer- sities in many countries: Abdiel Adames (Panama) LP. Blanchard (Moncton) D. Bok (Harvard) Cristovam R.C. Buarque (Brasilia) J.E. Chamberlin (New College, Toronto) Jill Conway (formerly Smith, USA) Ataulfo M. da Costa (Uberiandia, Brazil) José J.C. de Carvalho (Paraiba, Brazil) Berard Desbals (formerly Limoges, France) Marco A. Fiori (Londrina, Brazil) Margaret Fulton (formerly Mt. St. Vincent, Canada) John Godfrey (“farmexiy!. King \"s; College, Halifax) Pedro W. Guimaraes (Goias, Brazil) Keith Hancock (Flinders, Australia) Leslie Harris (Memorial, Newfoundland) Ron Ianni (Windsor, Canada) Donald Kennedy (Stanford, USA) Eva Kushner (Victoria, Toronto) A. LeRoy Greason (Bowdoin, USA) Sir J. Lyons (Trinity Hall, Cambridge, U.K.) Jair S. Madureira (Mato Grosso, Brazil) John Marburger (Stony Brook, USA) B.C. Matthews(“ormerly Guelph, Canada) Jacques Monet (”. Regis, Canada) George Pedersen (Western Ontario, Canada) Walter Pitman (OISE, Canada) Carlos Araya Pochet (Costa Rica) Klaus Ring (J.W. Goethe, Frankfurt) William Saywell (Simon Fraser, Canada) Brian Segal Guel phy Canada) Robert Smith (Western Australia) David Strangway (British Columbia, Canada) José Tafner (Blumenau, Brazil) Georges Verhaegen (Brussels) Donald Wells (Mt. Allison, Canada) Brian Wilson (Queensland, Australia) Di Yerbury (siacquatie, Australia) Inge Lonning( Oslo) Hubert LaForge(Chicoutimi, Canada) Arthur Kruger(Woodsworth, Toronto) Naomi Hersom(Mt.St.Vincent, Canada) Terrence White(Brock, Canada) Narve Bjorgo (Tromso, Norway) E. Bloustein (Rutgers) P.J. Boyce (Murdoch, Australia) Aldee Cabana (Sherbrooke, Canada) Eduardo J. Coelho (Catélica de Campinas) Richard Cyert (Camegie-Mellon, USA) Hildiberto R. de Albuquerque (Fluminense, Brazil) Fernando P. de Sousa (Maring4, Brazil) James Downey (New Brunswick, Canada) Lord Flowers (University of London, U.K.) Michel Gervais (Laval, Canada) Arnfinn Grave (Bergen, Norway) R.D. Guthrie (U. of Technology, Sydney, Australia) Evelyn Handler (Brandeis, USA) Theodore Hesburgh (Notre Dame, USA) Paulo Renato Costa Jonga (Unicamp, Brazil) Patrick Kenniff (Concordia, Canada) Alvin A. Lee (McMaster, Canada) M.L Logan (Monash, Australia) Hor&cio Macedo (Rio de Janeiro) John Mallea (Brandon, Canada) Sir Roy Marshall (formerly Hull, U.K.) P, Meincke (formerly P.E.I., Canada) Jorge Nagle (Sao Paulo, Brazil) Jose Pinotti (formerly Campinas, Brazil) Rodolfo J. Pinto de Luz (Santa Catarina, Brazil) Peter Richardson (University College, Toronto) José H. Santos (Minas Gerais, Brazil) , Edni O. Schroeder (Met. Bennett, Brazil) David Smith (Queen’s, Canada) Eliv Steinnes (Trondheim, Norway) John Stubbs (Trent, Canada) Jurgen Timm (Bremen, Germany) Ron Watts (formerly Queen’s, Canada) Jerome Wiesner (formerly M.LT.) Douglas Wright (Waterloo, Canada) Paul Olum(Oregon ) Ove Nathan(Copenhagen ) Jean-Guy Paquet(Formerly Laval) Robin Farquhar(Winnipeg G.R.Williams (Scarborough, Toronto) Marvin Goldberger(Inst.Adv.Study, Princeton) -7- (iv)Among the supporters are also many other scientists, scholars, writers, artists, musicians, trade unionists, clergymen, persons and organizations in all walks of life, in many countries; we list only a very few: ‘Field Medalists (for mathematicians, similar to the Nobel Prize): Sir Michael Atiyah, David Mumford, Laurent Schwartz, Lars Ahlfors, Stephen Smale, Charles Fefferman, Michael Freedman, Pierre Deligne, Lars Hormander, William Thurston, Alain Connes, Simon Donaldson. John G. Thompson, Heisuke Hironaka, Shing-T Yau Other Mathematicians (to name o afew): Jacques Dixmier, Dennis Sullivan, Edward Nelson, Robert Edwards, M. Kreck, W. Ambrose, B. Monteiro, W. Bloom, J . Seade, Joel Lebowitz, Hale Trotter, Lipman Bers, Leon Kushner, Ezio Stagnaro, Miles Reid, Jean Louis Verdier, Jean Francois Mela, Robert Anderssen, Carl Faith, Marcos Sebastiani, Udo Simon, Gyorgy Targonski, L. Amold, Dieter Kohnlein, Kurt Leichtweiss, Shokichi lyanaga, Kenichi Shiraiwa and 23 other senior mathematicians in Japan, Irwin Kra, John Mather, Raoul Bott, Nathan Jacobson, Jean Dieudonné, D. Woodhouse, Gustave Choquet, Henri Cartan, Jacques Tits, Barry Mazur., fF. Hirzebruch, T.W.Kormer Other Physicists in many countries, among them: Kurt Gottfried, Elliot Lieb, John Charap, Herman Feshback, Daniel Kastler, Rudolf Haag, William McGowan, J. Trumper (Pres. German Physical Soc.), Myer Bloom, Paul Kessler, J.P. Mathieu, Eduardo Amaldi (Pres. Accad. Nazionale dei Lincei), Rafael Sorkin, Fritz Rohrlich, S. Frankel, H. Rohrer, Per Hemmer, Walter Kohn, Peter Demos. Arthur Wightman, Charles Nelson, Peter Sigmund, John Blewett Other Musicians: John Weinzweig, Harry Freedman, Anton Kuerti, Louis Applebaum, Maureen Forrester, John Beckwith, Oscar Peterson, Oskar Morawetz, Leon Pommers, Gerard Kantarjian. Violet Archer Other Writers: Per Wastberg, Arthur Miller, Timothy Findley, Susan Sontag (Pres. PEN. Amer. Center), Northrop Frye, June Callwood, Marion Andre, Robert Fulford, Robertson Davies, Margaret Atwood, Francis King (Pres. International PE.N.), Edith Fowke, Pierre Berton. Graham Greene , Ariel Dorfman Clergymen: Rt. Rev. Paul Moore Jr., Bishop of New York Most Rev. Michael J. Peers, Primate of the Anglican Church of Canada Very Rev. Lois Wilson, President, The World Council of Churches Most Rev. Edward Scott, President of the Canadian Council of Churches Moderators of the United Church of Canada (present and former): Dr. Sang Chul Lee, Dr. Anne Squire, Clarke MacDonald, Bruce McLeod, Robert Smith, Lois Wilson Rabbi Dow Marmur, Holy Blossom Temple, Toronto _ Father William Addley, S.J., Provincial Superior Henri Goudreault, Bishop of Labrador City, Canada Sister Mary Rose Rawlinson, F.C.J., Provincial Superior R.F. Wooton, Associate Secretary, Uniting Church in-Australia John Sherlock, Bishop ot ‘London, Canada The Revd. The Lord Soper, Methodist Church, U.K. Rt Rev. A.C. Holland, Bishop of Newcastle, Australia Jean-Guy Hamelin, Bishop of Rouyn-Noranda, Canada Archbishop James Hayes, Halifax, Pres. Canadian Coriference of Catholic Bishops   Also: Lord Avebury (Chair, U.K. Parliamentary Human Rights Group); Supreme Court Justices Walter Tamopolsky, J.D. Amup (retired), Alvin Rosenburg, R.M. Rogers; Albert Shanker (Pres. Amer. Fed. of Teachers); Dr. J.L. Gonzalez (Pres. Colegio Medico de Chile); F.R. Gomez (Dean, Fac. Science, Nat. Univ. Mexico); Dr. Klaus Hansch (Member European Parliament)., Ramon Latorre(Pres. Latin Amer. Soc. Biophysicists), Yvon Boaulne {Former Ambassador from Canada to the Human Rights Commission of the United Natiena) , Mrs. Susan Baird(Lady Provost of Glasgow), E. Thijn(Burgomaster of Amsterdan ) Hon Nigel Griffiths, M.P.(U.K.) , © Senators and 14 M.P. in Canada, David Suzuki Adrienne Clarkson Organizations: The New York Academy of Sciences National Academies of Science of Bolivia, India The Committee of Concemed Scientists Can. Cttee. of Scientists and Scholars Math Section (Royal Soc. Canada) Comité des Mathématiciens (France) Comité des Physiciens francais\" The German Physical Society The Norwegian Physical Society The Chemical Institute of Canada Comité Yuri Orlov (Switzerland) Fellowship of Reconciliation Actors Equity of New Zealand, Actors Equity of Australia Aliance Can. Cinema, T.V., Radio Artists Canadian Labour Congress United Steelworkers of America Ontario Federation of Labour The Jesuits of Canada Dominican Friars of Toronto Ass’n Can. Univ. Teachers of English The Criminal Lawyers’ Association (Canada) The Law Union of Ontario National Math Societies of Calcutta, Canada, Costa Rica, Mexico, Norway, Portugal, Israel Faculty Associations Can. Universities: Toronto, Brock, Mt. Allison, Western Ontario, Moncton, Simon Fraser, Windsor, , McMaster, Laval, Guelph, Trent, Victoria, Manitobar Champlain College PEN., International Playwrights, Essayists and Novelists (Can. Centre) Social Responsibility and Justice Cee United Church, Australia) ACM Committee on Freedom of Science Inter-Church Cue. on Human Rights in Latin America (Canada) Asocidcion Universitaria y Cultural Andres Bello (Chile) Division of Worid Outreach (London Conf., United Church, Canada) Can. Centre for Victims of Torture The Pavlovian Society of North America Swedish Committee for Freedom of Science The German Commission for Rights of Chilean Women The Committee of University Scientists of Germany (FRG) The Ontario Chapter of the Chilean College of Physicians Faculty of Science of Univ. of Witwatersrand (South Africa) Brazilian Society for Mathematics, Applied and Computing (SBMAC) National Education Association of the USA Latin America Society of Biophysicists Fed. Australian University Staff Associations Mouvement internationale des Juristes Catholiques —~ Baptist Convention of Ontario and Quebec The Royal College of Psychiatrists   Please contact one of the regional representatives if you wish to be listed as a supporter of this Campaign, or if you can offer to distribute copies of the Bulletin (specify how many, to whom, can you make the needed copies or do you need them supplied to you), or if you can make a modest financial contribution (equivalent to 10 or 15 dollars), payable to: CHR. The work of this Campaign is done entirely by volunteers, there are no salaried employees. Our expenses are high, but they are exclusively printing, postage and telephone. We distribute a tremendous number of copies of the Bulletin, especially in Chile.   Regional representatives: Professor Elliott Lieb, Princeton University, Princeton, NJ 0854,USA Dr. Miles Reid/ Mathematics/ University of Warwick/ Coventry CV4 7AL/ U.K. Prof. Terje Oestvold/ Inorganic Chemistry/ University of Trondheim/ 7034 Trondheim/ Norway Prof. Kenichi Shiraiwa/ Mathematics/ College General Education/ Nagoya University/ Chikusa-ku, Nagoya 464/ Japan Prof. Dr. Udo Simon/ Mathematics/ Technische Universitat Berlin/ Strasse des 17 Juni 136/ D-1000 Berlin/ FRG Germany Prof. Anibal Faundes M.D/ Caixa Postal 6181/ CEP 13.081/ Campinas/ SP Brazil Prof. Paul Kessler/ Lab de Physique Corpusculaire/ College de France/ 11, Place Marcelin-Berthelot/ 75231 Paris/ France Prof. Mischa Cotlar/ Res. Paris, Ap.82/ Av. E] Mirador/ La Campina/ Caracas 1050/ Venezuela Prof. Israel Halperin/ Mathematics/ University of Toronto/ Toronto, Ontario/ MSS 1A1/ Canada   “The man dies in all who keep silent in the face of tyranny.’’ Wole Soyinka, Nobel Laureate", "Halperin, Israel ; International Campaigns for Human Rights", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2ifa-aqnu.n6kr", "00000000-0000-0000-DEE3-4E78B22BFDB8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Memorandum from Edward D. Korn [on FY1990 Congressional Justification]", "101584910X354", null, "1988", "18 November 1988", "The Director of the National Heart, Lung , and Blood Institute informs laboratory and branch chiefs at NIH that it is time to provide material to be used to prepare a narrative for the Congressional justification of fund allocation.", "Memorandums", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Date: November 18, 1988 From: Edward D. Korn, Ph.D. Acting Scientific Director, NHLBI Subject: FY 1990 Congressional Justification for DIR To: Lab/Branch Chiefs Now is the time that we must provide the material to be used to prepare the narrative for FY 1990 Congressional Justification of DIR. I enclose (1) a copy of last year's justification, and (2) a copy of your submission last year on which the narrative was based. Please prepare not more than 2 paragraphs in lay terms, which: - identifies a significant recent research finding or problem under investigation; - explains why this area is significant - identifies the next research steps; and - describes how this research may alleviate a health problem In addition to this material, we would like to have a separate sheet on research related to AIDS from each lab or branch that feels it is doing AIDS related research or will do AIDS related research. Rather than define AIDS related research, please describe the research that you think is AIDS related. The format for the AIDS narrative is as follows: Current Research on AIDS: FTEs: Prof.; Tech.; Support Planned AIDS Research: FTEs: Prof.; Tech.; Support I need this material in my office absolutely no later than Tuesday, November 29th.  I know the Thanksgiving holiday makes this a busy time, but we were not notified until today.", "Korn, Edward D.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6tpf~4qhj-jfru", "00000000-0000-0000-A47E-E3984738AB53", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from V. Richard Damerell to Marshall W. Nirenberg", "101584910X355", null, "1969", "7 January 1969", "Damerell informs Nirenberg that Dr. Robert B. Livingston has developed a \"Philosophy for Survival\" that may provide a philosophical basis for research.  The basic theory \"suggests that the human energy state, or more specifically, the cell energy state, should play a role in the incidence and prevention of cancer.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "January 7, 1969 Dear Dr. Nirenberg: As I was about to mail the enclosed material to Dr. Robert B. Livingston, the thought came that this philosophy might also hold some interest for you, since it seems to bring together much vital frontier work into a unification with nature. I will be honored if you, too, will read the article A PHILOSOPHY FOR SURVIVAL. If it helps in any small way to provide a philosophical base for your research I will be most happy that I sent it to you. Thank you for any interest this may arouse. Sincerely, V. R. Damerell P.S. The basic theory in the enclosed article suggests that the human energy state, or more specifically the cell energy state, should play a role in the incidence and prevention of cancer. I am sure the NIH has gone into this. VRD", "Case Western Reserve University. Department of Chemistry ; Damerell, V. Richard", null, null, null, "National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jefs_fn2t_86pe", "00000000-0000-0000-3609-BA2AE41C025E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Jorge E. Allende, Hermann Niemeyer to Marshall W. Nirenberg", "101584910X358", null, "1969", "February 1969", "Allende and Niemeyer invite Nirenberg to participate in the Interamerican Council for Culture's Multinational Training Project for Biochemistry, created by the Organization of American States.  A handwritten addition to the letter suggests that Nirenberg's presence as visiting professor would be invaluable for starting new programs in countries such as Argentina, Brazil, Chile, Venezuela, and Mexico.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Jorge E. Allende.", "Copyright may apply", null, null, "Dear Dr. Nirenberg: The Interamerican Council for Culture, created by the Organization of American States has approved a project to develop basic and applied biochemistry in Latin America, as part of the broad Regional Program for the Development of Science and Technology.  The countries Argentina, Brazil, Chile, Venezuela and Mexico were recognized to have nuclei of trained biochemists actively doing research. Biochemical centers in these countries, therefore, were named \"Responsible Centers\" and were given the task of training biochemists from other countries of the continent. In Chile, the Instituto de Quimica Fisiologica y Patologica of the Faculty of Medicine, University of Chile, was selected as Responsible Center, and myself, Hermann Niemeyer as the Responsible Director for the operation of a Multinational Project with special responsibility towards training people from Bolivia and Peru. In addition, two Active Participating Centers (Department of Biochemistry of the Faculty of Chemistry and Pharmacy, and Department of Chemistry of the Faculty of Medicine) will collaborate in this endeavor. The following committee elected by biochemists from Peru and Chile will advise the Responsible Director in the operation of this project: Drs. Jorge E. Allende (Chile, Secretary), Alberto Cazorla (Peru), Osvaldo Cori (Chile), Frank Marcus (Chile) and Marino Villavicencio (Peru). The approved project includes the participation of Visiting professors from the United States and/or Europe.  These professors would stay in the Responsible Centers for variable periods (one to twelve months) according to the arrangements that can be made and the requirements of the project. During this time, these professors will participate in training courses on a college or advanced level, and will collaborate and advise in the research activities of the existing groups. The approval of the Regional Program occurred this past December and this did not give us enough time to contact with appropriate advanced notice the people who might possibly be interested in participating. We hope, however, that some of our colleagues might accept to come during 1969.  The summer months of the northern hemisphere coincide with the middle of our academic year during which there is a month of winter holiday and therefore would be ideal for the training of advanced students. The visiting professors would receive their passage and US$ 1166 a month to finance their stay in Chile. When the stay is of six months or more, extra funds are provided for transportation of one accompanying person and of personal effects. We would appreciate it very much if you would consider the possibility of participating in this program this year or in the following years (1970 and 1971). In any case, please inform other members of your Department and advise us if any of them would be willing to come. When we have an estimate of the people that in principle are available to participate, we will extend the formal invitation.  The selection will be based on our current teaching schedule and the funds available. We are including a form in order to facilitate the arrangements and programming of activities. Please do not hesitate to request any further information that could help you to make a decision in regard to your participation in this project. Thank you for the attention that you could give to this letter. Sincerely yours, Jorge E. Allende Secretary Herman Niemeyer Responsible Director February 1969 [Handwritten note:] Dear Marshall: It would be wonderful if you could come even for a short while.  Your presence here could really help us to start this program right.  Best regards to Perola and the friends in the lab", "Allende, Jorge E. ; Niemeyer, Hermann", null, null, null, "National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qxem~d25s_pc2j", "00000000-0000-0000-3632-E2612DEB741F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Bob Dole, Joe Biden, Rudy Boschwitz, Chic Hecht to Marshall W. Nirenberg", "101584910X359", null, "1986", "11 April 1986", "U.S. Senate members invite Nirenberg to the U.S. Congress salute to \"Vision and Guidance\", sponsored in cooperation with the American Friends of Lubavitch to mark the completion of the 850th year since the birth of the great philosopher, physician, and scientist Maimonides and the ushering in of the 85th year of the Lubavitcher Rebbe Shlitoh.  Nirenberg's achievements and contributions to science were also scheduled to be recognized.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "April 11, 1986 Dear Marshall: You are cordially invited to join us at the U.S. Congress salute to \"Vision and Guidance\" on April 17, 3:00 p.m. at the Caucus Room of the Russell Senate Office Building in the Capital. This event, sponsored in cooperation with the American Friends of Lubavitch, will mark two extraordinary milestones which coincide on the calendar, the completion of the 850th year since the birth of the \"Maimonides\" and the ushering in of the 85th year of the Lubavitcher Rebbe Shlitoh. \"Maimonides\" Rabbi Moshe ben Maimon (1135-1204), who was a great giant in Jewish philosophy and codifier of Halacha, was at the same time a great physician -- a physician to courts, who was also erudite in mathematics, physics, astronomy and metaphysics.  He harmonized reason with revelation, resulting in his role as the \"Guide\". The Lubavitcher Rebbe Shlitoh, Rabbi Menachem Schneerson, today eight centuries later serves a parallel role.  The U.S. Congress has appropriately recognized and the President has accordingly proclaimed his birthday as \"Education Day USA\". Your achievement and contribution to science today will be recognized at this event. The group of \"friends\" who are sponsoring this event, will be happy to cover your travel and lodging expenses.  Kindly contact immediately the office of the American Friends of Lubavitch at (215) 725-2030 for arrangements. Hoping that your schedule will allow you to accept our invitation and looking forward to greeting you. We remain Sincerely yours, Rudy Boschwitz Bob Dole Joe Biden Chic Hecht", "Biden, Joe ; Boschwitz, Rudy ; Dole, Bob ; Hecht, Chic ; United States. Congress. Senate", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-a3pe-xz8r-rexb", "00000000-0000-0000-C6D5-B44BA870C216", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Memorandum from Sandy Cain, National Institute of General Medical Sciences to Marshall W. Nirenberg", "101584910X360", null, "1996", "1 May 1996", "This memorandum includes a copy of the description of Nirenberg's laboratory as it appears in the latest Pharmacology Research Assistant program catalog.  This letter is sent for Nirenberg's approval of the description.", "Memorandums", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "May 1, 1996 TO: Preceptors, Pharmacology Research Associate (PRAT) Program FROM: Program Assistant, Pharmacology Research Associate Program, NIGMS SUBJECT: Revision of the Pharmacology Research Associate Brochure Enclosed is a copy of the description of your laboratory as it appears in the most recent PRAT catalog.  Please review this carefully for accuracy and readability.  Remember that this description may be the deciding factor in a PRAT fellow choosing your laboratory over someone else's.  Try to convey in this summary of your laboratory's research the excitement you feel about that research. If you wish this description to appear just as it is, please check the box marked ACCEPTABLE AS IS.  If you wish to change the description, please check the box marked CHANGE.  You may make changes directly on the page or attach a new version. You may either fax this information to us at 301-480-2802 or e-mail us at  PRAT@GMl.NIGMS.NIH.GOV by Monday, May 20, 1996. Sandy Cain Enclosure [ ] ACCEPTABLE AS IS [ ] CHANGE NAME OF PRECEPTOR: Marshall W. Nirenberg, Ph.D. Laboratory of Biochemical Genetics Section on Molecular Biology 301-496-2401 Basic problems in molecular biology and biochemistry are studied, especially molecular mechanisms that regulate gene expression during embryonic development.  Current research focuses on characterizing DNA clones that  correspond to novel mouse or Drosophila homeobox genes, defining mouse genomic DNA sequences that regulate gene transcription, and identifying regulatory sequences in cloned DNA that correspond to the gene for the a-subunit of an L-type voltage-sensitive calcium channel.  Clonal lines of neuroblastoma and somatic hybrid cells are used in some studies.", "National Institute of General Medical Sciences (U.S.) ; Cain, Sandy", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tgtb-4aza-ktxz", "00000000-0000-0000-7C87-8F08704B580F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Claire M. Lathers, Albany College of Pharmacy to Marshall W. Nirenberg", "101584910X361", null, "1996", "15 March 1996", "Lathers informs Nirenberg that he has been invited to accept an honorary doctorate due to his knowledge and expertise in biochemistry, his accomplishments in the areas of physiology and medicine, and his position as a professional role model for students.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Claire M. Lathers.", "Copyright may apply", null, null, "March 15, 1996 Dear Dr. Nirenberg: On behalf of the Albany College of Pharmacy, I am pleased to formally invite you to accept an honorary doctorate at our commencement on June 2, 1996. We believe that your knowledge and expertise in biochemistry and your accomplishments in the areas of physiology and medicine commend you as a professional role model for our students and merit public recognition and acclaim. We will be honored to host your visit and have you participate in our graduation weekend activities. As a degree recipient you are welcomed to make brief comments, if you wish, however you will not be expected to make a speech at graduation. This past fall we instituted our \"Profiles in Leadership\" program wherein we provide forums for our most recent honorary degree recipients to address our students, faculty, staff, the community and the press. The two-day event included a breakfast with members of the Chamber of Commerce, a presentation to students and faculty, an informal lunch with faculty and a formal dinner with, among others, the members of the Board of Trustees. We found this provided a varied and meaningful opportunity for everyone to interact. We will look forward to your participation in our 1996 \"Profiles\" program which will be held on September 11th and 12th. We will be sending you a complete listing of events surrounding graduation and will make every effort to facilitate your visit. In the meantime if you have any questions please feel free to contact me or my assistant, Mary Van Ryn. Sincerely yours, Claire M. Lathers", "Albany College of Pharmacy ; Lathers, Claire M.", null, null, null, "National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cbyw_yx62-jwpw", "00000000-0000-0000-D869-973A714DE209", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Memorandum from Edward D. Korn", "101584910X372", null, "1996", "18 March 1996", "This memo is a report from Jack Crowley of the Massachusetts Institute of Technology on the issue of funding for biomedical research.  Crowley summarizes the comments of Rep. John E. Porter, Chairman of the House Appropriations Subcommittee on Labor, Health and Human Services at a rallying speech for Research!America.  Issues addressed include the report from Citizens Against Government Waste that NIH is \"foolish government spending\" and the suggestion for a national trust fund for research.. NOTE: The original text is cut off on the left side on page 1.", "Memorandums", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Courtesy of Jack Crowley, MIT There Are Many Lessons To Be Learned Here: At noon 13 March Hon. John E. Porter, Chairman of the House Appropriations Subcommittee on Labor, Health and Human Services, Education, and Related Agencies, an ardent proponent of biomedical research, gave a rallying speech to Research!America on this subject. Rep Porter said: What worked in FY 1996 won't be enough in FY 1997. Last year, not many know this, I called about ten university presidents (he named several institutions). All graciously agreed to energize their boards. We will do that again this year -- more broadly. We must balance the budget, make the government more efficient, program by program. However, \"there is no more vital endeavor\" for government than NIH. How do we make this contageous?  I need your help. The American people must make it a priority. You must do this. Infect the people with the needs. Three targets in Congress: 1. John Kasich and the members of the H Budget Committee. The House Budget Resolution may be on the House Floor in mid-April. 2. Bob Livingston and the House Appropriations Committee. Markups will occur in mid-May. 3. Every Member of the House and Senate. Set meetings in each District. Visit the Members in their local offices. Tell them what NIH means to them personally in their home districts. We need to do this because even some in the leadership don't understand. One leader in the House (not the Speaker) said, \"why do we need NIH?   Research is being done in all of our universities.\" We have to make the argument that in both sectors (universities and industry) we lead the world. It is the most efficient government spending of all. It makes longer, more healthy lives across the country and around the world. It provides for greater economic growth, more exports, more high technology jobs. We must get this message out to Congress, to newspapers, to local media and to the people. Speak at local community organizations. Urge others to write to their Congressman. I believe in the firepower theory of lobbying. If you fire enough bullets you will hit some targets. Research!America's suggested actions are right. They do make a difference. Popularize Research and what it does for our society. We cannot forget last year. The House Budget Committee began with a 5% cut which translated to a 25% cut on a static base over 7 years.  The Senate then suggested a 10% cut.  God bless Mark Hatfield who turned it around. However, he's retiring. The Senate provided only a 2.7% increase and then did not pass the bill. We may be looking at something similar again. Such cuts would be disaster. Awards would drop. Talented minds will move to other fields. We can provide a 6.5% increase for NIH this year if we all work aggressively in the home districts and home groups. We can accomplish it! Response to questions: 1. Citizens against Government Waste challenged NIH as foolish government spending. Sam Donaldson helped there by saying that such spending may appear foolish at first blush but it is good science. I am particularly worried now. Not many ideas in the FY '96 bill got funded like NIH.  There are 800 line items in the bill. All of the others will target NIH arguing that it is not fair to cut them in order to fund NIH. But it is not a matter of fairness. It is a matter of priorities and of where the dollars should be spent. What the Senate did yesterday (adding funds for several programs, including education) won't help next year because there will not be the money to sustain those increases. We need to be careful that the NIH is not targeted. Last year I took all 5 new members of the subcommittee to NIH for a day and then asked Dr. Varmus to bring a group of Nobel laureates before the Subcommittee. This year I'm taking our spouses with us to NIH. I called ten university presidents; this year we want to call 100!   I want to reach every single university president. There is nothing more important than having the president of the local university come into the Member's office and tell him that this is something important. Also, look ahead to the fall electrons. Ask the local candidates what they think about this issue. But talk with them ahead of time so that they give the right answer. If you do that the Member or candidate learns and then those in the room hear the right answer. And, do not forget the President and the Secretary of DHHS. They need to be lobbied. It will be much tougher, much tougher this year. 2. A national trust fund for research?  We ought to look at long-term solutions. Obviously, there was no peace dividend. With dropping award rates a 6.5% increase for NIH is minimal. We need to do much much better in the future. Senators Harkin and Hatfield have raised the question of a trust fund. Tobacco is a wonderful idea.  But, we will raise NO taxes. Maybe we can dedicate a percentage of existing taxes on tobacco to biomedical research. But don't get off the appropriations fight until it has run its course!  Last year's 5.7% increase was about the cost of one B-2 bomber. We don't need more SeaWolf submarines. And, we do not need a tax cut either! (Which does not make me popular within my party!)  And, do not make predictions of cuts in research. Do not accept cuts! The Speaker said that he would review not only biomedical research and NIH but all federally funded research -- and he did. But not much changed. Make appointments to see the Speaker and make the case for all government research. Republicans ought to support all research because it is essential to grow our economy, to jobs and to sustaining our high standard of living. We must not lose our technological lead. We must chose our priorities.", "Korn, Edward D.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ms7a_rm8y.yqki", "00000000-0000-0000-8FFA-E810EDE68F7D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Memorandum from Michael M. Gottesman", "101584910X373", null, "1996", "21 February 1996", "In this memo, Gottesman addresses the issue of care and use of animals in biomedical research.  He emphasizes the requirements to comply with federal law, regulations, and policies, as well as recognized standards and guidelines.  Instructions are given for reporting violations and assurance is provided for those who come forward.. NOTE: The original text is cut off on the top and left side on page 1.", "Memorandums", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "February 21, 1996 To: Addressees From: Deputy Director for Intramural Research, NIH Subject:  Reporting Animal Care and Use Concerns within the NIH Intramural Research Program The purpose of this memorandum is to reiterate my commitment to maintaining full and open communications regarding animal care and use in the NIH Intramural Research Program (IRP). While this memo was prompted by a finding identified during an Office for Protection from Research Risks evaluation of our program, I feel strongly that a clear and thorough understanding of NIH management and administrative practices by all IRP staff will enhance our research environment. The care and use of animals in biomedical research carries with it the requirement to comply with federal laws, regulations and policies, as well as recognized standards and guidelines. That collection of regulations, policies and standards governs all aspects of our animal-related activities, including our relationships with other NIH employees and contractors. Anyone in the NIH IRP who has any concerns regarding any aspect of the care and use of animals in research here at NIH is encouraged to voice that concern. At the animal facility level, each facility veterinarian has the authority and responsibility for ensuring compliance with all animal care and use standards and is the appropriate individual to receive initial concerns related to that facility.  The Chairperson of the ICD Animal Care and Use Committee (ACUC) is the most appropriate individual to field concerns about animal research procedures or techniques, especially those that might be performed in laboratory settings. Concerns that might not be directly related to a particular facility or ICD, or are not appropriately dealt with at a local level should be addressed to the Office of Animal Care and Use (OACU), Office of Intramural Research, or to me. Concerns received by any of these individuals or offices will be reviewed and corrective measures instituted if appropriate. Allegations of potential wrong-doing at the local level should be reported to the facility veterinarian or the Chair of the ACUC having responsibility for activities in that facility. The immediate responsibility for reviewing, and if necessary, investigating allegations rests with the ACUC Chair. In some cases, persons may be unwilling or reluctant to make allegations at the local ICD level. In those cases, such individuals may contact the Director, OACU or me directly, and I will decide on appropriate action. The Director, OACU, must be promptly informed of ail such allegations, and is responsible for informing me, as the Institutional Official, of these allegations; I will determine the level at which the allegation should be pursued, including activating the NIH Ombudsman, who would conduct an immediate assessment of the alleged conditions, as outlined in the NIH Animal Research Advisory Committee Guidelines (section on NIH Ombudsman attached). If I so direct, the ACUC Chair will assemble an investigative team made up of scientists, veterinarians and other administrative or management officials, as appropriate, to assure a balanced and thorough assessment of the allegation. That team will be made up of intramural, intramural and extramural, or totally extramural individuals with expertise in biomedical research, personnel management, and/or other specialty fields deemed appropriate to the situation at hand. Alternatively, I may decide to conduct an investigation out of my immediate office. In summary, any individual who has concerns related to our use of animals in biomedical research is encouraged to voice those concerns at whatever level of NIH authority that is appropriate. To that end, I wish to stress that the NIH will not tolerate any reprisal against an individual who has come forward with concerns or allegations of wrong-doing involving the care and use of animals here at NIH. Such reprisal is prohibited under the Department of Agriculture regulations implementing the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and perpetrators are subject to sanctions. Individuals who feel that a personnel action has been taken against them because of their reporting of an apparent violation of animal care and use requirements, should present their case to their supervisor, their ICD Director, the NIH Director, the Office of the Inspector General, or the Office of Special Counsel. If individuals allege any form of discrimination, they should file their complaint with the Office of Equal Opportunity. Any questions or comments regarding the intent or contents of this memorandum should be directed to me or to the Director, Office of Animal Care and Use. Michael M. Gottesman, M.D.", "Gottesman, Michael M.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ddxe-j33y~6484", "00000000-0000-0000-387D-615B33542F68", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Minutes from lab chiefs meeting", "101584910X374", null, "1996", "15 April 1996", "In the minutes from this meeting a number of administrative and managerial issues are discussed, including: animal research protocols, annual reports, termination of postdoctoral trainees, promotion and tenure procedures, authorized use of radioactive material, requests for support with green card applications, new performance appraisal systems, and traveling procedures.", "Minutes (administrative records)", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Lab Chiefs Meeting April 15, 1996 Minutes The meeting was called to order at 3:00 pm by Dr. Edward Korn. All Lab/Branch Chiefs were present except for Dr. Balaban and Dr. Burg, who was represented by Dr. Ken Spring. Also present were Mr. Soclof and Mr. Hanson. Guests: Dr. Don Fox and Mr. John Witt gave a presentation on the Clinical Center Essential Maintenance and Safety Utility Renovations project. The program mission is to \"extend the life of the original Clinical Center 15 years and maintain a safe and healthy environment for biomedical research until a replacement facility can be constructed\". A handout which summarizes the presentation was distributed to attendees. Dr. Cynthia Dunbar, Hematology Branch, gave a presentation on her current research. l. Outside Activities/Official Duty Activities: Dr. Korn summarized the differences between these two categories of activities. The criteria for determining whether an activity is \"outside\" or official is not based on whether you are paid but on how closely it is related to your current \"official duty work\". 2. Animal Research Protocols - The revised policy states that new protocols will not be implemented until it has been determined by Drs. Hoyt and Korn that adequate caging is available. This is in addition to approval by the Animal Care and Use Committee. Also, and for the same reason, collaborations with other ICDs that ultimately will utilize NHLBI resources for housing animals (for example, knockout or transgenic experiments) must not be initiated without approval of Dr. Korn. Lab Chiefs and PIs using animals will have to do more long-range planning before initiating certain kinds of animal research. The hope is that it will be possible to continue to support all animal research but resources are finite and needs continue to expand. 3. Annual Reports - Consistent with the common understanding that the Principal Investigator is the individual to whom the resources have been committed,  the Scientific Directors have agreed that only tenured and tenure-track scientists should be identified as the \"Principal Investigators' on Annual Reports. The ICDs commit resources (and scientific independence) only to tenured and tenure-track scientists. When justified, others may be listed as co-PIs. The concept of a PI is different than and unrelated to the concepts of \"first author\" or \"senior author\" of a manuscript. 4. Termination of Postdoctoral Trainees - Postdoctoral trainees are usually, but not always, given an initial appointment of two years with one year renewals, up to 5 years or, with special exception, up to 8 years. All time at the NIH is counted, irrespective of the mechanism or source of funding.  The trainee should be given approximately 12 months to find another position. Because of the 5/8-year policy, from now on every postdoctoral trainee beginning the 5th year will receive a letter from Dr. Korn stating that this is the final year, unless a special exception has been requested and approved.  Dr. Korn distributed a memo, summarizing the importance of discussions between the trainees and their mentors during the fourth year at NIH, if not sooner. Dr. Korn asked the Lab/Branch Chiefs to review the attachment which was an individual list for each Lab/Branch of trainees in their 4th year or later postdoctoral year at the NIH and to determine its accuracy. Dr. Korn will send appropriate memos to the trainees. Except under special circumstances, every effort should also be made to give 11-12 months notice to trainees whose appointments will be ended earlier than the original termination date. 5. Promotion and Tenure Procedures - For everyone's clarification, Dr. Korn summarized the promotion process to Grades GS-12 and above. The Lab/Branch Chief sends a memo to the Scientific Director (SD) requesting the promotion which, if not disapproved at that stage by the SD, is forwarded to the NHLBI Promotion and Tenure Committee which advises the SD on all promotions of scientists to GS-12 and above. The SD has final approval authority for promotions to GS-12. Promotions to GS-13 through GS-15 must also be approved by the Director, NHLBI, and the Deputy Director for Intramural Research, NIH. NHLBI now has 10 tenure-track scientists. Conversion to tenure is initiated by a memo from the Lab/Branch Chief to Dr. Korn and, unless he disagrees, the scientist is reviewed by the NHLBI Promotion and Tenure Committee which makes its recommendation to Dr. Korn. If Dr. Korn agrees with a recommendation to convert to tenure, the request is then forwarded to Dr. Lenfant for concurrence and then to the NIH Tenure Committee before final approval by the Deputy Director for Intramural Research, NIH. Dr. Korn noted that, even though the Lab/Branch Chief is requested to submit names of references to the NHLBI Promotion and Tenure Committee, it is the Committee's prerogative to decide from whom to request letters of recommendation. Suggested names should be national and international leaders in the scientific field of the candidate and it would be helpful to the Committee to identify any relationships between the suggested references and the candidate. 6. Authorized Users for Radioactive Material - Dr. Korn told attendees that he is now requiring that every tenured and tenure-track scientist in DIR be certified by the Radiation Safety Branch as an authorized user. Individual lists regarding authorized users in each Lab/Branch were distributed for review and correction. The most up- to-date information regarding authorized users and those persons assigned to them is being obtained from Radiation Safety. Dr. Korn will request Radiation Safety to provide more training opportunities. 7. Requests for Support of Permanent Resident (Green Card) Applications - An increasing number of requests for letters of support for non-NIH scientists requesting permanent resident status or other changes in visa status are being received by NIH staff. Dr. Korn suggested that, before writing such a letter, one may want to seek the advice of Ms. Sylvia Funk, FIC. In any case, it is inappropriate to write such a letter on NIH letterhead and it should be made clear that one is supporting the application as a private individual and not in one's official capacity. Often, what the applicant needs is an official letter from a government agency stating that granting the new visa status is in the nation's interest. 8. Small Business Innovative Research (SBIR) - This program was discussed previously (see February, 1996 minutes); Dr. Ken Spring, DIR representative on the NHLBI Committee, gave an update on this program. Guidelines for intramural staff are being developed and hopefully will be available by the next Lab Chiefs' meeting. In summary, the proposals must be for a project which will lead to development of a product which can be sold by the company which receives the contract. Proposals should be submitted by early June for announcement in September. They will be reviewed in February and awarded in July.  The cost should be in the range of $500,000 to $850,000 and may involve more than one related project. Unlike other contract processes, the individual making the proposal can invite contractors to respond to the announcement requests, attend the Study Section meetings as an observer, be involved in the selection process for the contract award, and act as the project officer with the authority to establish interim deadlines and bench marks. 9. NIH and NHLBI New Performance Appraisal Systems - NIH has approved a new system which will be initiated this year. A near-final draft of the proposed NHLBI system has been distributed to every GS or WG employee for review. The current system will be replaced by a two-level system: \"acceptable\" or \"unacceptable\" performance. In order to implement this new system, it may be necessary to revise many position descriptions (PD) because the new appraisal elements will be derived directly from the PD. All appraisal elements will be \"critical\" and performance must be acceptable for all elements to obtain an overall rating of acceptable. Performance awards will no longer be directly linked to the annual appraisal period but will be given throughout the year as appropriate for the individual award. Thus, there will be more opportunities to reward deserving employees with cash awards than in the past. All employees have been asked to comment on the plan and comments should be submitted to Dr. Korn by April 26th so that he may send a consolidated response to the NHLBI committee that will prepare the final plan. If it seems desirable to do so, a meeting of all DIR employees will be held to discuss these new programs. 10. Sponsored Travel (348s) Update - Carroll Hanson reminded attendees that the HHS 348 for sponsored travel will not be forwarded to Bldg. 31 unless there is an attached copy of a letter from the traveler to the sponsoring institution which includes a statement that reimbursement must be made in the form of a check made out to NHLBI. Next Meeting Will Be Held On Monday, May 13 At 3:00 PM - 7S235", "National Heart, Lung, and Blood Institute", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-t5ga~vi8p_5fj2", "00000000-0000-0000-17DD-F0984EA8B9C5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from David Moushine, Weizmann Institute of Science to Marshall W. Nirenberg", "101584910X375", null, "1978", "23 February 1978", "Moushine congratulates Nirenberg on receiving the Ph.D. Honoris Causa by the Weizmann Institute of Science in Israel.  Logistical details are worked out in the letter and additional information is requested from Nirenberg.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of the Weizmann Institute of Science.", "Copyright may apply", null, null, "February 23, 1978 Dear Dr. Nirenberg, May I congratulate you on the conferment upon you of a Ph.D. honoris causa by the Weizmann Institute of Science and let you know that we are looking forward to having you with us in Rehovot in May. As you know, the conferment ceremony will take place on Monday, May 15 at 4.00 p.m. at the Wix Auditorium on our campus. Tentative reservations have been made for you on campus from May 13 to 17 and they may, of course, be changed according to your wishes. Your travel expenses will, of course, be borne by the Institute. Ms. Martha Loewenstein of the American Committee for the Weizmann Institute will contact you in regard to flight arrangements. The address of the American Committee is: 515 Park Avenue, New York, N.Y. 10022, telephone 212 Plaza 2-1300. In order to have a well-fitting gown, hood and cap made, I am afraid I must ask you to divulge your measurements, namely, height, width and particularly, size of hat. We would appreciate receiving these details at your earliest convenience. Should you wish to invite a few special Israeli friends or relatives to the ceremony, please let us have their names and addresses. Please let us have flight details so that we can arrange to meet you on arrival. Yours sincerely; David Moushine Director of Special Affairs and Secretary of the Association", "Moushine, David ; The Weizmann Institute of Science", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3bdf~gbhv~utfi", "00000000-0000-0000-700A-5667D8C16288", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Press release from the Union of Councils for Soviet Jews", "101584910X376", null, "1972", "3 March 1972", "NOTE: Attached to the news release, but removed for scanning, is a note from Jack Cohen that reads \"Dr. Nirenberg, I wanted to bring the situation of these Russian Jewish scientists to your attention.  I would like to discuss this with you.  I will try to call you Tuesday.\"", "Press releases", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "7", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "March 3, 1972 For Immediate Release [Photo = \"Roman Rutman\"] \"I Have Stopped Building Cities For Pharaoh\" -- Says Soviet Jew Following is the text of a message received today by Dr. Louis Rosenblum, Chairman of the Union of Councils for Soviet Jews from Roman Rutman, Pokrovsky Bulvard 14/5, apt. 47, Moscow, USSR. \"On Feb. 18, OVIR, the office dealing with the departure from the Soviet Union, informed me that my family was refused the right to repatriation to Israel. The OVIR official justified this refusal by the nature of my work:  I am a scientist in the field of automated controls and refusals in such cases are connected with alleged knowledge of 'State secrets.'  However, I have not dealt with classified work since 1960 and my wife had never anything to do with such work. \"Thus the reason for the refusal is quite apparent -- they do not wish to let scientists go. It seems that if I had not done my best during my professionally active career, if I had not been chief of the Laboratory in the Institute of Machinery of the Academy of Sciences, if I had not been a lecturer in the Institute of Radio Engineers, and if I had not obtained my B.S. and Ph.D. degrees, but had been spending my time collecting stamps, then OVIR would not have had reason to detain my family. \"The approach used to refuse me the right to leave is also used against many professional people wishing repatriation to Israel. The most transparent expression of this, in my case, came from a statement provided to OVIR by the Management and Trade Union Committees of the Central Scientific Ministry of the Cotton Industry where I was employed most recently. They objected to 'Rutman's departure to Israel as he is a specialist of the highest abilities. Such a high evaluation of my abilities did not stop the Institute of Machinery management from removing me from the post of chief of the laboratory, nor of prohibiting me from teaching in the Institute of Radio Engineers. \"Since February 21, I have begun a strike. Following the refusal by OVIR to permit my family repatriation to Israel, I have given up my present job where I am not allowed to work in my profession, where they keep trying to fire me under contradictory pretexts, and where elementary norms of behavior toward me axe violated. It is written of my ancestors in the Book of Exodus that the Egyptians 'did set over them taskmasters to afflict them with their burdens. And they built for Pharaoh store-cities, Pithom and Raamses.'  Now, I have stopped building cities for Pharaoh. I demand simply that my wife, my 12 year old son, and I be allowed to go to Israel, our homeland.\" Union of Councils for Soviet Jews 14308 Triskett Road Cleveland, Ohio 44111 February 23, 1972 The following appeal was received by phone from Moscow today, l2:l5 p.m. E.S.T.: To:  Commission on Human Rights, U.N. International Labor Organization Federation of Trade Unions of Israel International Lawyers Commission Israel Association of Engineers and Architects Today in the session of Prunzelsky Regional Executive Committee of Moscow, in the presence of the Regional Procurator, Vladimir Slepak was told that he was a parasite. This \"parasite\" is a highly qualified engineer, former chief of the Laboratory of the Science Institute for TV Research, a man who had worked in the State enterprises for over 20 years, and who was compelled last September, as a result of the persecution, to leave his job at the Institute of Organic Chemistry of the Russian Academy. At present, Vladimir Slepak earns his living as an officially registered tutor in physics and mathematics. According to Soviet law, this is considered a \"socially useful\" activity. However, those who enforce the law do not abide by the law. Vladimir Slepak is well known for his struggle for Jewish repatriation to Israel and for defending the rights of the Jewish minority in the USSR. For all this he was repeatedly cautioned, searched, and arrested. Now a further reprisal is being prepared based on the law on parasitism (intended originally to clear the cities of vagrants, alcoholics, and workers guilty of absenteeism). This so-called parasitism is punishable by imprisonment up to one year. We appeal to everybody who treasures human rights. To Jews and non Jews: Do Not Let The Reprisals Against Vladimir Slepak Take Place. (64 signatures) Please send a cable or night letter to those listed below. Ask that the reprisals against Vladimir Slepak stop and that he and his family be allowed repatriation to Israel. Mr. V. A. Kirillin, Chairman State Committee of the USSR Council of Ministers for Science and Technology Kremlin Moscow, USSR Dr. M. V. Keldysh, President USSR Academy of Sciences Leninskiy Prospekt 14 Moscow V-17, USSR [Photo = \"Vladimir Slepak\"] Soviet Jewry -- Some Legal Aspects by Leonard W. Schroeter Word received from Moscow of a People's court decision, highlights a new and ominous problem in the unrelenting effort of Soviet Jews to secure their right to leave the Soviet Union. The case--unique in the annals of Soviet Law and the Jewish repatriation movement--was filed on November 25th, and decided in peremptory fashion on the same day by the Court of the Kirovsky Region (Moscow). One of four identical suits brought by Vladimir Slepak, Victor Polsky, Ilena Polskaya, and Mikhail Klatchkin, it placed squarely in issue the Soviet effort to prevent some Jews from being granted their right to leave on the grounds that their departure would constitute a security risk for the USSR. Although the court's decision applied only to the petition of Mikhail Klatchkin, similar results are imminently expected as to the other three. Klatchkin, a highly trained scientist, contended that in 1966 he signed a routine security agreement in connection with his engineering work at a Moscow area industrial plant. Such agreements provide that the employee will not discuss technical details associated with his work and will not reveal any \"secrets\" connected with the plant. No provisions were included, and no oral warnings were given, that upon completion of employment there were any restrictions on leaving the USSR. However, when he requested an exit visa from OVIR (The Department for Visas and Registrations of the Ministry of the Interior) he was advised that he had no right to leave because he had signed an agreement clearing him for \"secret work.\"  Klatchkin contended that he had no way to become aware of the restriction on his freedom of movement, it not having been included in the agreement or published in government regulation, and that he had never been advised of such a consequence until he applied to OVIR. Thus the agreement was invalid under Section 57 of the Civil Code of the RSFSR (Russian Soviet Federative Socialist Republic). This provides that \"a transaction entered into under the influence of a mistake of a substantial kind\" will be declared invalid at the instance of the party acting under the influence of the mistake. [Photo = \"From left to right: Vladimir Slepak, Michael Zand (now in Israel), Victor Polsky, Vladimir Prestin, P. Abramovich.\"] The background of the case, and the problem it poses, has grave significance for Soviet Jews as well as the scientific community. It is not coincidental that scientists comprise much of the leadership of the Jewish repatriation movement and the democratic movement in the Soviet Union. Among the foremost activists of the Jewish movement must be numbered 4 Moscow scientists--Vladimir Slepak, Victor Polsky, Vladimir Prestin, and Pave1 Abramovich. Yet all four, and three of their wives are present victims of the Soviet claim that their \"secret work\" prevents them from leaving. Others prominent in the Jewish resistance such as Klatchkin, and Gabriel Shapiro, are similarly situated. Slepak, a 44 year old radio (electronic) engineer, has worked in planning and use of control equipment for TV research. From 1957 to 1968, while at the Scientific Institute for TV Research, he signed a security agreement. (In the USSR, there are three categories of security status, in all of which there are typical security prohibitions against discussion, document usage, etc. However, since applications for exit visas commenced, the KGB has insisted that associated with each class are restrictions against leaving the USSR even after completion of the work or resignation. These restrictions, although unpublished, are said to run from 3 to 5 years). Slepak, as chief of a laboratory for development of TV and impulse apparatus; as the author of nine articles in \"closed\" Soviet journals; and the person responsible for issuance of a patent, worked in an installation where his work was classified as Class I Security. In 1968, he resigned, also choosing not to finalize his doctorate because of his involvement in the Jewish movement. He worked in the Geophysics Trust, a non-security position from which he was fired in March 1970 for requesting a character reference -- a pre-requisite for OVIR application. He then did non-security work in the Special Design Bureau of the Institute of Organic Chemistry of the Russian Academy, planning nuclear magnetic resonance, until in September 1971, he was forced to resign. One of the organizers and signers of the historic letter of 39 on March 10, 1970 (in which Moscow Jews openly criticized and attacked a stage-managed press conference of Soviet Jews denying anti-Semitism), Slepak has participated in virtually every Moscow petition and demonstration since that time. He was jailed from March 26 to April 10, 1971 after demonstrations and sit-ins at the USSR Procurator-General, concerning detention of Jewish political prisoners, and maintained a 12-day jail hunger strike. On June 15, 1970 (the day of the Leningrad hi-jacking arrests) the KGB searched his apartment. Interrogated numerous times by the KGB, he was summoned in September 1970 as a witness against the Leningrad defendants, but refused to cooperate. Although no reason was given by OVIR when he was first refused an exit visa, the 8 subsequent refusals have all orally been stated to be security regulations. Originally, KGB advised that he would be issued a visa 3 years from the date of his 1968 resignation from his \"security\" job. That time having now passed, the KGB still cites security reasons. These appear no more valid than the classification of his original employment as top secret. Slepak's case is similar to Victor Polsky's. Polsky, a 41 year old physical engineer with a doctorate in photo electronics, has taught at the Institute for Energetics and, while chief of a laboratory doing research in non-destructive tension, signed a security agreement. Demoted after asking for his OVIR required character reference, he was forced to resign in March 1971, and since then has taught at one of Moscow's dozen burgeoning Hebrew ulpans. Like Slepak, he has signed all recent petitions, demonstrated, been arrested, and has led delegations of protest to Russian officialdom. Refused exit visa permission, he has been given the routine \"security\" explanation, even though he had originally been told that his restrictions ceased when he terminated his laboratory work, and though he has presented evidence that his research didn't involve security issues. Like Slepak, Prestin, Abramovich, Klatchkin and Shapiro, he was one of 11 signers of a November 10th letter to the Committee on State Security which criticized the \"stereotyped refusal (that) you are all working in places handling secret work.\"  Labelling this decision as \"irresponsible\" the signers state that their being permitted to leave will not disturb USSR security and that they will prove with reasoned evidence that this is the case. They have had no answer. In a recent telephone conversation, Polsky said: \"We are in their power. If they want to keep us a year, they keep us a year. If they want to keep us three years, they will keep us three years. We can do nothing about it. But we want our situation to be known throughout the world. We need support to keep up the fight.\" Polsky's wife, a 36 year old electrical engineer, also signed a security agreement in connection with her work on radio relay lines at the Institute for Communication Research. Thirty-seven year old Vladimir Prestin, an electrical engineer; the author of numerous articles and the holder of three patents, worked in \"closed\" (security) institutions until 1969 when he resigned because of his Jewish involvement. Since then he has worked at the Geophysical Trust and at the Computer Centre, where he was forced to resign because of his activities. He, too, now teaches Hebrew in a Moscco Ulpan, and has joined the petitions, demonstrations and jail terms of his colleagues. He has been refused four times by OVIR on security grounds, and though originally told that he could leave in April 1972, the KGB has recently extended that time. His wife, Ilena, also an electrical engineer is in a similar situation. So is Pave1 Abramovich, a 32 year old radio engineer and expert in computers, who worked from 1962-1970 in a closed institute of computer research. Forced to resign from a non-security computer job in September, he too teaches at an Ulpan. Although he signed a security agreement, Abramovich, whose Jewish-activist record is similar to his companions, has vigorously insisted that the KGB is wholly unable to prove that his work was secret or had top security implications. Much of the research has been done in parallel or advanced form in Western countries and has appeared in public scientific journals, available throughout the world. It is commonly believed in Jewish circles, as well as in scientific ones in the Soviet Union, that the \"security\" reason is a pretext to prevent emigration. Soviet scientists are concerned about official use of such an explanation because of their efforts to liberalize Soviet science from its blanket of secrecy and obscurantism. Deprived for years of contacts with their foreign colleagues, and arbitrarily assigned to military-oriented research, some Soviet scientists, in recent years, have valiantly sought to limit KGB security control of their activities. Some eminent Soviet physicists like Andrei D. Sakharov, Andrei N. Tverdokhelbov, and Valery N. Chalidze, founded and led the Soviet Committee on Human Rights which has energetically championed the right of Jews to leave. To them, the right of free repatriation is part of the recognition of the community of world problems and human rights. Sakharov, and others have authored an appeal of scientists stating: \"It is in seeking exchange of information and ideas that we come up against the greatest stumbling block in our country. Truthful information about our shortcomings and negative phenomena is classified as secret. Exchange of information with foreign countries is restricted . . . . Freedom of information and creativity are necessary to the intelligentsia because of the very nature of its work, because of its social function. The State, however, counteracts this and brings to bear all kinds of restrictive measures, administrative pressure, dismissal from work, and even court trials.\" Little wonder then, that there is deep concern about Soviet limitations on the mobility of scientists. These restrictions amount practically to detention and are akin to arrest. When they are justified in the name of \"secrecy\" (something scientists doubt exist and, in any event, abhor), or \"security\" (which scientists view as a term impeding research into questions openly discussed, and further advanced, in other countries), the complications for both freedom and science are ominous. There are those who defend restrictions on the right to leave when the restraints are for reasons of State Security, as a justifiable limitation of the human right. The classic 1963 UN \"Study of Discrimination in Respect of the Right of Everyone to Leave Any Country, Including His Own\" by Judge Jose D. Ingles, acknowledges that such restrictions are not unusual. Many countries refuse permission to leave on the grounds of national security, and a few countries prevent the departure of persons with high technical or scientific skills or qualifications. Judge Ingles criticizes such limitations, asserting that the national security claim can only be made where the person's activities are punishable under penal law. As to scientists working on vital defense projects, the UN study requires that any limitation on the right to leave must be part of the contract of employment and must end with the termination of employment. As to the contention that people may possess military or state secrets, Judge Ingles places a heavy burden upon the State to justify any restraint by meeting \"the test of clear and pressing danger to the national security.\"  If this cannot be done, Article 13 of the Universal Declaration of Human Rights has been violated. Gavriel Shapiro is one of those detained. He worked as a chemical engineer without access to secret documents. He was given the \"secrecy\" reason long after OVIR had rejected his earlier applications for \"bad behavior\", for \"being involved in demonstrations\" and only after he advised OVIR that he had received a grant of Israel citizenship. Shapiro has complained to the UN Commission on Human Rights. He has forwarded his petition to Tamar Eshel, Israel's representative on the Commission. One might hope that Israel will make the petition public; raise the \"security\" question with its important human rights implications; and protect its citizen. Shapiro, Slepak, Polsky, Prestin, Abramovich, and Klatchkin all await concerned voices everywhere in the world, insisting that a minimum standard of humanity would permit them to prove that their repatriation could not adversely affect the land where they are detained. January 1972 Mr. Schroeter is principal legal assistant to the Attorney General of Israel, an expert on Human Rights issues and the Soviet Jewry problem. He is a free lance writer.", "Union of Councils for Soviet Jews", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wip6-nctv.8azv", "00000000-0000-0000-355D-E4AD20DF149E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from John N. Whitaker, American Neurological Association to Marshall W. Nirenberg", "101584910X377", null, "1996", "31 May 1996", "Whitaker, President of the American Neurological Association, urges his colleagues to respond to the publicity surrounding World Animal Awareness Week by delivering a \"clear message about the benefits and importance of biomedical research, as well as the indispensable part animals play in critical projects.\"  Included is a draft letter to send to Congressional representatives against pending bills that would impede research.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of the American Neurological Association.", "Copyright may apply", null, null, "May 31, 1996 Dear Colleagues: The animal rights community has designated June 18-24, 1996 \"World Animal Awareness Week.\" Organizers are anticipating a \"six figure crowd\" of participants to converge on Capitol Hill, June 24. The entire biomedical research community must take advantage of the media and other attention that will be created during this week to deliver a clear message about the benefits and importance of biomedical research, as well as the indispensable part animals play in critical projects. Enclosed is a draft letter which the National Association for Brain Research urges you to send to your U.S. Representatives urging them NOT to co-sponsor H.R. 3393 \"The Family Pet Protection Act of 1996\" or the \"Pet Safety and Protection Act of 1996\". These bills do not protect pets, but would severely impede research. These bills ban research facilities from obtaining dogs or cats from dealers who do not breed and raise the animals themselves; make it more difficult for research facilities to obtain unwanted municipal pound animals that would likely be killed; disallow private animal shelters from voluntarily choosing to make animals available for research; and limits individuals from donating cats or dogs to research facilities to giving only those animals they bred and raised as well as owned for not less than 1 year before donation. For your convenience, the draft letter may be sent to you via e-mail by contacting Linda Wilkerson at 103053.222@compuserve.com. Sincerely, John N. Whitaker, M.D. President Sample Letter For H.R. 3398 and H.R. 3393 The Honorable (Use NABR Congressional Directory) U.S. House of Representatives Washington, DC 20515 Dear Representative I am writing to urge you NOT to cosponsor H.R. 3393, the \"Family Pet Protection Act of 1996\", and H.R. 3398, the \"Pet Safety and Protection Act of 1996\", introduced by Representatives Jon Fox (R-PA) and Charles Canady (R-FL), respectively. Please do not be swayed by the innocuous titles of these bills; neither measure protects pets from theft. Instead, H.R. 3393 and H.R. 3398 play on every family's real fear of losing the pet. Both bills would hinder research by eliminating a vital source of dogs and cats used in lifesaving medical research such as cardiovascular, neurological, and orthopedic disorders and diseases. H.R. 3393 and H.R. 3398 are based upon the false and undocumented premise that one million dogs and cats are being stolen from homes across the nation and sold to research facilities. According to the USDA's documented figures for FY 1994, an approximate total of 100,000 dogs and 30,000 cats were used for research and education. About 50% of these animals were purposely bred for research. Research facilities acquire the other 50,000 from USDA-licensed animal dealers (Class B dealers) who obtain animals from pounds or other individuals.  Depending on state or local law, researchers may also acquire abandoned pound animals. Researchers do not want or need to use pets. The research community wants the public to be confident that research animals are not pets and strives to assure the public of this fact. Researchers, however, now find themselves in an impossible situation of proving that none of the 50,000 random-source dogs used for research are stolen or lost pets. The chance pets will be found in a laboratory is extremely remote. It is a thousand times more likely animals will end up at the pound, where 10-16 million animals are killed annually because they are not claimed or adopted. Under H.R. 3393 and H.R 3398, the 50,000 random-source animals would be illegal, if not impossible, to obtain. H.R. 3393 and H.R. 3398 would put all Class B dealers doing business with research institutions out of business. The bills would prevent research facilities from obtaining pound animals by imposing impossible restrictions on the pounds. Additionally, only pounds making animals available to research would be required to register with USDA and subject to federally mandated holding periods. H.R. 3393 and H.R. 3398 would disallow private shelters from making animals available to research. I hope that you will oppose H.R. 3393 and H.R. 3398. The bills do not protect pets, but would severely impede research. If you have any questions or would like more information, please contact me at or the National Association for Biomedical Research at (202) 857-0540. Thank you for your consideration. cc: Rep. Fox Rep. Canady NABR Office", "American Neurological Association ; Whitaker, John N.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bzye~3psp-7k7r", "00000000-0000-0000-B2EA-7386FEF63CF7", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Interview with Marshall W. Nirenberg", "101584910X378", null, "2001", "13 June 2001", "This interview covers Marshall Nirenberg's transition from genetics to neurobiology and includes discussions of neurobiological research in the 1960s, communication between fields, the genetic-neural analogy, and Nirenberg's thoughts on his three major contributions to neurobiology.", "Interviews", null, "Neuroblastoma Research, 1967-1976", "9", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Interview with Marshall W. Nirenberg Wednesday, June 13, 2001 Topic: Nirenberg’s transition from genetics to neurobiology Interview by Jim Tabery Jim Tabery (JT): There’s been some wonderful historical analysis of your work on the genetic code, but that analysis often stops in the late 1960’s when you turned to other areas of research. I’m hoping to look at your transition from genetics into neurobiology because you’ve now amassed 30 years studying neurobiology, and to approach that phase of your work you have to start at the beginning. I’m interested in roughly the years 1966 to 1973. It’s your transitional period plus when you got the ball rolling experimentally. I hope I’m not catching you off guard here. I’m sure you haven’t thought about much of this work for quite a while Marshall Nirenberg (MN): Well, that’s true, but I can tell you that I was always interested in neurobiology from the time I was a graduate student. When I got a position as a post-doctoral fellow in Gordon Tompkin’s office here as an independent investigator I thought long and hard about what I wanted to do, and it ultimately came down to one of two areas: one, either molecular biology (which I eventually went in to), or neurobiology. I decided that neurobiology wasn’t advanced enough at that time to really be able to do something important in it, so I picked molecular biology which was the most exciting area in biology at the time. This was in the late 1950’s. I’ve always been interested in the nervous system because there are really only two systems in biology that process information: genetic information and the brain, the nervous system. So that was the connection, and that was the rational, and actually a number of molecular biologists decided to go into neurobiology: Seymour Benzer, for one. JT: And then you also find Adler, Stent... MN: That’s right. JT: I found an interesting letter from Jacob to you from 1970 saying that he’s fed up with nematodes and that he’s decided to move into the mouse nervous system, so he wanted some guidance with techniques in the area. Did he ever actually turn to neurobiology? MN: I don’t really remember. I applied to him for a post-doctoral fellowship before taking this job. I wanted to go to his lab to study. He’s a terrific investigator and a terrific scientist and always has been. But he didn’t have space for me, and he later kidded me that his contribution to science was that he turned me down because then I went ahead and did the genetic code work here. But I don’t know if he ever went into neurobiology. JT: Well, it was an interesting letter because he obviously saw that you were having success with neurobiology, and he was interested in maybe doing the same. MN: Well, you know, neurons don’t divide, and so it’s logical to assume that tumor neurons might retain the properties of differentiated neurons and then could be used as a model system to study information processing in neurons and synapse formation. JT: So this is what took you to neuroblastoma? MN: That’s the idea. I thought we might be able to devise a model system that we could use to study synapse formation in cell culture, and we could also study properties of neurons in cell culture. At this time, the prevailing opinion (up to a year before) was that once you put cells in culture and clone them that the clonal cells dedifferentiated. But a year before there was a paper that appeared that showed that they didn’t really dedifferentiate, but instead retained some properties of differentiated fibroblasts (I think). This was a paper that had a real affect on me, and I thought that maybe we could get tumors of neurons and clone them and establish them in culture, and then maybe they would retain enough neural properties to make it extremely interesting and an extremely valuable model system to study neurobiology, so that’s what I did. I had never done any tissue cultures before, but I just jumped in with both feet...feet, hands, and everything. There was a visitor here from the Weizmann Institute, [in Israel], at the time who was a highly experienced tissue culturist, and so I did the first experiment with him. That was helpful because I had never done tissue cultures before. We started and found a mouse neuroblastoma that was a solid tumor that was passed from animal to animal that was known. And, independently, Gordon Sato cloned the same mouse neuroblastoma and established clones of this, and we actually published in the same PNAS journal. JT: In your first article on neuroblastoma research, you mentioned that you had first spoken with Sato at an NRP meeting about the neuroblastoma work. MN: I don’t remember, but I’ll tell you on thing: the NRP/MIT neuroscience program played a very important role for me. I later became a member of the MIT neuroscience program. They had summer courses held in Boulder, CO. I attended and participated in two of them, and they were absolutely wonderful for somebody that was just entering the field and didn’t know anything about the thing. The NRP had people who were world famous experts in various topics and would give lectures on the work that they would do. I actually gave a lecture. I don’t remember the years exactly, but I attended two of them in Boulder. Boulder’s a wonderful place. It’s the gateway to the Rockies, and so we drove out into the mountains on the weekends, and it’s just so beautiful and gorgeous: the blue skies and wild flowers over everything and the mountainous terrain. It’s lovely, and it was a wonderful experience for me. This was right towards the end of my work on the code. I had been working for six years as hard as I could, and all during this time I had to get out papers every month or every other month, and so it was just the most wonderful sense of freedom. Freedom from doing things that had to be done instead to learning and talking to people that were really knowledgeable about neurobiology. It was a time of tremendous mental activity and thinking and fun. JT: What was neurobiology like when you entered it in the 1960’s? MN: Well, in the 1960’s nobody was doing tissue culture of neurons or any kind of cell lines. The literature in molecular biology is so beautiful. Intellectually, it’s gorgeous. The experiments, the design of the experiments, and the work is clean. That work was really breathtaking and really beautiful. Neurobiology, on the other hand, was like walking up to your neck in a swamp. It was horrible! The literature was awful: controls were missing, the people didn’t do the experiments properly. But you still had to learn, and so I read everything, but what a difference. It was really in an awful state. You know, actually, during the coding years, around 1962 or 1963 I gave a talk at the Brain Institute in Los Angeles and wrote a paper on genetic coding, but it was in a Brain Conference. So all during this time I was very conscious of neurobiology. JT: As I was going through your experimental day books and journals, the earliest mention you make of actively pursuing neurobiology came on New Year’s Eve 1965. Later in your journals, everything is titled very specifically, but this one just says “Brain”. It was almost like a New Year’s resolution because you were really thinking things through for the first time. MN: [laugh] JT: Then on January 1“, 1966, you write about where you want to go in the next 6 months, how you want to attack the field, the different systems you want to use. It was really interesting. MN: Well, I tried two things almost simultaneously at the beginning. First, we set up the neuroblastoma cells, but then we started to work with nematodes. This was before Brenner published anything. Ruth Pertel was a graduate student at Berkeley. You know, I forgot to tell you: when I was a post-doctoral fellow, I found an old book that was published in 1905 or 1906. This was on tropism in microorganisms: paramecium, bacteria, etc. It was a fantastic book, and the author had made wonderful observations. I got so interested in it that I gave a seminar to our journal club in the lab. I also became interested in an article written by Edward Land from Polaroid, which was published in Scientific American on vision. He had a theory of color vision. He took two projectors with black and white slides of the same view. On one projector he put a red filter and on the other one a green filter was placed, and then he combined the images on a screen, and they came out in perfect color. From this, he provided a theory of color vision. I became so interested in that article that I gave another seminar in our journal club on Land’s work and demonstrated that it really did work. So I was interested in neurobiology all along. One of the major reasons for my interest was that it was a black box for me. I didn’t know anything about the nervous system. I thought that this was an area where tremendous discoveries were going to be made eventually, and I was really, truly interested in it, so studying it and jumping into it was fantastically fun. I knew I didn’t have enough time to do both [genetics and neurobiology]. I couldn’t really carry the stuff I was doing (This was around 1966.). I was still working on the code, and there were fantastic problems that still needed to be attacked and that we were attacking at the time, but I had a wonderful associate, a former post-doctoral fellow, by the name of Tom Casky who was in the lab at the time. I gave all of my post-doctoral fellows to him. They had come to me because they were interested in protein synthesis and coding work, and here I was leaving the field, so I gave them all to him, so that I was free to do what I wanted. JT: How did you communicate that decision to the lab? Did you call a meeting? MN: Oh yeah. I just told them. Of course Tom was delighted to take them all on, and they were all in problems of their own, so it really worked beautifully. It freed me up. I didn’t have any responsibilities then; I was free to explore simple systems in the lab at that time. Shortly after that time, the lab became a little zoo with all sorts of little invertebrates that we took into the lab to see if they would make a good system to study. We had rotifers in the lab, brine shrimp...all kinds of different invertebrates that we could put in culture. Have you ever seen a living rotifer? JT: Not since biology lab. MN: [laugh] Well, they’re beautiful organisms. Actually, I still have little chambers that have two cover slips that you can screw together. So if you put water in there with a rotifer, you can immobilize the rotifer by pressing down a bit with the top cover slip and so you can see all the internal organs. I even got some ascaris, parasitic worms, from a slaughterhouse in Baltimore, and Oh my God! They were awful. They smelled so bad. I got a bucket of them and took them to the lab, and Norma Heaton almost quit then and there. They threw up their hands. The smell was horrible. And you can get infected with these things. They’re not safe to work with. But somebody by the name of Goldstein around the turn of the century had published these beautiful anatomical drawings of ascaris’ nervous system which I had xeroxed. It looked like the inside of a submarine with nerves going in all different directions like pipes. Although Brenner later told me that Goldstein was wrong. I tried all kinds of simple systems before we finally settled on neuroblastoma. Ruth Pertel, as I began earlier, was a grad student at Berkeley with Dougherty. When you went through the literature, you found that the only invertebrates that you could raise on a defined medium were nematodes, and that was because Dougherty had put years of effort into getting a fully defined medium that would support the growth of nematodes. So nematodes were the ideal invertebrate to approach because they were the only ones with a defined medium. Dougherty, around that time or slightly earlier, committed suicide. And Pertel was just getting her degree, so I invited her to come to the lab, and we decided to start working on nematodes. She was his student, so she was familiar with all the techniques. We still have a couple of racks of nematode mutants that are in the liquid nitrogen. But then I decided that when Brenner came out with his work on nematodes (he was working on the same nematode actually that we were working on because that was the one you could grow). So nematodes were a logical choice for him and for us, but I felt after working a few years with nematodes (We got many mutants, most of them motion or nervous system mutants) that I couldn’t really handle nematodes and neuroblastoma simultaneously. It was just too much, and so I felt that I should focus on neuroblastoma cells since Brenner was working with the nematodes. JT: Was there much communication with the others, such as Brenner, Benzer, and Delbruck...the group that migrated? MN: There was communication. [Interruption from post-doc about current research] MN: We’re doing something now that is really exciting. We’re screening virtually the entire Drosophila genome looking for genes that affect the assembly of the nervous system. And that’s using a new technique that has just come up recently called double-stranded RNA interference. If you inject double-stranded RNA for a particular mRNA into the early embryo, there’s an enzyme that grabs hold of it and cuts it into pieces about 22 base pairs in length--two turns of the double helix. And a part of that enzyme probably then unwinds the strands. Then they go about searching for mRNA that will base pair with the oligoribonucleotide. If it base pairs correctly, then another enzyme is utilized that will then destroy the mRNA. It’s a quick way of getting a mutant phenotype for any gene. And it’s an amazing technique. That’s what virtually everybody in the lab is doing right now. And we hope to screen most of these genes. There are 13,600 genes in drosophila, and this technique is so much faster. We were doing the same kind of thing before using an enhancer trap method, which has transposable genes that has a betagalactosidase reporter gene, so that it will transpose into another gene. Then the expression of the betagalactosidase, which we can stain for, is determined by the regulatory region of the gene it’s inserted into. We made 500 transgenic lines of flies and found many interesting mutants that affected the nervous system. Anyway, this new method is so much faster than the enhancer trap method, but it’s also very laborious because you have to inject embryos with double-stranded RNA, and you have to infect about 40 embryos for each double-stranded RNA. So when you have 14,000 genes it gets to be very time consuming and difficult. But, thus far, we’ve screened about 350 genes and found 3 mutants, so there’s going to be about 1% of the genes that affect the assembly of the nervous system. But, anyway, we’ve gotten sidetracked. JT: Well, I asked you before whether or not there was communication between the molecular biologists that made the neurobiological shift. MN: Yes, absolutely, and particularly at the beginning. I talked to Adler who I haven’t seen for a long time, but was a good friend at the time and Seymour Benzer, who is one of the best scientists in the world. I mean, he is really a terrific scientist. He was the first to actually go into neurobiology with Drosophila. And we had long discussions about doing it. You know, I remember him saying at the time that this was what he really wanted to do, but he also added that he didn’t want to fail. Because when you go into something like this you’re an absolute beginner, and you have to learn from scratch, and he was at the top of the field in molecular biology. He was leaving something that he was a world-recognized expert in and going into something that was very uncertain and with an uncertain future. Everybody that made the switch went through exactly the same thing. It took a lot of guts and a lot of courage to do it. I think Benzer is such a superb scientist. He’s done so much so well. And he’s still at it. I have great respect for him. JT: And do you think it was the same motivation for them: they were working on the one information processing system, so it naturally led to the other one. MN: Yeah. The thing was that the big black box was the nervous system. It was there for everybody to look at, and none of us understood what was going on in the nervous system, and we were all interested in information processing, so it was very natural to make the move. Sure we talked about it, and I talked to Julius Adler about it. I didn’t really talk to Brenner about it because I didn’t really have the opportunity. He wasn’t at any of the meetings that I normally went to. Adler has done beautiful work with £.coli chemotaxis. JT: What about Delbruck? MN: [laugh] Delbruck. I only met him once actually. I attended a meeting at an abbey in France with him where I presented the work on poly-U, so this was the early 1960’s. He was the chairman of that particular session. This was the first time I had ever seen him. This was only months after I had initially reported [the poly-U discovery], and when I finished he looked at me and said that he tried to use poly-U to direct the synthesis of polyphenylalanine and it doesn’t work. So I looked at him and said, “Well, I've made probably 75 preparations of cell-free extracts. There’s a little variation between the activity of one prep and that of another prep. Some are better than others. But every one of them worked. So make these solutions. Throw out all your old solutions and try it again.” I said, “I’d be glad to help you. If you have trouble with it, come to my lab or send somebody to my lab and I'l run through it with them.” And that was the end of it. I never heard anything more. JT: He seems to be famous for making those kinds of abrasive comments. MN: Very, very abrasive. This was before the entire audience. I asked him a few questions about his methodology, but it was clear that there was some trivial kind of error that they had made, and I’m sure that there was no problem trying to figure it out. JT: Now, was there any communication with Stent. MN: Stent used to come to these NRP meetings. JT: So, when you talk about having opportunities to meet and speak with the others, was it often at the NRP meetings? MN: With Stent, yes. Have you read his new book? This is a book he just published recently, and it has the worst title ve ever heard in my life. It’s autobiographical: Nazis, Women, and Molecular Biology. I mean, it’s the kind of thing you want to get in a brown bag. I ordered it through Amazon.com, and it came to the lab, and my secretary looked at me when she handed it to me and didn’t say anything. It is interesting, though. He’s from Germany originally, and his family was refugees that came to the United States, so he spoke German. Immediately after the war, he was hired by the army as a civilian to go into Germany and find papers and scientists on various scientific projects. And so the book is about his experiences going back to Germany immediately after the war. I heard he had it privately printed. It’s an interesting book, but it’s got a terrible, terrible title. JT: So there was communication with him at the NRP? MN: Oh yes. He and his wife were there, and I spoke with him and everybody there. Those NRP meetings were terrific. They lasted a month, and it was a wonderful way of getting a solid introduction to the field. But once we had these neuroblastoma cells, it was clear that I had to learn electrophysiology to sample them and also a lot about the methods of tissue cultures, of cloning them. Takahiko Amano came to the lab at the time as a post-doctoral fellow. He was an expert at tissue cultures and cloning. He hada green thumb. And total dedication to the work, so he took over the cloning, and he cloned all of the neuroblastoma cell lines. He’s really a wonderful tissue culturist. And, you know, one of the great things about the NIH is that it’s easy to have collaborations with people: Phillip Nelson, who is an expert neurophysiologist, used to be in the basement of this building. We started a collaboration to do electrophysiology of cultured neuroblastoma clones, and that was wonderful. That was the reason I moved from building 10 to this building: because he was here, and because the space had just become available in this building. This building is full of the neurosciences. JT: Was there any communication or collaboration with Axelrod? MN: I knew Axelrod. I’ve known him for many, many years, but I’ve never collaborated with him. He was mostly working on trying to find new enzymes in the nervous system. I see him about every month. We have lunch together. JT: So as far as actual collaboration, Nelson is the only one at the NIH? MN: Well, Nelson is the major one. There have been two major collaborators...actually three that I’ve collaborated with on neurobiology. Phil Nelson was the first one, and for years we worked together and it was very productive. The major problem was learning a new scientific language. I had to learn electrophysiology, and he had to learn molecular biology, and so we were teaching one another. He gave me things to read; I gave him things to read, and so it proved to be a wonderful collaboration that made us both better scientists. It was wonderful. Later, we collaborated on a whole series of papers with Werner Klee, who used to be in this building. Werner had wanted to know if any of our neuroblastoma cell lines have morphine receptors. JT: So was he the catalyst that turned the work over to the morphine studies? MN: Yes. He came to me and wanted to know if we had anything that had [morphine receptors]. We had so many cell lines. We have probably more cell lines than anybody else in the world. We have a cell bank here. What we did later, after we established the initial neuroblastoma clones, was then to try and characterize them to see if they had neural properties. That meant that we then had to set up assays. We studied them electrophysiologically. We looked for electrically excitable membranes for ion channels, and found that they could generate action potentials, and that the excitable membranes were regulated, and we could shift populations of cells from a dedifferentiated to a differentiated state by upping intracellular cAMP levels. Then we simultaneously set up all these enzymatic assays to look for neurotransmitters. This took a couple of years. It was a major project in the lab at the time as we tried to establish all of these assays that could be applied to the cultured cells. We devised methods for tyrosine hydroxylase and many others. JT: There was also the dual regulation of adenylate cyclase. MN: That was with Werner Klee. To rescue gene expression from the nervous system we used somatic cell fusion with neuroblastoma cells. We found that most of the neuroblastoma cell cultures are relatively dedifferentiated if you grow them in log phase. They don’t have excitable membranes; they don’t express neural properties. So we had to first learn how to grow the cells by upping intracellular cAMP, so that they shifted to a more differentiated state. But to try and rescue gene expression from the nervous system we fused neuroblastoma cells with cells from the normal nervous system (retina, hypothalamus, peripheral nervous system, etc.) to make somatic cell hybrids. We got many cell lines generated, and we characterized about 30 that looked like they had the most neural properties. And one of them was NG108-15, a somatic hybrid cell (neuroblastoma X glioma). This was a glioma that actually Gordon Sato was the first to publish and describe. This hybrid cell line turned out to be loaded with morphine receptors, and so we asked ourselves if we could make the cells dependent upon morphine if we cultured the cells in the presence of morphine. Would they become dependent upon morphine? That lead to a whole series of really interesting papers where we showed that adenylate cyclase was regulated, that morphine inhibited adenylate cyclase, and that if you cultured the cells for a day in the presence of morphine they become dependent upon morphine because adenylate cyclase activity increases, so that the morphine inhibits it and it becomes the control level. So then if you remove the morphine the enzyme becomes sky high and tremendous amounts of cAMP are produced, so that it takes a long time to return to normal. JT: And that’s the withdrawal? MN: That’s the withdrawal. JT: The NIDA (National Institute on Drug Abuse) came about in 1974. Was there any collaboration with them? MN: No. We got some drugs from them, but nothing else. JT: Really? You would think that they would be interested in that work. MN: Well, one of the wonderful things about the NIH is that people are very helpful, so I talked to a lot of people during this whole time, and people were helpful if they knew how to do something that you didn’t know how to do. Anyway, we worked for a number of years on the opiate dependence problem, and we also found the same phenomenon with both musc. receptors--if you place them in the presence of a compound that activates the receptors. And noradnergic receptors, that if you cultured them in the presence the compound it will do exactly the same thing: inhibit adenylate cyclase and then become dependent on the neurotransmitter that’s present. So it’s a very basic phenomena that doesn’t only work with opiates but also works with normal neurotransmitters like acetylcholine and norepinepherine. JT: I want to turn the conversation back a few years because one of the first things that I saw in your work that got me interested in your transition was the phase where you were comparing and contrasting the neural code with the genetic code. You seemed very interested in their analogies. Can you tell me a little bit about that? MN: Well, I had come from spending years intensely thinking about genetic information transfer, so the obvious thing was to contrast how genes work with how the nervous system works. So I was interested in that. I had a lot of interesting ideas and a lot of wild ideas. JT: Did any of them evolve into anything else that was productive? MN: Well, when you say “productive”, I think everything is productive in the sense that it shapes you and makes you ask more questions. Maybe people work differently, I don’t know, but for me, the way I work is to generate hundreds or thousands of questions. Some of the questions are awful; they really stink. Others are mediocre, and others are okay or pretty good. And then a few of them really hit the bull’s eye. So, if you like throwing darts at the dart board, then you cover the wall with dart holes, but a few of those questions are really terrific questions. That’s just part of generating ideas. JT: So where does the genetic-neural analogy fall into your hierarchy? MN: Well, there are major differences between the nervous system and the genes, and they’re obvious, but I still think it’s interesting to think about it and to compare them. I spent a lot of time doing that. It’s a lot of fun to do it. You know, we found in the cell lines that Amano had established a number of neuroblastoma cell lines that made acetylcholine, and this was totally novel and new at the time. It was thought before then that only adenergic sympathetic neurons did this, but we found cholinergic neuroblastoma cell lines that could do it. Later on people looked for cholinergic cell lines in children and found them. JT: If you had to think of your 3 big contributions to neurobiology could you list those or would it be too difficult? MN: Sure I can list them. I'll tell you what the areas were that we worked in that I think are really important. First, we established simple systems/clonal lines of cells that have neural properties, and our objective was to find cell lines that form synapses, and we did that. We found 5 cell lines that synthesize acetylcholine and that form synapses with muscle cells. We could innervate every muscle cell on the plate. And so they’re wonderful systems for studying various questions relating to neural information transfer. They’re electrically excitable; they have neural transmitters and receptors that form synapses, and they’ve been used by many people throughout the world as simple systems. We characterized lots of these properties including the opiate receptors and dependence. So that’s one area. People write to me all the time and ask for cultures of different cell lines which we send them. We send them all over the world. Another thing that we studied was the generation of monoclonal antibodies in retinal cells. We spent a lot of time studying the retina, and found that with a chick embryo retina you can disassociate the cells in the retina. Then, when you reassociate them, they form synapses during development that look exactly like the normal synapses, so we used this as a model system to study synapse formation for a long time. You know Roger Sperry, the grandfather of neurobiology, in the early 1960’s wrote an interesting paper hypothesizing that you cold give every cell in the retina a molecular address by two gradients of different kinds of molecules at right angles to one another. So we looked for molecular gradients by using monoclonal antibodies. We took little pieces of retina from different positions in the retina (dorsal, ventral, etc.) and injected them and made monoclonal antibody cell lines that synthesize antibodies, and each cell line would synthesize a different kind of antibody. And then we tested them and looked for antibodies that would recognize an antigen more abundantly in one part of the retina than in another part of the retina. And, low and behold, we found one. We found an antigen that we turned “top” for “topographical” that was much more abundant in dorsal retina than in ventral retina. And, in fact, there was a concentration gradient throughout the entire retina. This was a membrane protein. We purified the membrane protein. And it was the first time anybody had ever found a gradient protein in any system, and so we had a lot of fun with that. I tried to clone the antigen, but failed. We couldn’t detect it with the antibody we were using. Maybe we didn’t try hard enough. But then I figured that it was too complicated; that trying to do it in the chick embryo was just too complicated, and that’s why I went into drosophila. So the idea was to look for new homeobox genes in drosophila. We found 4 new ones. One of them, which was NK2, seemed to be expressed at such an early time in the nervous system that I thought that maybe it’s the first step. And it turned out to be true; it is the first step. The expression of this gene initiates neural development in part of the ventral nerve cord in the embryo. JT: Have homologous genes been found in other organisms? MN: Yes, the mouse has 7. And, actually, there’s something called the NK2 family of homeobox genes that are found in all kinds of organisms from Xenophis to zebra fish to planaria, tape worms, chicken, and the mouse, as I said, has 7. We study many aspects of NK2 because it really initiates neural development in the most ventral part of the medial ventral nerve cord. And it turns out that there are 3 different homeobox genes. Each one initiates neural development. Like an anterior/posteri", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-zky8~7rgi~qqvd", "00000000-0000-0000-E3D6-20A5FAC5DA70", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Jean Jadot to Marshall W. Nirenberg", "101584910X379", null, "1974", "1 July 1974", "Jadot informs Nirenberg that Pope Paul VI has nominated him for membership in the Pontifical Academy of Sciences.  Congratulations are offered along with a request that the news be kept confidential until published in Rome.  He thanks Nirenberg for the \"valuable contributions to the welfare of mankind\" that he made through his research.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of the Most Reverend Gabriel Montalvo, Apostolic Nuncio to the United States.", "Copyright may apply", null, null, "July 1, 1974 Dear Doctor Nirenberg: I am pleased and honored to inform you that His Holiness, Pope Paul VI, has nominated you for membership in the Pontifical Academy of Sciences. The announcement of your nomination will be published in the July 4th issue of the Osservatore Romano which will appear in Rome on Thursday afternoon. Until that time, the news of your nomination is to be kept confidential. Within a few days, you will receive formal notification of your nomination. Meanwhile, may I respectfully request that you acknowledge receipt of this letter. If you wish to do so by telephone, the number of the Apostolic Delegation is (202) 333-7121. I wish to offer my personal congratulations on your nomination to the Pontifical Academy of Sciences. It is a fitting recognition of the valuable contributions to the welfare of mankind that you have made through your scientific research. With cordial regards and best wishes, I remain Sincerely yours, Jean Jadot Apostolic Delegate", "Jadot, Jean", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yg5d~65jf_kt7z", "00000000-0000-0000-7CEA-A4056795933C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Edwin C. Whitehead to Marshall W. Nirenberg", "101584910X380", null, "1986", "15 July 1986", "Whitehead informs Nirenberg that he has been elected to receive the National Library of Medicine Award honoring America's Nobelists.  Whitehead provides logistical details and requests Nirenberg's confirmation that he will attend the gala reception where President and Mrs. Reagan are expected to attend.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Susan Whitehead.", "Copyright may apply", null, null, "July 15, 1986 Dear Dr. Nirenberg: We are delighted you have elected to receive the National Library of Medicine Award honoring America's Nobelists. The response to this event has been so great that the original site was far too small.  We have had to postpone the dinner and festivities to November 18 to accomodate the larger numbers. We are planning a late afternoon reception at the White House with President and Mrs. Reagan who will be our hosts. The reception will be followed by a gala celebration at the new and very beautiful J. W. Marriott Hotel, 1331 Pennsylvania Avenue, N.W., where we will be joined by members of Congress and other prominent citizens. Travel and lodging expenses, of course, will be provided by the Friends of the National Library of Medicine. We apologize for the inconvenience of this late date change, it was beyond our control.  We sincerely hope that you can make the 18th of November and look forward to hearing from you. Sincerely, Edwin C. Whitehead", "Whitehead, Edwin C.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ubmd_77df-aa57", "00000000-0000-0000-928F-41A1163B7C00", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Simcha Dinitz to Marshall W. Nirenberg", "101584910X381", null, "1976", "20 January 1976", "Ambassador Dinitz invites Nirenberg for a private meeting with Prime Minister of Israel, Yitzhak Rabin.  Dinitz informs Nirenberg that the meeting \"will provide an opportunity for exchange of views on the current Middle East situation.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Simcha Dinitz.", "Copyright may apply", null, null, "January 20, 1976 Dear Professor Nirenberg: I take pleasure in inviting you to a private meeting with His Excellency Yitzhak Rabin, Prime Minister of Israel, on Thursday, February 5th from 12:30-2:00 pm,  at the Waldorf Astoria Hotel in New York,  Jade Room. The meeting will provide an opportunity for exchange of views on the current Middle East situation. Kindly confirm attendance by calling (202) 483-4100 x 251.   This invitation is personal and non-transferable.   A buffet luncheon will be served. I look forward to seeing you there and extend to you my best personal wishes. Sincerely, Simcha Dinitz Ambassador", "Dinitz, Simcha", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jduf-e6gs.e7yq", "00000000-0000-0000-8AEE-0F125CFA5171", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Interview with Marshall W. Nirenberg", "101584910X371", null, "2002", "26 August 2002", "The topics of this interview are research, genetics, and social/political activism.. NOTE: The Digital Manuscripts Program (DMP) referred to in this interview is a program of the National Library of Medicine.", "Transcripts, Interviews", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "10", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Interview with Marshall Nirenberg Tuesday, August 26, 2002 Topic: Research, Genetics, Social/Political Activism Interview by Eric Boyle Eric Boyle (EB): Since a lot of the Digital Manuscripts Program collection covers the latter portion of your career, I'd like to discuss briefly the general trajectory of your research from the neuroblastoma and hybrid cells and chick retina work to Drosophila and homeobox genes in the 1980s and beyond. Now in your interview with Jim Tabery last year you touched on your earlier work with Drosophila and homeobox genes. Can you discuss this transition and how or why it took place. Marshall Nirenberg (MN): Well the first transition really took place between the time we deciphered the genetic code and the time I decided to go into neurobiology. And the logic to that was that there are two systems in biology that process information, genes and the nervous system. And I was always interested in the nervous system anyway, although I didn't know much about it. So, that was really one of the big factors that made me interested in it is that I didn't know much about it. So, that was the logic for switching from basically genes and protein synthesis in the genetic code to neurobiology. Now when you make a change like this it was a big change. It was so easy to do the work in molecular biology and protein synthesis related to messenger RNA and the code. It was like turning a crank. I thought I could do it with one hand tied behind my back. But I gave all of my postdoctoral fellows and all of the problems we had going to Tom Cassidy who was a former postdoctoral fellow. He took over all of that work so I was free then to explore neurobiology. Basically the thinking behind it is that it is really fun to explore and the only way to really discover things is to jump in and ask question and begin to explore the field. It's a risky business though. I mean you're giving up all of the things that took you a long, long time to set up -- all of the procedures, the methods, an established laboratory, basically. So you do it with the knowledge that there is a lot of risk involved in doing it. Nobody wants to fail. I don't want to fail. And yet there is a real possibility of doing that. So you weigh different thoughts and ideas in doing it. So, anyway when I decided to go into neurobiology it was a fantastic and wonderful experience because I didn't know anything. You're starting from the beginning, really from the beginning, and it was just a wonderful sense of learning, exploration, of freedom. You know I didn't have the responsibilities then because I had given away all of my responsibilities. It was a wonderful learning experience. I decided, eventually, to explore two different paths. One was neuroblastoma cells. Neurons are non-dividing cells and I thought it would be nice, it would be wonderful, to have a simple system of clonal cells lines that had the properties of neurons and which grew synapses in culture. And so I switched to, started to study, a tumor of neurons called the neuroblastoma -- a mouse neuroblastoma transplantable tumor. We did a lot of work with the neuroblastoma cells. Gordon Sato, also about the same time cloned the same mouse neuroblastoma, the C-1300 that we did. And I also began to explore C. elegans, nematodes, as a simple system. It turned out that the only invertebrate that you could culture in a known medium, a defined medium, was a nematode. There were a number of species of nematodes that had been used at the time and this was before I knew that Sydney Brenner had also picked nematodes to study. So, we worked a couple of years on nematodes making mutants and so forth. I never published anything about the nematodes and eventually I thought that both systems were very promising but Sydney Brenner was doing very beautiful work with the nematode system and I thought that I could only handle one or the other, not both simultaneously. So I switched to neuroblastoma cells and our initial objective was to clone mouse neuroblastoma cells, characterize there neural properties, and determine whether they could form synapses. If they had sufficient neural properties, they made neural transmitters for example, to be able to form synapses and use it as a model system for studying synapse formation and information processing by neurons. We spent a lot of time doing this. It proved to be a very fruitful thing. Takehiko Amano came to the lab about that time. He was a superb tissue culturist from Japan and he was the one who basically cloned all the lines of neuroblastoma cells. And we had many, many lines of neuroblastoma cells. And then I began to set up with some postdoctoral fellows who came to the lab who wanted to work in this area, set up biochemical assays for various neural properties, like choline acetyltransferase that catalyzes the synthesis of acetylcholine, or acetylcholinesterase which catalyzes its breakdown, or a number of other assays, also some enzymes of catecholamine biosynthesis. We also began to study the electrophysiologic properties of the cells and we soon found that with the many different clones that we had that some clones synthesized norepinephrine, whereas other clones synthesized acetylcholine. This was the first time that acetylcholine had ever been found as a neurotransmitter in neuroblastoma cells and that was exciting. Later it was found that in humans, also, some neuroblastoma cells synthesize acetylcholine. So we had both cholinergic and ademergic neuroblastoma cells and many neuroblastoma lines that would synthesize either transmitter. And we explored the properties of these cells in great detail and we found that the neuroblastoma lines that would synthesize acetylcholine could form synapses with striated muscle cells in culture. Later we found a clonal line of muscle cells that we could use with clonal neuroblastoma cells to form synapses. We could innervate every muscle cell on the plate. We found that many of the neural properties were regulated by the level of cyclic AMP in the cells. We found that if we elevated cellular cyclic AMP for a number of days, like five days, we could shift the cells from a relatively undifferentiated state to a differentiated state where they behaved like neurons. They acquired voltage sensitive ion channels, sodium channels, potassium channels, calcium channels, and this then could from action potentials. We showed that they could release transmitters and they innervated muscles cells. If you reduced the level of cyclic AMP you could reduce the effectiveness of the synapses. You could regulate the efficiency of the synapses by the level of cyclic AMP that the cells possessed. We used a number of techniques to elevate cellular cyclic AMP. Like we found that the cells had prostaglandin E-l receptors that were linked to activation of adenylate cyclase when they were stimulated. So, we would add prostaglandin E-l to the culture medium that they would then activate the endogenous adenylate cyclase, which would catalyze the synthesis of cyclic AMP. You could raise cyclic AMP levels over a period of days. These effects were slowly acquired and long lived. I've always thought we were looking at some form of memory, some type of memory, although I couldn't prove it and still can't prove it. But I think the ability to turn synapses on and turn synapses off at will and the length of time, the slow acquisition of the ion channels and the other machinery that are necessary for transmitter release and effective synapse communication, all indicated that this was a form of memory. That if you lowered the cyclic AMP levels gradually this cell became less able and finally unable to communicate synaptically. This was a very interesting type of system to work in. You could use these cells to study the biochemistry as well as the electrophysiology of the process -- a good model system. And that's what my aim was, to make model systems that were useful for neurobiology. We used another technique to rescue gene expression. Many of the cell lines were defective in synapse formation and so we fused the neuroblastoma cells to other cell types from the normal nervous system hoping to rescue gene expression for neural properties. We made somatic cell hybrids by fusing cells of different kinds and we made many kind of somatic hybrid cells that proved to have interesting properties. We found that many cell lines had specific defects in synapse formation and we characterized those defects. These are useful system, I've sent these cell lines to investigators all over the world for many years. EB: One of the things that was most striking from looking through your papers is that you have letters from Japan and Europe, all over the world. MN: We sent hundreds and hundreds, thousands probably, of cell lines to thousands of investigators all over the world over many, many years. They've been used in many ways so that's, I think, really been a very useful system. One of the things we did I did with Werner Klee who was an investigator at the NIH and a superb biochemist. Werner was interested in opiates and he wondered if the neuroblastoma or the somatic hybrid cells had any opiate receptors. We screened many of the lines, actually I've got thousands of cell lines, and really it's really a cell bank. It's probably more neuroblastoma or hybrid cell lines than exist anywhere else in the world. And so we screened many of cell lines looking for opiate receptors. And lo' and behold we found one of our most well studied cell lines, the NG-10815 cells, which is a neuroblastoma-glioma hybrid cell line, was loaded with opiate receptors. Once we found that and we were able to count the density of receptors on the cells we wondered what would happen if we cultured the cells in the presence of morphine. For example, would they become addicted, would they become dependent on morphine. This led to some really interesting experiments. We found that when you culture the cells in the presence of morphine that morphine inhibits adenylate cyclase activity, so the level of cyclic AMP drops precipitously. But then gradually if you continue to incubate the cells over the course of a day the level gradually comes back to the normal level. Now, the reason that it comes back to the normal level is because the activity of adenylate cyclase increases during this day's incubation. But then the cells are dependent upon morphine to inhibit the enzyme because if you withdraw the morphine, the level of adenylate cyclase activity shoots way up and the amount of cyclic AMP synthesized really goes way up and then only gradually falls back to the normal level. So, we proposed this dual regulation of adenylate cyclase-morphine inhibits adenylate cyclase and the cells gradually become dependent upon inhibition of adenylate cyclase and the basal level comes back to normal-and this is equivalent to withdrawal phenomenon of the opiates. Other have confirmed these findings and we also extended the findings to other neurotransmitters.  We found exactly the same thing with the muscarinic acetylcholine receptors. If you treat with carbachol, it inhibits adenylate cyclase. Gradually the level of adenylate cyclase will come up to the normal level. Then the cells are dependent upon carbachol to inhibit the enzyme. The same thing with the alpha receptors. I think it is a general phenomenon. We don't truly understand everything about this phenomenon even today because we asked the question: are we synthesizing more enzyme or simply activating the enzyme? The results showed that it's mixed. Mostly you're activating the enzyme but there is some synthesis of the enzyme, increased synthesis that does occur that counts for about a quarter of the increase in adenylate cyclase activity. Three quarters of the increase is simply activation of the enzyme. I don't know how the enzyme is activated like that, that remains to be determined. EB: That's what I was going to ask you. MN: I don't know. And I really wondered at the time. That work really raised questions in my mind about possible treatments for opiate addiction and dependence. I never pursued the clinical aspects of the findings but I always regretted not having pursued that because I think that might have been a very fruitful pharmacologic approach to the problem of opiate addiction. That problem really interested me for a number of years. Then I became interested in a different kind of a problem. In the sixties it was proposed by the grandfather of neurobiology, Roger Sperry, in a theoretical article in PNAS, strictly theory, but an interesting theory, that you could label, you could give every cell in the retina an address, a molecular address with two kinds of gradients of molecules in right angles to one another. That was a very intriguing suggestion. The reason he suggested this is because it's known that in the brain there are various retinal topographic maps that topographically reproduce the position of neurons in the retina so that you can recreate a cohesive picture of the outside world. There are multiple topographic maps like this that are produced and nobody knows how they're produced, or what the molecular bases of the maps really are. So, I thought of a way of asking: is there a molecular topographic map that exists in the retina? And to study this we used monoclonal antibodies. Monoclonal antibodies are produced with a clonal cell where each clone makes a different kind of an antibody in culture, and so we used as an antigen cells from dorsal retina, basal retina, temporal retina, or ventral retina -- different topographic regions of the retina -- and made monoclonal antibodies and then made thousands of different clones, screened these clones and asked the question does this antibody recognize an antigen that is preferentially distributed in one region of the retina rather than the other retina (what Sperry had predicted, had suggested). This was a test of Sperry's prediction. And lo' and behold we found an antibody that recognized a molecule that we called the topographic that was distributed in a dorsal-ventral gradient all the way across the retina. And it was a big gradient, like a thirty-five fold gradient. We purified the antigen, the molecule that the antibody was recognizing, and found it was a small membrane protein that's present in highest concentration in the dorsal retina and lowest concentration in the ventral retina. This was really a fascinating affair and the interesting thing to study, because the way the retina develops is concentrically, the oldest part of the retina is the center of the retina and then you have rings of neurons that are laid down, concentric rings as development proceeds. And how you form a dorsal-ventral gradient with that kind of developmental history was really, I didn't understand it, couldn't understand it at all. We also found out that the cells remember the amount of protein that they're supposed to synthesize because we could trypsanize the retina, separate the cells (when you treat them with trypsan, a protealytic enzyme that destroys the antigen) and then we would culture the cells and we would take cells from dorsal retina or ventral retina or in between and do the separating we found that they remembered how much protein to synthesize. And it didn't require cell-cell contact, it didn't require contact with cells that had less or more of the antigen. Nor was it regulated by mixing cells from ventral and dorsal retina-cells had a memory of how much protein to synthesize. That was really exciting because this was the first time a protein had ever been found that was distributed in a concentration gradient across the entire tissue. I didn't understand and still don't truly understand how a grade of synthesis or accumulation of this protein could occur, what the mechanism regulating the synthesis and or degradation of this protein was. What did I understand? What the function of the protein was. We tried to clone DNA for this protein. We purified the protein. That was quite a job, to get enough retina to purify the protein and identify the protein. One interesting thing, during the course of this study we found a chick embryo with three eyes. Two eyes in the regular place and the third eye in the middle of the forehead and we asked whether the third eye also formed a gradient and it did. All three eyes had exactly the same gradient. But we failed to clone DNA for this protein and that was an important question because if we could have cloned it we could have identified the amino acid sequence of the protein and would have given us a tool to use, a very important tool, for further studies. For some reason we were not successful in cloning it. At that time, soon afterward actually, there were a lot of reports of gradients of proteins that had been found in Drosophila. I thought that to really understand this problem you have to go to a simpler system where you have genetics that you can use. You can use genetics as a tool. Drosophila has been studied for a hundred years almost, ninety years, and there is a tremendous amount of genetic information that is known and wonderful genetic tools that can be used with Drosophila. And that's the reason I switched to Drosophila. Plus the fact that I saw a paper by Michael Levine and his co-workers on evenscate, which is a homeobox protein that was distributed quite remarkably in some neurons in the developing embryo and not in other neurons (highly specific expression of the gene regulation of homeobox genes), genes that bind to DNA and recognize the sequence in DNA that turn genes on or off, so they're gene regulators. So this is an important class of genes and to find them quite specifically distributed in specific sets of neurons was quite a remarkable observation. At that time there were seventeen homeobox genes that were known, that had been found in Drosophila and it was a burgeoning field of study. I was really intrigued by this expression of a specific gene regulator in a specific subset of neurons because we had been trying to find things like this with monoclonal antibodies as a tool and here it was in Drosophila. I had never worked with Drosophila before but when Yongsok Kim came to my lab as a postdoctoral fellow immediately after he got his Ph.D. in Korea, I suggested to him that we look for new homeobox genes in Drosophila. I designed a whole set of all the nucleotides that could be used as hybridization probes to screen the library for new homeobox genes. These were oligonucleotides to highly conserved regions of the homeobox. The homeobox is a hundred and eighty base pair region that encodes a conserved sequence of sixty amino acid residues that fold in a characteristic way that have a turned helix confirmation. And this is the part of protein that actually binds to DNA and recognizes the sequence of DNA. And so they are relatively constant, conserved regions within the homeobox. And so Yongsok is a superb scientist and in a very short time he had discovered four new homeobox genes in Drosophila which he called NK-1, 2, 3, and 4. And they all proved to be extremely interesting. I've continued to work with NK-2 because first of all it was distributed primarily in the developing nervous system and it was expressed so early that I thought it might turn on neural development, might even be the first step, the commitment step and that later was shown to be true. It turns on neural development in the ventral part of the neuroectoderm in a stripe, an anterior-posterior stripe of cells, and it initiates neural development in this stripe. It's really interesting the way the nervous system is formed in Drosophila. There are four anterior-posterior stripes which are next to one another, of cells, and they all originate differently, in different mechanisms of origin. Eventually they form neurons that will form the ventral nerve cord, which is like the spinal cord in higher organisms. It's a really interesting story how they arise, how they regulate one another. NK-2 initiates neural development in the most ventral portion of the neuroectoderm. Another homeobox gene, a different one, initiates neural development in the intermediate neuroectoderm. Still a third homeobox gene, a different one, we don't know what initiates neural development in the dorsal neuroectoderm but MSH is required for specification of the identity of some neuroblasts in the dorsal neuroectoderm. So, there are four different genes or proteins, they're all gene regulatory proteins, that are required to initiate neural development to form the ventral nerve cord. In higher organisms, in the early neural development, the homologs of these genes are found in the same relative position during early development in what gives rise to the spinal cord. I think the basic mechanism has been conserved in evolution. So, anyway, that's how I got into Drosophila. EB: It seems like Drosophila, again, is giving you that model system that you were talking about earlier. MN: That's right, that's exactly right. EB: So, you mentioned sequencing a couple of times with the oligonucleotides and again some sequencing was involved with the homeobox genes themselves that were discovered in Drosophila. MN: Yes, right. EB: So, I'm wondering if there is a relationship between this sequencing work and what we've been hearing so much about in the genome project and their sequencing in the 1990s. MN: Well, I think the genome project and the project on other organisms are extremely important. My goodness, the only instructions for how you build an organism are really present in the genome. The Drosophila genome has been sequenced also. This information is stimulating research in all directions, all fields, so it's really an amazing time right now where there are thousands and thousands of genes that have been discovered and in many cases nobody knows what the function of these genes are. So, the functional genomics is really important now. Currently we're screening the Drosophila genome using RNA interferons which is a new method which will give you the equivalent of oligonucleotides by injecting double stranded RNA corresponding to a specific messenger RNA. This results in the destruction of that messenger RNA and it looks like a mutant phenotype then. We're looking for genes that are affecting the development of the nervous system and we've found quite a few genes thus far. EB: How do you feel about some of the forecasts that have been made for biomedical applications in genomic research? Do you think the distance between the research that is taking place now and those biomedical applications to come is perhaps greater than some people promise? MN: Well, when you're doing basic research, to translate that into useful products that have therapeutic usefulness takes a lot of work and a lot of effort. There's a big gap between the two. But I think, as I said, with the sequencing of the genome that information is available now is stimulating work all across the board. Virtually everybody's work is stimulated by it, by the availability of all this information on the genome, and so I think that this is having a very large effect on research that is being done currently throughout the world. And it will continue to do so. EB: Another interesting parallel that I think there is between your own work and some of the discussion related to the genome project is these concerns about the direction of research and potential ethical and moral implications, reminiscent of your editorial from Science in 1967. One of the striking things about recent genetic research seems to be, again, these ethical questions. From what I've seen, you've taken a firm stand on some of these key debates in recent years. In 1988, you signed a letter against human cloning from the American Society for Cell Biology, to the President and Members of Congress. In 2001, you also supported a statement in favor of stem cell research to George W. Bush. MN: Absolutely. I think that stem cell research has tremendous possibilities for future research. It's not quick. Progress won't be tremendously fast, but in the long run I think that it holds enormous promise for future research. And I think that Bush compromised on this by allowing research to be conducted with federal funds on the cell lines that were already established at that time. That's not enough by any means. I think that that kind of restriction ties the work down tremendously. We need many, many more cell lines. First of all, there aren't sixty cell lines really available and useful, which he thought that there was at the time. And you need many more cell lines. I think that's an artificial restriction that should be done away with. And I think that this approach, using stem cells, offers a lot of hope as potential therapies for various kinds of diseases-Parkinson's disease, for example, Alzheimer's disease, even repair of heart attacks, infarcts, that kill cardiac muscles cells, and other things as well. Ron McKay here at the NIH has shown that he can culture pancreatic cells that will form islets and will release insulin on demand, although at a much lower rate than is found in mice. But it works. This has to be improved, the efficiency has to be improved, even for diabetes. For repair of all kinds of cells, potentially, they could be done by this stem cell research. But the therapies will be a long time coming, it's not going to be a rapid crack or something, in fact I think it is going to be a slow, gradual, incremental thing. But I do think it has tremendous promise. And I do think that research should be pushed and that the government should support it, without the restrictions that are currently on that. In terms of human cloning, nobody I know is in favor of human cloning. I think that's a very bad approach on the instinct and I don't know anybody who is in favor of human cloning. EB: Could you talk a little about the difference between human cloning versus stem cell research. MN: Sure, I mean to get stem cells you take very early embryos that are only a few cells, very small, small cells, very early, early embryos and these are usually obtained as a byproduct of in vitro fertilization therapies typically. And most of them are ordinarily destroyed. They are routinely produced and most of them, the ones that aren't used for in vitro fertilization to help parents that can't have children, are destroyed. Normally. And so to take some of these tiny embryos consisting only of less than a hundred cells, fifty cells or something like that, that would ordinarily be destroyed and could be used for research purposes, stem cell research. Stem cell research is trying to use cultured cells and allow them to differentiate in different ways that they wouldn't normally differentiate, to make muscle, or nerves, or pancreatic cells, or what have you. To find out all the steps that are needed for normal differentiation of the cells you need local hormones, stimuli of various kinds, and many other reactions that we know very little about right now. Basically, you're asking how do you form an embryo. What are the molecular reactions that occur between the first cell and the final baby that is forming, the entire organism. There is a tremendous amount of unknown information in there that really has to be obtained to have effective stem cells which could ultimately be used for replacement tissues. You know, I think that whether the work proceeds in this country or elsewhere in the world it will proceed. It's going on in other countries right now that don't have the restrictions that Bush has placed on stem cell research in this country. I think the United States should be the leader in this work instead of being unduly restricted. EB: Another interesting thing that I think pops up that is related to a lot of these ethical or moral questions related to science specifically, but maybe exist on a broader level in some of the correspondence in the collection at DMP, is that you seem to receive a lot of letters from a lot of different organizations supporting a lot of different causes -- humanitarian organizations, animal rights organizations, human rights coalitions, things like this -- requesting your support. I was wondering if you could talk a little bit about your role, or participation, or support of some of those groups and what some of your ideas are about that relationship between scientists and social or political activism. MN: Well, I think I've always tried to support causes that I believe in, that I think are good causes. So, when I receive requests of this kind, or to join with others in favor or opposed to specific ideas or things, I always try to participate. And I think it's important to do so. I normally receive lots of letters from various organizations to support different things. Those that I believe in I always support, I always try to lend my support. I think that's important to do that. Some people I think, like Linus Pauling, for example, who was very outspoken in trying to put the ideas that he believed in forth to the public, and I think that's really important to do this. At the time that he was doing it, for example, concerning the radioactivity and the bomb, things of this sort, he took at that time what was really an unpopular view and publicized the reasons why he believed in what he was saying. And I think he did a tremendous service to the country and to the world actually by doing this. I think it's very important to do it. EB: Do you think, possibly at times there have been expectations that might have been put on you as a result of your being a Nobel laureate as far as your involvement in these types of things. MN: Well, sometimes. I mean sometimes I get requests that are completely out of my expertise and if I really don't understand the topic then I won't either join it or oppose it because it's really truly out of my area of expertise. But some of the questions really are social questions and I don't think anybody can say that they're right all of the time. I mean I know that I've been wrong some of the time but you do the best you can. EB: Do you think this role of being the expert or having an expertise is the only reason why people sometimes look for the support of scientists in particular, or is there a greater sense of responsibility that you feel a scientist has outside of just expertise. MN: Well, I think that if you're knowledgeable about a particular area, more knowledgeable than most people about this area, then you should use your knowledge to impart that knowledge to the public and also it may be necessary for politicians to understand your thoughts and the reasons for your thoughts, for your particular stand on things because I think that studying science you have special knowledge that maybe the politicians don't have that would be useful to them. So, that's the reason to support or oppose particular projects. EB: One of the things that is impressive about the body of work that is represented in the collections is how you are able to balance your responsibilities as a lab chief, as a practicing scientist, as a figure who is involved with work within the community, and also different humanitarian organizations. MN: Well, there is never enough time to do everything I think you want to do and you have to make priorities, you have to set priorities. But within the constraints of time, certainly the bulk of my time is spent in trying to do research in the lab and in working within the lab but these other things sometimes don't take much time and if they're useful we try to be useful and we try to be helpful wherever we can. EB: Great, thank you. It's been very helpful. I really appreciate you taking the time to meet with me today. MN: Thank you.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, "Boyle, Eric", null, null, null, null, null, null, null, null, null, null ]
, [ "row-qrhx-aqa3-s3ai", "00000000-0000-0000-98DF-8EF73BE8E14A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Robert C. Gallo to Marshall W. Nirenberg", "101584910X383", null, "1972", "31 May 1972", "Gallo asks Nirenberg to present an open seminar or a more informal laboratory group meeting for the Department of Health, Education, and Welfare.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "May 31, 1972 Dear Marshall, I enjoyed the conversation with you last week.  I do hope you can seriously consider presenting a seminar at some time to our laboratory.  I would be most happy to make this at any time possible for you. If you prefer we can make this an open seminar or a more informal one, which would be at our usual laboratory group meeting.  The latter involves our own lab and Bob Ting's group and some other people at NIH who come fairly regularly.  If you do decide that you will be able to do this and would like to make it at our regular lab meeting, they are usually held on Thursdays at 3 p.m. at Litton-Bionetics, Inc., 7300 Pearl Street, Bethesda, Maryland.  We could, of course, arrange to pick you up and drive you there.  On the other hand, we could obtain a conference room at NIH and make it an open seminar.  (My fear of that is that you would bring in all of NIH.) The only time that would be impossible for me would be between June 8 and June 28. Thank you very much, and I hope you will be able to fit this into your schedule. Sincerely, Robert C. Gallo, M.D.", "Gallo, Robert C. ; United States. Public Health Service", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-u6xp-a9k8~yirt", "00000000-0000-0000-E564-3462D5B76EC9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Mike Kapis to Marshall W. Nirenberg", "101584910X384", null, "1987", "7 January 1987", "In this letter, Kapis describes an organization that he and a fellow graduate student founded, called Alternatives to Animals, which aims \"to encourage and promote the use of alternative methods to reduce or replace the use of animals in education, testing, and research.\"  Kapis asks for Nirenberg's opinion on the use of animals in research.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Mike Kapis.", "Copyright may apply", null, null, "January 7, 1987 Dr. Marshall W. Nirenberg National Institute of Health Bethesda, MD 20205 I am writing to you in regards to our lecture on synapse plasticity at the F/81 symposium, Trends in Bioassay Methodology. Last May, a fellow biology grad student, Kevin Winterfield, and I, Mike Kapis, formed ATA to encourage and promote the use of alternative methods to reduce or replace the use of animals in education, testing, and research. Would you please comment on the following questions. Since the time of the symposium, what is the status of your research in clonal lines of cells?  Has the amount of time been reduced in defining the neural properties of the clonal system? What is the current situation of monoclonal antibodies in connection with your work? How does the factors regarding sensitivity and validity of your research match up to similar research on whole animals? Have you reach any conclusions as to whether your particular research has reduced use of animal research? Is funding difficult to obtain in your research compared to in vivo? What were your thoughts on the symposium? Your insights will be valuable in our research information gathering process! Please keep us updated on your research. Thank You. Sincerely, Mike Kapis", "Kapis, Mike", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hvf5.6s7e-4vvp", "00000000-0000-0000-2765-CB67182E1444", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Ronald LaCoss to Marshall W. Nirenberg", "101584910X382", null, "1989", "December 1989", "LaCoss thanks Nirenberg for meeting with a group of students from the Landon School.  He mentions Nirenberg's enthusiasm for his work and suggests that the students were impressed with his views on working in the field of science.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Ronald LaCoss.", "Copyright may apply", null, null, "Dear Dr. Nirenberg, I wanted to take this opportunity to thank you for taking time out of your schedule to speak to a group of my students. The boys were most impressed with your views on working in the field of science. Your enthusiasm for your work was infectious. I particularly felt that one comment, about making your life's work your hobby, left an indelible message. I wish we could bottle your philosophy and send it out to science classes across the country. Again my thanks, and all the best these holidays and in 1990. Sincerely yours, Ronald LaCoss", "LaCoss, Ronald", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-zdnc_pmbp-n88h", "00000000-0000-0000-46B8-F18ACAB9F661", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Quach Thanh Tam to Marshall W. Nirenberg", "101584910X385", null, "1986", "29 December 1986", "Tam requests Nirenberg's assistance in resolving a problem with his political refugee status, which prohibits his departure from France.  In order to obtain a green card he needs the written support of \"scientific personalities.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Dear Marshall. I am researcher, purifying a neurotrophic factor with Bruce; my wife and me, we would like to thank you for allowing us to share this admirable scientific environment in your laboratory. Right now, I am in Paris.  I would like to return in U.S.A.  Unfortunately, I have some problems to leave Paris because of my political refugee status.  This status does not facilitate a rapid return to Washington before a delay of 3 months to 1 year. May be more. Fortunately, an american lawyer living in Fairfax county can help me to get rapidly a green card, if my request for the green card is supported by several scientific personalities.  This support consists to write a letter describing rapidly what I am doing, and point out that this research is original and important in Neurobiology.  Also it can have many possible applications in the health care. Your help is essential for my green card request, and consequently for my scientific life.  Opportunity in Europe is so rare with all the economical crisis.  Practically we cannot find any interesting job.  I have written a long letter to Bruce explaining my whole problem.  If you need more informations, please don't hesitate to ask him or me, directly. During this period of holiday, let me wish you and your family a happy New Year full of successes and satisfactions. Paris, 12.29.1986. Quach Thanh Tam", "Tam, Quach Thanh", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-vgie~am8f.at5i", "00000000-0000-0000-AFC7-B12D52447B36", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Wallace T. Drew to Marshall W. Nirenberg", "101584910X386", null, "1986", "December 1986", "In this letter to Nuclear Age Peace Foundation members, Drew outlines the Foundation's objectives, which include promoting peace over armed conflict, and presents alternatives to a policy of mutually assured destruction.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "December 1986 Dear Fellow Members: The Biblical story of David and Goliath gives cheer to those of us who wonder how relatively weak peace organizations can successfully challenge the powerful political and industrial organizations behind the expansion of nuclear weaponry. The monstrous MAD policy sustained by the U.S.S.R. and the U.S.A., implemented by nuclear armaments in many forms, and now supplemented by the SDI engines, is an idea whose time has passed. Now is the time that the Nuclear Age Peace Foundation, with its noble motives and its clear thoughts, can present better alternatives to the American people. Using the same leverage David employed in defeating Goliath, we are taking important ideas, applying a little money, and skillfully advancing the ideas of peace over those of armed conflict. The timing of our actions could not be better: -- the world is sickened by the nuclear threat to all nations: -- the two major antagonists -- the U.S.S.R, and the U.S.A. -- are losing heart for the struggle; and -- prayers for peaceful solutions are heard worldwide. The Nuclear Age Peace Foundation -- now seeking $200,000 to support its 1987 action campaign -- can make a difference. The tax climate for charitable giving to a non-profit institution such as the Nuclear Age Peace Foundation will probably never again be as good as it is in 1986. Why not make your Gift for Peace today to the Nuclear Age Peace Foundation?  Don't forget:  Ideas + Energy + Money = Important Results. Sincerely, Wallace T. Drew Vice President and Treasurer", "Drew, Wallace T.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mcid.yjnq_thkh", "00000000-0000-0000-7238-F16351E1865C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from G.B. Marini-Bettolo, Pontifical Academy of Sciences to Marshall W. Nirenberg", "101584910X387", null, "1988", "28 November 1988", "In this letter to members of the Pontifical Academy of Sciences, Marini-Bettolo outlines the primary objectives of the Academy as outlined by the Pope in 1985.  The main points emphasized for future action include: \"the meaning of the universe, the relationships between man and nature, the new biological techniques and the dignity of the human person.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "5", "pages", "Text", "English", "Reproduced with permission of the Pontifical Academy of Sciences.", "Copyright may apply", null, null, "November 28, 1988 Dear Professor Nirenberg, The great honor conferred on me by His Holiness Pope John Paul II, i.e. the task of guiding the Pontifical Academy of Sciences for the next four years, is a source of joy, but also a cause for concern, in view of the great responsibilities facing me. I am deeply aware of the honor of being chosen to continue the work undertaken for the Academy by my distinguished predecessors, Father Agostino Gemelli, Monsignor George Lemaitre and Father Daniel O'Connell, and continued during the last sixteen years by Professor Carlos Chagas, who has guided the Academy with enthusiasm, capability, authority and dignity during one of the most exciting and fascinating periods of our recent history, when science and its applications brought about the great changes which characterize our world today. I hope that the Pontifical Academy of Sciences will continue to develop its activity at the same high level as in the past, with the participation and the active collaboration of' the Academicians,  and yours in particular, as well as the support of the Director of the Chancellery. The directives for future action were clearly indicated by the Holy Father two years ago, on the occasion of the Solemn Plenary Session for the Fiftieth Anniversary of the Pontifical Academy, and again today proposed to our attention. Three main points have been emphasized for our future action: the meaning of the universe, the relationships between man and nature, the new biological techniques and the dignity of the human person. The daily advances in astronomy and astrophysics represent a challenge regarding their interpretation, and require the full attention of the Academy. Progress in genetics, embryology, neurosciences, molecular biology and immunology should be examined also from an ethical point of view in order to defend the dignity of the human person. Ecology should be based on an ethics aiming to indicate new relationships between man and nature, in order to protect our environment and promote the quality of life in a sustainable development. We should also consider the unity of science, now split between the humanistic and the experimental, including mathematics. This will be possible through an interdisciplinary collaboration and a stimulating discussion between the scientists of the Pontifical Academy of Sciences and the distinguished theologians, philosophers, economists and sociologists of the Pontifical cultural institutions. The Pontifical Academy of Sciences, which represents a significant sample of the world scientific community, will study these topics thoroughly and suggest the line to follow, bearing in mind that science is the search for the truth and that it should always be at the service of man and never against man. We can now add to these indications those given by the Holy Father to the whole world in the Sollicitudo Rei Socialis which, in line with Populorum Progressio, calls all of us to the reality of a world divided among wealthy and poor people. As scientists we have the moral obligation to eliminate, with the help of science and technology, the discriminations due to insufficient resources, severe environmental conditions, diseases, malnutrition and poverty. It may be useful to this purpose to remember briefly the tasks assigned to the Academy since its foundation, in different historical and social periods, by the continuous and clear directives given by the reigning Popes. The Academy was founded by Pius IX in 1847, over 140 years ago, with the name of Pontificia Accademia dei Nuovi Lincei. The name Lincei was a recognition of the merit of the work done by the Accademia dei Lincei, founded in 1603, the first Academy of Sciences of the world, where the experimental method was established, opening the way to modern science. Pius IX had a clear idea about the role of science in his time -- having also a personal experience as a student when he published a monograph on optics -- and he established the functions of the Nuovi Lincei. He proposed to the Academy three main tasks: \"to study the progress and spreading of Science\", \"to promote the technical disciplines, the arts and industries which rely on 'Science'\", and \"to assess the Government and the society, when requested, on its works and with its wisdom\". This was the first model of institutions assessing the State in the field of science and technology which were established in the world a century later as National Research Councils. The Academy was re-established in 1936 by Pius XI with the present name of Pontifical Academy of Sciences, becoming also an international Academy.  The scope of its activity was given in the Pope's Motu Proprio announcing the event. The Academy should be \"the Senate of the Holy See in the field of sciences\" and the \"Pontifical Academicians should devote themselves more and more to the advancement of science.  We do not require anything else from them because their noble work in the search for truth represents the contribution we expect from them\".  The Academy is formed by high level scientists from all over the world without any religious or ethnic discrimination, and at this stage it is an Academy of prestige. The Pontifical Academy of Sciences, the Catholic Church being present everywhere in the world, has no boundaries to its activity.  Because of the fact that its members come from all over the world, a traditional academic activity is impossible. The only solution was to hold periodical Plenary Sessions. Father Gemelli, in order to open a constructive dialogue with the world scientific community, after consultation with the Academicians proposed extraordinary meetings: the Study Weeks, with the aim of discussing particular topics with the participation of Academicians and invited scientists who were experts in the field under consideration. In the years 1945-1970 the Academy thus became also an Academy of action.  The subjects of the Study Weeks are focused not only on the findings of basic science, such as those on astrophysics or molecular forces, but also on problems of interest for humankind, such as agricultural production, world hunger, and even on questions at the interface of science and philosophy such as that of the brain and conscious experience. President Carlos Chagas since 1972 has these developed these lines, increasing the role of an Academy of action.  He has given, with his deep knowledge and sensibility, a new dimension to the problems of the Third World, of the environment and of the questions raised by the rapid evolution of biology at every level. Moreover, with the aim of obtaining the direct opinion of experts on specific questions arising from the most recent scientific advances, he established a new type of meeting, the Working Groups, restricted to the participation of a few scientists and experts. The horizon of the Academy's scope was broadened by the new Statutes, issued in 1976: the Academy should promote not only \"the progress of the mathematical, physical and natural sciences\" but also the \"study of the related epistemological problems\". The results of basic and applied research in the last fifty years have meanwhile influenced, through the new technologies, the present society.  They have made possible the exploration of outer space and even of the planets, but have also opened up new possibilities to manipulate embryos, perform fecundation processes in vitro and to realize incredible graftings in man. In the unrestricted use of scientific progress and new technologies, society may lose the sense of moral and ethical limits.  Even the results of scientific advances in basic research, i.e. the laws governing the universe, the origin of life, the evolution of humans, require special attention by the Holy See, which has to be duly and timely informed and provided with documentation. If you read with attention the discourse addressed to you by John Paul II at the Plenary Session of 28th October 1986, you will find not only the guidelines for our future activity but also the indication of a new interdisciplinary effort in the search for truth as stated in the following words:  Philosophical and theological studies of man and nature need your contribution, so that the common knowledge of the inanimate, world of the living universe, and of the human being may advance\". These words invite the Academy to open a new direction in its activity.  It will be another step out of the ivory tower of pure research that will give to our work a new dimension.  I wish to   remind you that in many countries in the last few years a discussion with philosophers and humanists on the impact of science and technology on our society has also been sought by agnostic and free-thinking scientists.  The object of these discussions was the concept of the knowledge of truth, the ethical consequences of the indiscriminate use of scientific discoveries, from cosmology to the origin of life, from the use of destructive weapons to the manipulations of the human person. I wish to recall that in these years groups and associations of scientists, philosophers and jurists have been formed spontaneously all over the world in order to discuss the self-control and the limits that scientists, on a general ethical basis, should establish for their research. The recent discoveries need a continuous rethinking by philosophers and scientists for the implications they may have on our present knowledge of matter, from sub-nuclear particles to the universe, of the fundamental laws, of the questions arising from the relationship between brain and mind, matter and spirit. The Pontifical Academy of Sciences must play its role not only in science but also in the present culture.  We are a part of it and we cannot ever forget the responsibilities of the misuse of science, always bearing in mind that science should always be at the service of man. The first steps in this direction have already been taken by President Chagas, who a few years ago had the merit to abandon agnostic science and the purely academic debates, moving   towards a brighter concept of science at the service of man. I am sure to have also your full collaboration in this particular task. Your suggestions for the Academy's activity will always be very useful.  I also rely on you for the participation when necessary on committees and in assessing the Academy's general or particular problems related to science and its applications.  I think that closer contacts should be maintained by you with the Academy on several issues: e.g. submitting every year the names and the curricula of scientists to be considered for election to the Academy, and sending in your more relevant publications. Last year at the Synod of Bishops the Academy presented some considerations about the role of science as the main factor of the present progress, but also about the fear of the extraordinarily destructive power acquired (weapons, degradation of the environment, uncontrolled use of natural resources, etc.), and its capacity to act on the human person. I may now ask: is science becoming, as anticipated three centuries ago by Sir Francis Bacon, only power?  Scientia est potentia.  Perhaps so, if we consider the present capacity of self-destruction of humankind.  We can stop this issue if we succeed in giving a human and a moral dimension to science. The Holy See needs to be continuously and precisely informed and assessed regarding the evolution of science and its impact on our world. No single scientist can cope with this task, but a body of scientists of different countries and disciplines, such as our Academy, is in a position to give this contribution. I believe that for us it is a great honor and even a great challenge in our work, as well as a recognition of our scientific skill and also of our wisdom.  It also represents a further engagement for us in the work of the Academy as a collective body.  It may require closer and more frequent contacts among us, and I am sure that the Academy can rely on your full collaboration in the accomplishment of our duties as Scientific Senate of the Holy See in promoting science and assessing the impact of scientific advances on humankind. I am grateful to President Chagas, to all of you, and to the Director of the Chancellery, Ing. Dardozzi, for what has been done so far for the progress of science, the use of its results for  developing a  world  without  inequalities, for a better understanding among the world scientific community of the great problems of the future world. I apologize for the length of this letter, but at the beginning of my work at the Pontifical Academy of   Sciences I thought it most important to let you know my ideas.  As I am asking for your collaboration, I hope to continue in the future to maintain with you frequent contacts, not only through correspondence but also, when possible or necessary, by direct talks with you.   On the other hand you will be informed in advance if I plan to travel to your country.  Therefore please inform me whenever you are coming to Italy and to Rome, in order that I may arrange to meet with you.   I trust in your advice, collaboration and support for the Academy's future activity. Now let me send to you personally and to your family, also on behalf of Ing. Dardozzi and the personnel of the Academy, best wishes for a Merry Christmas and a Happy New Year. With best regards, I am Sincerely yours, G.B. Marini-Bettolo", "Marini-Bettolo, G.B. ; Pontificia Accademia delle scienze", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-95i4~ayp9.rz8a", "00000000-0000-0000-1DDF-EE1213BB6477", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "International Campaign", "101584910X388", null, "1985", "[October-November 1985]", "This bulletin from Henri Cartan and Israel Halperin outlines the details of an \"International Campaign\" to free imprisoned and ailing scientists Yury Orlov and Anatoly Shcharansky.  Amnesty International also \"adopted\" Orlov and Shcharansky as prisoners of conscience; cases of \"persons brutally punished for speaking out for freedom of thought.\"", "Newsletters", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "8", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "1981-1984: INTERNATIONAL CAMPAIGN - PROFESSOR J.L. MASSERA (URUGUAY) 1984 - : INTERNATIONAL CAMPAIGN ~ ORLOV AND SHCHARANSKY (USSR)   39 Elm Ridge Orive Toronto, Ontario, Canada, M6B 1A2 Telephone (416) 782-8087 BULLETIN of October-November 1985   These International Campaigns are directed by: Henri Cartan, Membre de 1'Academie des Sciences de 1]‘Institut de France Foreign Member The Royal Society, Foreign Associate National Academy Sciciices, Ancien president de ]'Union mathematique internationale Israel Halperin, Fellow of The Royal Society of Canada Secretary of The Canadian Committee of Scientists and Scholars Former Vice-President of The Canadian Mathematical Society   A statement published in Science, vol. 229, by the American physicist Thomas H. Sti correctly expresses what is in the minds of millions of persons: We are driven on each side by existing fear and mistrust. From International Campaign-Orlov-and Shcharansky to Mikhail S. Gorbachev, Secretary-General of the Communist party of the USSR, September 18, 1985: We can understand that the Soviet people fear that the military forces that oppose them might some day fall under the control of a fascist madman. You should under- stand that we fear that the armed might of the USSR may come under the control of the KGB. fhe irihumane treatment by the KGB of Soviet persons does such harm to the interests of the USSR that it seems that the KGB is more concerned to strengthen its hold on others in the USSR than it is concerned with the best interests of the USSR. We are not among those who want to use the issue of human rights as a trick for political purposes. Mr. Vsevolod Sofinsky, the Head of your delegation to the recent Ottawa Conference on Human Rights, published in our newspaper, the Globe and Mail of August 7, 1985, the statement that, in the Soviet Union's view, human rights are a significant element of relations between states. Until this statement is matched by a change in the way persons are treated in the USSR by the KGB, we are left to feel that our concern to achieve a serious basis of mutual trust is treated by the USSR with contempt.   The International Campaign for Orlov and Shcharansky focuses the attention of persons and organizations in many countries on this basic issue: will the USSR, in its own best interests, recognize that it can only obtain the trust of others by humane treatment of people?   From Article 3 of the Universal Declaration of Human Rights: All human beings are entitled to freedom from torture or cruel, inhuman or degradi ng punishment. 2. The Soviet authorities make it almost impossible to get reliable news concerning Orlov and Shcharansky. Both continue to be in dangerously poor health and their release is urgent. Helsinki Watch, New York summarizes: Born August 13, 1924. Physicist. Arrested February 10, Yury Orlov. ayy, Sentenced to seven years strict regime labor camp and five years exile. Currently in Siberia, in Kobyay, Yakutskaya ASSR 678310, USSR. Chronic cystitis, nephritis, other chronic illnesses. Anatoly Shcharansky. Born January 20, 1948. Mathematician and cyberneticist. Arrested March 15, 1977, sentenced to three years prison and ten years strict regime labor camp. Vision deterioration, headaches, heart ailments. Or. Yury Orlov and Dr. Anatoly Shcharansky, both adopted as prisoners of conscience by Amnesty International, are key cases (among many, many others) of persons brutally punished for speaking out for freedom of thought and conscience. This Campaign will continue to gather more and more supporters in many countries. We will persist with publicity and regular Bulletins in several languages until Orlov and Shcharansky are free and allowed to emigrate. ORLOV TRUST FUND In several countries, including Canada, scientists seek to arrange that permanent residence be offered to the ORLOVS, when they are released (Canada has already offered permanent residence to Anatoly Shcharansky). Organizations and individuals are invited to send donations payable to: ORLOV TRUST FUND, sent to Canadian Committee of Scientists and Scholars, 39 Elm Ridge Drive, Toronto, Ontario, M6B 1A2 Canada. The Fund will be used exclusively to help rehabilitate Dr. and Mrs. Yury Orlov wherever they reside, when released by the USSR. If it turns out finally that the Fund cannot be used on behalf of the Orlovs, the donations will be returned to the donors.   YOU_CAN HELP For Orlov and Shcharansky, this is a critical time. We appeal to you: write urgently to Mikahail S. Gorbachev, Secretary-General of The Communist Party, Kremlin, Moscow, USSR, and express your determination to persist with this Campaign until Orlov and Shcharansky are free. Please help this Campaign by distributing copies of this Bulletin to friends and colleagues. We are building a mighty voice of world-wide public opinion, which with dignity, without violence, simply by persistence, will effectively oppose the plague of terrorism and torture and oppression which is the horror of our time. If you can, please make a financial contribution, payable to: ICOS (abbreviation for International Campaign - Orlov and Shcharan to help pay expenses of postage and printing. — From 85 Nobel Laureates to the USSR Academicians: The undersigned Nobel Laureates ask you to use your influence to persuade your Government to release Dr. Yury Orlov and Or. Anatoly Shcharansky. P. Anderson M. Eigen Linus Pauling C. B. Anfinsen A. P. Esquivel A. Penzias K. Arrow P. Flory (since deceased) M.Perutz J. Axelrod W. Fowler Sir G. Porter D0. Baltimore M. Friedman R. R. Porter Sir D. Barton Donald Glaser E. Purcell Samuel Beckett S. Glashow J. Rainwater B. Benacerraf R. Granit T. Reichstein P. Berg G. Herzberg A. Sakharov (representative) H. Bethe R. Holley P. Samuleson K. Bloch DO. Hubel. A. Schawlow N. Bloembergen Sir A. Huxley F. Sanger B. Blumberg F. Jacob R. Schrieffer H. B&1! (since deceased) Sir B. Katz K. Siegbahn H. Brown Brian Josephson H. Simon Sir M. Burnet A. Klug G. Snell Adolf Butenandt A. Kornberg R. Sperry S. Chandrasekhar F. Lipmann H. Taube C. Cori (since deceased) W. Lipscomb H. Temin Leon Cooper A. . Lwoff J. Tinbergen A. Cormack A. Martin N. Tinbergen Sir J. Cornforth James Meade J. Tobin A. Cournand Sir P. Medawar Lord A. Todd F. Crick S. v.d. Meer C. Townes J. Dausset C. Milosz G. Wald G. Debreu P, Mitchel] P. White R. Dulbecco Sir N. Mott T. Wiesel Sir J. Eccles D. Nathans R. Wilson G. Palade Six Foreign Members of the Academy of Sciences of the USSR now support International Campaign-Orlov and Shcharansky. We urge all Foreign Members of that Academy to speak out, to use their influence with the Soviet authorities. Who supports this Campaign? Persons and Organizations listed as supporters of this Campaign are not responsible for material printed in the Bulletins. They are simply expressin publicly and collectively, their strong opinion that the USSR ought to release Bricy and Shcharansky. Among the thousands of supporters are the 85 Nobel Laureates who sent a message to Soviet Academicians and the following organizations:   Swiss Academy of Sciences American Academy of Arts and Sciences New York Academy of Sciences Norwegian Academy of Science and Letters Human Rights Committee of the National Academy of Sciences (U.S.A.) The Israel Academy of Sciences and Humanities American Association For the Advancement of Science © and.... 4, Supporting Organizations continued... National Physical Societies of: Norway, France, Germany, Israel, U.S.A., New Zealand, Denmark, Switzerland National Mathematical Societies of: Norway, Sweden, Italy, Israel, Chile, Denmark, Irelan U.S.A., Brazil, New Zealand, Edinburgh, Calcutta, Canada, Switzerland National Applied Mathematical Societies of: Canada, Brazil, U.S.A. (SIAM) Mathematics Departments of: Federal University Rio de Janeiro, Warwick University, Newcastle (Australia), Clausthal (Germany), University of New South Wales (Australia) Mathematics Division of the Royal Society of Canada American Statistical Society Chemical Institute of Canada Classical Society of Canada Canadian Ethnology Society Canadian Educational Researchers Association American Educational Researchers Association European Working Group on Statisticians and Human Rights Committee on Scientific Freedom (Association of Computing Machinery) Swedish Committee for Freedom of Science Committee of Concerned Scientists (U.S.A.) Canadian Committee of Scientists and Scholars Yury Orlov. Committee (Switzerland) German Committee of University Scientists Sakharov, Orlov, Shcharansky Committee (U.S.A.) Comite des Physiciens (France) Comite des Mathematiciens (France) British Mensa Human Rights Group . Department of linguistics and philology, University of Montreal Committee For The Defence of Unjustly Accused Movement international des Juristes Catholiques Alpha Omega (dental) Fraternity (Toronto) Physicians Against Psychiatric Torture (Canada) Canadian Labour Congress Ontario Federation of Labour Amalgamated Clothing and Textile Workers Union (Canada) United Steelworkers (Canada) Legislature of Ontario Senate of Memorial University (Canada) Universidade Estadual de Campinas Canadian Psychiatric Association American Psychiatric Association The Canadian Physfological Society The Council of the Swiss Psychiatric Society Canadian Association of University Teachers American Association of University Professors U.K. Association of University Teachers Australian Federation of University Staff Associations Faculty Associations of Canadian Universities: Toronto, Waterloo, Brock, Carleton, Calgary, Bishop's. McMaster, Mt. Allison, Memorial, Winnipeg, Regina, Technical University of Nova Scotia, British Columbia, Manitoba, Mt. St. Vincent, Windsor, Trent, Western Ontario, Victoria, Queen's, St. Thomas (N.B.), Lethbridge and also.... (i) (ii) Leaders in the Churches The Cardinal, Louis Albert Cardinal Vachon Most Rev. Or. John Habgood, Archbishop of York, U.K. Most Rev. Archbishop Edward Scott, Primate of the Anglican Church of Canada Most Rev. Archbishop Trevor Huddleston Bishops: Eric Kemp (Chichester), David Sheppard (Liverpool), Keith Arnold (Warwick), M.A, Feughen (Chester), P.K. Walker (Ely), John Trillo (Chelmsford), John O'Mara (Thunder Bay), Adolphe Proulx (Hull, Canada), Rt. Rev. J. Bickersteth (Bath and Valls) Ronald Bowlby (Southwark), J.B. Taylor (St. Albans), J.A. Baker (Salisbury), Colin James (Winchester), Jean Guy Hamelin (Rouyn-Noranda, Canada). Moderators of United Church of Canada: Robert Smith, Clarke MacDonald President of The World Council of Churches: Lois Wilson President-Designate of the British Methodist Mission: Hughes Smith President of The Lutheran Church (Canada): Robert Binhammer National Superior of English Jesuits, Canada: William Addley, S.J. President of the Canadian Council of Churches: Russel] Legge Baptist Convention of Ontario and Quebec: Rev. A.E. Coe Methodist Church of U.K. : The Rev. The Lord Soper Chairman Birmingham Methodist District (U.K.): C. Hughes Smith Presidents, Principals, Rectors, Vice-Chancellors of Universities   Edward Bloustein (Rutgers) Lloyd Barber (Regina) Derek Bok (Harvard W.. Beckel (Carleton) Jill Conway (Smith J. Daniel (Laurentian) R.M. Cyert (Carnegie-Mellon) R. Farquar (Winnipeg) Lord Brian Flowers (Imperial, U.K.) Gilles Boulet (Quebec a Sainte-Foy, Canada) Manfred Fricke (Tech. Univ., Berlin) E.M. Gulton (Mt. St. Vincent, Canada) E. Handler (Brandeis) Leslie Harris (Memorial) T. Hesburgh (Notre Dame) M. Horowitz (Alberta) H. Kelm (Frankfurt) R. Tanni (Windsor Sir Roy Marshall (Hull, U.K.) P. Keniff (Concordia) Roy Miller (Royal Holloway) A.A. Lee (McMaster) Paul Olum (Oregon) G. Massicotte (Rimouski) J.A. Pinotti (Campinas, Brazil) 8.C. Matthews (Guelph) Michael Sovern (Columbia) P. Meincke (P.E.I.) Naimark (Manitoba) Julius Stratton (Emeritus, M.I.T.) A. G. Pedersen (Western Ontario) H. W. J. Timm (Bremen) Maurice Shock (Leicester) W. Alexander Mackay (Dalhousie) Petch (Victoria, B.C.) Saywell (Simon Fraser) James Downey (New Brunswick) J.T. Spinks (Emeritus, Saskatchewan) J.H. Woods (Lethbridge) B. Segal (Ryerson) Aldee Cabana (Sherbrooke) N. Wagner (Calgary) Louis Philippe Blanchard (Moncton) D. Wright (Waterloo) D. Strangway (elect, British Columbia) B. Shapiro (0.1.S.E.) Robert Smith (British Columbia) D. Theall (Trent Harry Arthurs (York) P. Garigue (Glendon, Canada) Harry Woolf (Director, The Institute for Advanced Study, Princeton, New Jersey) 6. (iif) (iv) (v) (vi) (vii) Other Members. of L'Academie des Sciences (France)   Alain Connes (Fields Medalist) J. H. Oort Henri Cartan Laurent Schwartz (Fields Medalist) Jacques Tits Edgar Lederer Pierre Deligne (Fields Medalist) Rene Lucas W.T. Koiter Rene Thom (Fields Medalist) Francis Perrin Robert d'‘Aubigne Jean Ojeudonne Pierre Lepine Alfred Jost Andre Lichnerowicz J.C. Pecker Etienne Baulieu Gustave Choquet Fellows of the Australian Academy of Science: F.W.E. Gibson, FRS S.J. Redman J.A.F.P. Miller, FRS James Michael G.E. Rogers Alan Sargeson Bruce G. Hyde G.B. Sharman B. Newnann, FRS G.M. Kelly William Levick, FRS A. Kerr 268 Fellows of the Royal Society of Canada, including: The President Prof. Alexander G. McKay, and Z.S. Basinski, FRS Paul De Mayo, FRS Robert Langlands, FRS R.E. Bell, FRS O. McLaren, FRS A. Litherland, FRS H.S.M. Coxeter, FRS John Polanyi, FRS Louis Siminovitch, FRS R.G. Gillespie, FRS J.H. Quastel, FRS H.G. Thode, FRS C.S. Hanes, FRS D.A. Ramsay, FRS Tuzo Wilson, FRS Donald Hebb, FRS Harold Copp, FRS Gerhard Herzberg, Nobel Laureate, FRS (September 12, 1985, from President McKay: Council encourages the President to suggest to all Fellows of the Society that they consider joining the Campaign in support of these persecuted academics) 190 Fellows of The Royal Society including the President Sir Andrew Huxley, Nobel Laureate, and the past-president Lord Alexander Todd, Nobel Laureate and 40 other Nobel Laureates. Members of The Royal Irish Academy Thomas J. Laffey, J.R. McConnell, O.J. Sims, T.T. West (viii) Other Mathematicians (to name only a few) Fields Medalists: Lars Ahlfors, Paul Cohen, Charles Fefferman, David Mumford, Stephen Smale, William Thurston, John G. Thompson Nathan Jacobson, Jacob Schwartz, Dennis Sullivan, Leopoldo Machbin, Jean Cert, R.D. Grigorieff, Robert Edwards, Lipman Bers, Barry Mazur, Edward Nelson, D. Woodhouse, Gyorgy Targonski, Beno Eckmann, William Lambert, Bertrand Mond, Mischa Cotlar, Leon Kushner, Bruce Calvert, Shokichi Iyanaga, H.H. Kairies, Q. Penrose, Pierre Samuel, John M. Howie (Vice-President London Mathematical Society), Robert S. Anderssen (President, Australian Mathematical Society) Kenichi Shiraiwa, Udo Simon, Ralph Bradley, Roberto Conti, Carl Faith, Ubiratan D'Ambrozio, Tang Che-hong (ix) Other Physicists (to name only a few) (x) Robert Marshak Elliott Lieb Paul Kessler Kurt Gottfried Eduardo Amaldi John Ziman 8B. Bleaney Brian Josephson J.F. Nye Mildred Oresselhaus Herman Feshback A.D. Yoffe Victor Weisskopf Pierre Hohenberg Grenville Turner V. Heine G.D. Rochester Sir Brian Pippard Rudolph Haag Maurice Goldfarb Norman Ramsey J.P. Mathieu David Tabor John S. Bell J.S. Mitchell H. Lipson Charles Nelson W.T. Koiter Ephriam Katzir (former President J.H. Oort A.J. Leggett of Israel) Thomas H. Stix In other walks of life (to name a few) Lord Avebury, England Lord Butterworth, England Or. Thomas Bewley, President Royal College of Psychiatrists (U.K.) Francis Low, Provost, M.I.T. Noam Chomsky, linguistics, M.I.T. Dr. Fred Lowy, Dean of Faculty of Medicine, University of Toronto Or. V. Rakoff, Chairman, Dept. of Psychiatry, University of Toronto Simon Levin, Ecology, Cornell Charles Yanofsky, Biology, Stanford R.H. Haynes, Biology, York University, Canada Gregory Viastos, Philosophy, Berkeley Tom Stoppard, CBE, FRSL, playwright Arthur Miller, playwright Marion Andre, playwright W.C. Royster, Dean, University of Kentucky Ernst Deutsch, geophysics, Memorial University Ursula Franklin, metallurgy, University of Toronto Sir Clive Sinclair, U.K. ° John Beckwith, composer, Canada Stewart Smith (President Science Council, Canada) FRSC Pierre Juneau (President C.B.C., Canada) FRSC. Pierre Bois (President Medical Research Council, Canada) FRSC Leo Yaffe, chemistry, McGill, FRSC Jean Dresch, Paris (Foreign Member, USSR Acad. of Sciences) M.P, C.K): J.F. Pawsey; Alan Haslehurst; Robert Hicks; Rt. Hon. P.J. Thomas, Q.C.; t n. Reg. Prentice; Rt. Hon. Sir Bernard Braine, DL; Hon. Greville Janner, Q.C.,¢.P Canadian Mayors: Al Gleeson (London), John Murphy (St. John's), Norm Jary (Guelph), Angus Campbell (Pembroke), Marion Dewar (Ottawa), Robert Morrow (Hamilton), Michel Leger (Hull), Michael Harcourt (Vancouver), Arthur Eggleton (Toronto), Manning MacDonald (Svdne William Smeaton (Niagara Falls), Bob Barker (Peterborough), Audrey Green (Refrew), Gilles Beaudoin (Trois Rivieres), John Gerretsen (Kingston), Ron Wallace (Halifax). Yvon Beaulne, former Canadian Ambassador to the Human Rights Commission of the U.N. Gisele Cote-Harper, former Canadian Member of the Human Rights Committee, U.N. Margaret Atwood, President of Canadian Centre of P.E.N. Hugh MacLennan, former President of Canadian Centre of P.E.N. Writers: Pierre Berton, Robertson Davies, June Callwood, Susan Sontag Pianists: Anton Kuerti, Leon Pommers John Meisel, former President of CRTC, FRSC Robert Kates, NASc., geographer Sigmund Skard, Academician (0slo) Peter Reddaway (London School of Economics) Gershon Iskowitz, artist Harold Town, artist 8. (x) continued..... Johnny Lombardi, President Radio Station CHIN Roger Coull, P.C. Gallaway, Martin Thomas, David Curtis, (Coull String Quartet) Dennis McDermott, President, Canadian Labour Congress Gerard Docquier, National Director, United Steelworkers of America Clifford Pilkey, President, Ontario Federation of Labour Sam Fox, Co-Director, Amalgamated Clothing and Textile Workers Union Antonio Maria Pereira (lawyer, Lisbon, Portugal ) John Humphrey (professor of law, McGill University) Group Captain Paul Harris, D.F.C. Professor H.L. Price, Ph.D., C.Eng., F.R.Ae.S., F.I.M.A. Northrop Frye (Canada) Guido Clemente (Italy) . Moshe Sadfie, architect, Harvard Or. Per Wastberg, President, International Writers Organization, P.E.N. Karen Kennerly, Exec. Director, American Center P.E.N. TO ASSIST THIS CAMPAIGN: Increase circulation of the Bulletin. Use your own initiative in publicizing the cases of Orlov and Shcharansky. Ask persons and organizations to list as supporters. Write to the Ambassador of the USSR in the capital city of your country about these cases. You_can correspond with our Toronto address or with: i. ii. iit. iv. v. vi. vii. viii. ix. X. xi. xii. xiii. Dr. Miles Reid, Math, University of Warwick, Coventry CV4 7AL, England Prof. Paul Kessler, College de France, 11 P1. Marcelin-Berthelot, 75231 Paris, Fran Prof. Kenichi Shiraiwa, MATH, College Gen. Education, Nagoya University, Chikusa-ku Nagoya 464, Japan Comite Orlov, C.P. 335, 1217 Meyrin 1, Switzerland Prof. A. de Oliveira, Rua Dos Arneiros, 28-3 C. Lisboa, Portugal Prof. Carlos Coimbra, SBMAC, Rua Lauro Muller 455,22290 Rio de Janeiro, Brazil Prof. Ubjratan D'Ambrosio, UNICAMP, Campinas, Brazi1 Prof. Warren Ambrose, MATH, M.I.T., Cambridge, MA 02139, U.S.A. Prof. Shlomo Sternberg, MATH, Harvard University, Cambridge MA 02138, U.S.A. Dr. K. Solomon, M.D., 6501 N. Charles St., Baltimore, MO 21204, U.S.A. Prof. Robert Edwards, 86 Vasey Cresc., Campbell, ACT 2601, Australia Wilfred D'Costa, 344/1 Hall Road, Kurla, Bombay 400070, India Prof. Dr. Udo Simon, Tech. Univ. Berlin, Strasse 17 juni 135, 1000 Berlin 12, FRG. When Orlov and Shcharansky are free, but only when they are free, we will launch an International Campaign for the actress carmen Bueno, who was abducted and disappeared in Chile in November 1974. Her case symbolizes the horrible practice by which official governments use unofficial agents to murder persons not in favour. May we list you as a supporter of the Campaign for Carmen Bueno, when it begins?", "Halperin, Israel ; Cartan, Henri", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8zgp-wbua.nhtk", "00000000-0000-0000-167F-F23913D98E1C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Alois G. Englander, World Congress Alternatives and Environment to Marshall W. Nirenberg", "101584910X389", null, "1985", "29 October 1985", "Englander outlines the position of the Weltkongress Alternativen und Umwelt (World Congress Alternatives and Environment), an international organization comprised of Nobel laureates and other distinguished participants.  Included is an appeal to President Ronald Reagan and Secretary General Mikhail Gorbachev to create a comprehensive plan for disarmament.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "3", "pages", "Text", "English", "Reproduced with permission of Juno Sylva Englander.", "Copyright may apply", null, null, "Vienna, October 29th, 1985 Dear Sir: Auspices 1st World Congress: The Nobel Laureates and other distinguished participants of the World Congress \"Peace - The Best Environment\" that has just been held in Maastricht, Holland from October 25th to 28th, in conjunction with \"KAOS Kongres\", have unanimously passed the enclosed appeal. They have decided to present it through delegations of Nobel Laureates to President Reagan in Washington and to Secretary General Gorbachev in Moscow, before the Summit will be held in Geneva on November 19th and 20th, 1985. Due to the pressure of time, please cable your agreement to have your name added to this appeal to: World Congress, 27-28 Graben, Wien, and return both copies signed. (2 appeals!) If you are interested in going to Washington and/or Moscow as part of one or both of the delegations, please let us know immediately. To cover the expenses any donations would be highly appreciated. We remain, with kindest regards, Yours very sincerely World Congress Alternatives and Environment Alois G. Englander Secretary General An Appeal To President Reagan And Secretary General Gorbachev Maastricht, Holland, Sunday, October 27th, 1985 You have an unparalleled opportunity to change the course of human history. We call upon you not to leave Geneva until you have agreed to a concrete plan for comprehensive disarmament, including the following immediate steps: We call upon you not to leave Geneva until you have agreed to a concrete plan for comprehensive disarmament, including the following immediate steps: 2. A Comprehensive Nuclear Test Ban Treaty, starting with a mutual moratorium on nuclear explosions; 3. Progressive de-militarization of outer space; 4. Meaningful reductions in present nuclear stockpiles as a beginning toward their early elimination: 5. Establishment of a joint framework for assessing the dangers of the accidental initiation of nuclear war. As President Eisenhower said, the arms race is daily taking food from the mouths of the hungry. Today security can be found only in disarmament and meeting the needs of a dignified human existence. The future demands a cooperative redirection of scientific and technological know-how. World peace will require less emphasis on ideologies and more on tolerance as well as on the solution of problems which depend upon international cooperation. Courage today is required not in war, but to make peace.  We call on you to begin the Peace Race. Xavier Perez De Cuellar: Message From The Secretary-General To The World Congress Peace - The Best Environment Maastricht, the Netherlands, Oct. 25-28 1985 Presented through the Assistant Secretary General Dr. Robert Muller. It is a great pleasure for me to send my greetings to the distinguished scientists and other participants in the World Congress on Peace: the Best Environment.  I congratulate the organizers of the Congress on their most timely choice of the topic for its discussion. You gather precisely at a time when, on the fortieth anniversary of the United Nations, its Member States are engaged in the vast exercise of drawing the balance sheet of peace.  The focus is on questions as to what has been accomplished during the last four decades in the effort to build a better world for all and what has not and why.  Answers to them involve an assessment of the fundamentals of the current human situation. I attach very great importance to the scientists' part in this exercise.  There are gratifying signs of a process of earnest self-examination in their ranks.  Many eminent leaders of science are disturbed by the misdirection of the scientific talent that is represented by the ceaseless development of the technologies of destruction.  The relentless arms race has now reached the absurd limit of threatening the very existence of the human species and perhaps, the continued habitability of the planet even.   If man is not to suffer the fate of the dinosaur, the necessity of re-orienting the scientific and technological outlook towards the goals of averting the collapse of civilization and of enhancing human dignity and alleviating human suffering needs to be recognized - first of all, in the scientific community itself.  Gatherings of scientists like yours, imbued not only with compassion for the human race but also with a sense of urgency, can stimulate this process. The proposition that peace provides the best environment for human betterment is itself axiomatic but its persuasive effect can be greatly strengthened by analytical exposition.  Scientists can make a contribution to it no less important than that of statesmen.  They know better than laymen what great advances for the reduction of disease, the elimination of poverty, the bringing about or an equilibrium between human society and its surroundings are within the reach of scientific ingenuity and how these advances can be realized only in conditions of just and stable peace.  Their observations on these questions, made with their characteristic regard to cold facts, cannot fail to have a beneficial impact on the political leadership of all societies. In extending to you my warmest wishes for a productive congress, I am voicing the hope that your discussions will lead towards this most desirable goal.", "Englander, Alois G. ; World Congress Alternatives and Environment", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j8gw.4chi.v5hs", "00000000-0000-0000-7E31-A427965915AD", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The International Campaigns for Victims of Torture and Oppression", "101584910X390", null, "1985", "[1985?]", "This letter to Nobel laureates requests support to end all torture and oppression in Chile in the wake of successful campaigns to free Soviet scientists Yury Orlov and Anatoly Shcharansky.  Included is a brief handwritten message from Halperin to Nirenberg.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "The International Campaigns for victims of torture and oppression directed by Henri Cartan, Membre de l'Academie des Sciences (France) and Israel Halperin, Fellow of the Royal Society of Canada Address: c/o Department of Mathematics, University of Toronto Toronto, Ontario, Canada, M5S 1A1 Telephone:  416-978-4156 Dear Dr. Nirenberg.  If you co-sign this letter from Nobel Laureates, this does NOT imply any further commitment.  Israel Halperin To all Nobel Laureates: Nobel Laureates gave leadership in the successful effort to free Prof. J.L. Massera in Uruguay, then Anatoly Shcharansky and Yuri Orlov in the USSR, (98 Nobel Laureates for ORLOV).  This influenced many thousands of individuals and organizations in many countries to support the International Campaigns.  So strong is the international support for these Campaigns that we can now set out to end all torture and oppression in CHILE. The message below from Nobel Laureates will be transmitted to every member nation of the Human Rights Commission of the United Nations.  This Commission meets in February-March each year.  We will ask our many supporters to flood the Commission with their individual letters, referring to this message, and expressing the strongest support for it. Message to every member nation of the Human Rights Commission of the United Nations: The inhuman practice of abduction, torture and oppression in Chile has been condemned by world-wide public opinion.  The Human Rights Commission has rebuked the Government of Chile but has failed to take effective measures to stop the practice.  This failure undermines the credibility of the Commission and weakens the United Nations.  The undersigned Nobel Laureates call on the Commission to take action that will be effective in stopping torture and oppression in Chile. If you agree, please return this coupon as soon as possible to Professor Israel Halperin, University of Toronto, Toronto, Ont. Canada, M5S 1A1 Coupon Name Address (i) Do you agree to be a co-signer of above message to Human Rights Commission, U.N.? (ii) May we list you in our Bulletin as a supporter of the objective of the Chile Campaign?", "Halperin, Israel ; Cartan, Henri", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ak7t~q7uq-yi9q", "00000000-0000-0000-F78A-2C9D050EDC87", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marcia Kerstein Zerivitz to Marshall W. Nirenberg", "101584910X391", null, "1988", "9 March 1988", "Zerivitz, the state coordinator for the Mosaic project for research on Jewish life in Florida, praises Nirenberg for being a role model for Jewish children and asks him to participate in an exhibit the organization is planning.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Marcia Kerstein Zerivitz.", "Copyright may apply", null, null, "March 9, 1986 Dear Dr. Nirenberg: I am so glad I was able to speak to you yesterday to talk about MOSAIC-Jewish Life in Florida. The project is generating so much excitement around the state as we progress in our research and discover interesting early history and prominent Jews who have contributed so much to the development of Florida and to society. You are a role model for our young people; your achievements, when documented and more widely known to this and future generations, can inspire them to higher levels of success and can help develop pride of what fellow Jews have accomplished. I am enclosing some fact sheets about MOSAIC. We are now assembling a preview mini-exhibit to take to Tallahassee and Washington to co-ordinate with our funding requests of the Florida Department of State and the National Endowment for the Humanities. We would like very much to include a photo of your receiving the Nobel Prize in Medicine and Physiology in 1968 and one of you as a young student in Orlando. Please send these as soon as possible to me at: 635 Mariner Way, Altamonte Springs, Fl. 32701. I will be in Washington the week of May 22 to set up the exhibit in the Senate Rotunda and would like very much to do an oral history of you for our archives. If this is possible with your schedule, please let me know. When I was in Pensacola speaking a few weeks ago and mentioned your name as a prominent Jew who grew up in Orlando and was educated in Florida, Sam Goldenberg jumped up and said he was your room-mate at Univ. of Florida. I also met an Aronson gentlemen who knows you. The exhibit is scheduled to travel state-wide in 1990-92 to coincide with the Columbus Quincentenary. It is scheduled for Orlando in December, 1991, and January, 1992. We would be honored if you and your wife could attend our gala opening.  I'll be in touch closer to the date! We will I be very happy if we receive your photos in the next week so that we can include you in our preview. I look forward to meeting you personally. If you have any questions or suggestions, please call me collect at (305) 834-8576. Most sincerely, Marcia Kerstein Zerivitz", "Zerivitz, Marcia Kerstein", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-k6fr.2w3f-n3nw", "00000000-0000-0000-EBCA-2A26A629F5E3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Walter A. Heiby to Marshall W. Nirenberg", "101584910X392", null, "1988", "23 March 1988", "Heiby developed a theory (similar to homeopathy's law of infinitesimals) known as the reverse effect, that states that \"there is a good probability that a vitamin, mineral, or drug that causes or exacerbates a certain disease at one concentration may reverse its role and molify or cure that same disease at another concentration, and vice versa.\"  This is the third letter sent to Nirenberg asking for his comments on the theory, which Heiby promised to include on the jacket for the book.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Walter A. Heiby.", "Copyright may apply", null, null, "[Handwritten note: The jacket design will soon be completed.  I'd like very much to include your comment. Walter Heiby 3-23-88] March 1, 1988 Dear Dr. Nirenberg: I have just completed my latest book and am enclosing an unjacketed copy. The theory of the reverse effect states that there is a good probability that a vitamin, mineral or drug that causes or exacerbates a certain disease at one concentration may reverse its role and molify or cure that same disease at another concentration, and vice versa.  I speculate (on pages 57, 72, 75-76, 81, 250, 276, 453, 857 and elsewhere) that mutagens, at different concentrations, may assume either a carcinogenic or a cancer-curing role.  There are 52 entries under Cancer, reverse effects on index pages 1098-1099.  The book discusses a total of over 250 reverse effects. Will you do me a favor?  Will you write a brief comment about my book The Reverse Effect to appear on its jacket?  My book will receive much better attention from the scientific community as well as from intelligent laymen if you, as a Nobel laureate, would give it favorable comment. If you believe the theory of the reverse effect may lead to nutritional and medical revolutions I hope you will write accordingly.  If my work can be compared, even in just a very minor sense, with any of the concepts or books of the past that sent science in a new direction I'd be pleased if you would make the comparison.  If you believe, as I do, that successful therapies for cancer and for other diseases may result from application of the theory of the reverse effect I hope you will say so. I know that the demands on your time must be very great indeed. May I, therefore, suggest that you read just the Preface, Introduction and chapter one plus anything relating to your own special interests.  (The indices should be especially helpful.) Then, before reading on, may I ask you to mail your comment for use on the jacket?  In just a few weeks I hope to send you a second copy of The Reverse Effect -- this one with your statement featured on the jacket. [Handwritten note: Your work is cited on pages 580-581.] Yours in admiration, Walter A. Heiby P.S. Perhaps you'll want to encourage a colleague to write a book possibly titled The Reverse Effect in Medicine. Pages II (not numbered), 73-74, 164, 260, 326, 786, and 868 suggest the possibilities.  I am awaiting receipt of comments from you and other distinguished scientists to complete the jacket.  I hope that, if at all feasible, I can have your comment in about two weeks. [Handwritten note: Dr. Nirenberg: Please -- I do, so much, want your words on the jacket]", "Heiby, Walter A.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ib5c~kcde~u6sa", "00000000-0000-0000-4FB7-CB043DBCD499", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from William Cuatico to Edward F. MacNichol Jr.", "101584910X393", null, "1969", "6 April 1969", "Cuatico asks MacNichol for advice on where and how he could learn more about the molecular basis of malignant brain tumors as a physician.  Attached to the letter is a return slip with remarks from Nirenberg in which he mentions the current work of James Watson, Francis Crick, Sidney Brenner, and Robert Spiegelman.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of the State University of New York.", "Copyright may apply", null, null, "April 6, 1969 Dear Dr. MacNichol, I read with intense interest your address to the program directors's workshop for neurosurgical training held in Denver Colorado, Jan. 22-24, 1969. I agree that there is need for neurosurgeons who are better versed in the basic research areas. My increasing frustration in clinical neurosurgery specifically, the treatment of malignant brain tumors, goads me to assume increasing interest in the more basic areas i.e. molecular biology where, hopefully, one may find the solution to this increasingly frustrating problem. It has reached the point that I am willing to spend 2 or more years as some kind of a fellow in order to delve into the problem in depth. I wonder if I could get some advice from you in this regard specially the where, how and what in terms of fellowship application.  I am a board-certified neurosurgeon holding a full time academic appointment at the above institution. I have had 1 l/2 years research experience in neuropathology with some electronmicroscopy and for the past 1 l/2 years, have spent almost full time working in the laboratory of micro-neurochemistry, I have in mind working with either Dr. Francis Crick or Dr. James Watson, the former in Cambridge. I would appreciate your opinion regarding the matter. Thank you for your consideration. Sincerely yours, William Cuatico, M.D., FACS Watson has recently become interested in oncogenic viruses so this would be good.  Sidney Brenner rather than Francis Crick is working on the nervous system in Cambridge, Bob Spiegelman will be moving to Columbia in July and is interested primarily in the cancer problem.  Probably Watson and Spiegelman's interests are closest to Cuatico's. You asked me to hold this so you can work it over some more.", "Cuatico, William", null, null, null, "MacNichol, Edward F., Jr", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xiit-8nty_9n6i", "00000000-0000-0000-901A-EE72A0127115", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to Edward C. Rosenow, Jr., American College of Physicians", "101584910X394", null, "1966", "[November 1966]", "Nirenberg thanks Rosenow and the American College of Physicians for their award.  Included are expenses for which Nirenberg was reimbursed.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Dear De. Rosenow: I would like to express my gratitude to you and to the American College of Physicians for selecting me as the recipient of the American College of Physicians Award. My visit there was a most pleasant one and Mrs. Nirenberg and I enjoyed it immensely. We only regret that we were not able to be there for all the gala events. In accordance with your letter of November 17, 1966, I am herewith submitting the expenses which were entailed during our visit to San Francisco. Round trip for two = $549.00 Miscellaneous expenses $75.00 Total $624.00 Thank you again for a most enjoyable visit in San Francisco. Sincerely,", "Nirenberg, Marshall W.", null, null, null, "Rosenow, Edward C. ; American College of Physicians", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-gjnj~y2u4-q6d2", "00000000-0000-0000-6941-AAE0E7294A89", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from William B. Cook to Marshall W. Nirenberg", "101584910X395", null, "1969", "17 April 1969", "Cook asks Nirenberg to serve as a member of the Steering Committee for an International Symposium on Education in Chemistry to commemorate the 50th anniversary of the American Chemical Society's Division of Chemical Education.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of William B. Cook.", "Copyright may apply", null, null, "April 17, 1969 Dear Doctor Nirenberg: I will state the purpose of this letter immediately. I would like to ask you to devote a bit of your valuable time to serve as a member of the Steering Committee for an International Symposium on Education in Chemistry which will commemorate the 50th Anniversary of the American Chemical Society's Division of Chemical Education. The objective of the Symposium is to bring together outstanding research chemists from industry, government and academae to consider chemical education at the college and university level. Chemists from around the world will be asked to participate.  Research chemists are not always as intimately involved in the educational process as those who are more \"teaching\" oriented, yet it is essential that the research oriented chemist contribute his thinking to the process of educating students in chemistry. Support for this Symposium, to be held at Snowmass-at-Aspen, Colorado during the summer of 1970, has been guaranteed by the Division of Chemical Education.  The ACS Board of Directors, at its December 8, 1968 meeting, voted to \". . . . endorse the concept of a 50th Anniversary Symposium on Education in Chemistry sponsored by the Division of Chemical Education and offer its assistance to the Division in the preliminary planning of the Symposium.\" Robert L. Silber, Director of Membership Activities of ACS together with the Division's Executive Committee will assist the Steering Committee in every way possible. The 1970 Symposium will be the first of a continuing biennial series of symposia on education in chemistry.  All symposia speakers, panel members and colloquia leaders will be selected on the basis of the quality of their work.  The Division will continue to sponsor technical programs at ACS national meetings as has been practiced in the past. The \"Westheimer Report\" of 1965 called attention to the dissatisfaction of chemists with support of research in chemistry.  Scant attention was devoted in the report to the dissatisfaction of chemists with the scope and quality of chemistry instruction.  In one part of the document, the authors admit that the report failed to examine in sufficient detail the \". . . . balance in teaching and research\" and suggest that, \". . . . chemistry could well profit from a further study of educational needs and opportunities, and perhaps from long-range research in education.\"  Research in chemical education has a better chance for success if chemists such as yourself participate. The steering committee will be responsible for developing the symposium program.  The committee probably will convene only two or three times for one day.  We hope to hold the first meeting during May, 1969.  Your advice and counsel may be sought on other occasions via phone or letter. Please give serious thought to this proposal. Bob Silber will call you soon to discuss personally your anticipated participation as a steering committee member.  All of your expenses will be covered. Sincerely yours, William B. Cook, Chairman Division of Chemical Education", "American Chemical Society ; Cook, William B.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-48rr~6hvv~utab", "00000000-0000-0000-62F1-731D84A529DC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Asen Stefanof to Marshall W. Nirenberg", "101584910X396", null, "1969", "12 February 1969", "Stefanoff was one of hundreds of people who requested autographs from Nirenberg.  In this case, Stefanoff includes a news clipping discussing Nirenberg in his native Bulgarian.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Asen Shabanakov.", "Copyright may apply", null, null, "HocaeABaHMATa B TASH O6AACT SaToUHAXA UpeAM CTO TOANER, worato maghiapcKesT XEMEK.ODUAPAX MamOD oTAeAM oT o0Ze ma K2eTKA Ha ECTSppa SGexkeSHNKABEA Dpax wa cy6cTaHnMATA, Keato Toll mapeyve HyKJeNmMOnA KNCeANHAa /EHCeEANHA HA SAPOTO, Ipaxtr ua Mumep octawa we3a6ein3ak B NpoxbiAxenme Ha Ae ceruzeTun, Eqna Bp cpezaTa wa YermpuAeceTTe TOANEN yUCHE-— Te OTKpRxa, we MOCHTeANTe HA HacdeAcTBeHETe KavecTBAa Bre EHETe CO CBCTOAT OT HYKACHHOBH KMCEABHH, NO-TOWHO OF Ae30 RcupuGonyKAeCNNOBA ENCCANKA /s cbepamennme wua/. Hpes 1953 roxmma Gpmrancxure 6Hoxmmunn Mopuc YExEEEC, , | Spamcuc Kpxx m amepuxauenst Jxeitu CBN AeCMM>pEpaxa MO WekyAapuua ctpoex ma JHA, 2a Koere SebeT FOARBE.UleRSLNO | HOLY UEX G6exzyonaTa war a, Penrrenosute cHAMEN DOTBSp—- xa, Ue rennre ce chcTros® ot Seskpaiun MoACKYANA BepEre . AHA BoB BEA Ha cumpasa, Benxa MoxeKyaa Ha Tena ce cBcTon oT XEARAm* aTouE, YueNuTé paSkKpuxXa eC ARE MEK por ocMoc c ruranrces pasMepn: Bepurute ma JHA camo H& eANO-CARHCTBEHO SAPO NA KACTKaTA MOFAT AM AOCTRPNAT ADARENA OT NOUTH ABA METPA, & HNMEETE wa JHA ma nakoto TaxO Omxa MOraH, AKO ce CbCANHAT, AA NOK puar pascrosume, 600 usTm nO-roaaMe OT pascTOsaHHeTO Mex~ ay Semuava uw Casnnero. a a Lor e wf . - wen pow Vie ' . a “4 ‘ g, Ox! tra od 564 LF! eo ae aang Se f CLA “iy 7 VY (ok 7 hie ASEN STEFANOFP Bul. ,Makedonia“ 57 VARNA, FOSEBARY 12TH. ,1969. VARNA - Bulgarig MR, MARSHALL We. N | R EN BERG ee ew ne nme ane ee me ee te Oe ee CEAR SER, To am Suwey HAPPY THAT I HAVE THE OPPGRYUNITY TO PETE you. Io amv A STAMP COLLECTOR. I COLLECT POST<STAMPS ANG STAMPS WETH THE PORTRAITS OF *NOBEL@PRIZE* WUNNERS. IN MY COLLECTION THE PLACE OF THE STAMP WETH YOUR FACE 85 EMPTY. UP TO NCW NOT ONE STAMP OF THIS KING HAN BEEN ISSUED BY ANY COUNTRY. INSTEAG OF A STAMP JT HAVE TC PUT A PICTURE OF YOURS & ATH YOUR SIGNATURE CN THE FIONT SIDE. WOULEG YOU Sf SO KING AS TO SENC ME A PICTURE OF YOURS.? I KNOW YCU ARE TCO SUSY WITH YOUR SC LENT IFIC WORK AND AM SORRY FOR DISTURBING You. I CAN’T SAY HCW MUCH OfL1GEC I WOULD BE AND IN THE Save TIME HOy HAPPY T wouLl BE tf I Get A PHOTO- PICTURE OF YousSs. SENCERELY YpuURS:", "Stefanoff, Asen", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-578u-ii29_hmjm", "00000000-0000-0000-57F2-5231E96740AD", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Heinrich Muldner to Marshall W. Nirenberg", "101584910X397", null, "1969", "3 November 1969", "This letter is an example of the collaborative nature of Nirenberg's work and his accessibility to other scientists.  Muldner, working at the Max Planck Institute for Physiological Chemistry, includes details from recent experiments with corresponding sketches.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of the Max-Planck-Institut fur Biophysikalische Chemie.", "Copyright may apply", null, null, "Dear Dr. Nirenberg, With separate mail I will send you next week new preparations of the commercially available Vibrio cholera-sialidase and different test substances. At the moment we have two pure sialidases; the one I have already mentioned which hydrolizes both 2 - 6' and 2 - 3' linkages of sialic acid to carbohydrates. And another one from influenza A2 virus which readily splits only 2 - 3' linkages. This is the characteristic feature of myxovirus sialidases. In all (7) isolated acidic oligosaccharides we find 4 types of bonds through which the sialic acid residues are linked to the carbohydrate: Type I 2 - 3 linkage to galactose II 2 - 6 linkage to galactose III 2 - 6 linkage to hexosamine IV 2 - 8 linkage from sialic acid to sialic acid Type I in gangliosides, fetuin (JbC 239 567 1964) and acidic 2-glycoproteins from humans (BBA 49 250 1961). Type III in submaxillaris mucin (BBA 38 513 1960) Type IV in ganglioside III and IV and colominic acid (polymeric sialic acid) (Biochem. 3 247 1964). As you can see the sialidase we will send splits all these bonds. Only type IV makes some difficulties because in addition to the 2 - 8 linkage a 1 - 9 labile ester linkage can easily be formed and prevent the enzyme to split the 2 - 8 linkage. In these cases one has to open the labile ester linkage first with n/100 NaOH (few minutes). There are of course other difficulties we are just looking for and I shall tell you later. I came back for some weeks to prepare larger amounts of our ATPase which is active on bilayer. I think I will return in spring and hope to see you then and give a talk. Thank you very much again for this valuable introduction to the nerve growth factor. Sincerely yours (Heinrich Muldner) Manfred Eigen sends his regards.", "Max-Planck-Institut fur Physikalische Chemie ; Muldner, Heinrich", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mkms_gahq-6g67", "00000000-0000-0000-4970-C8E20651070B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Don E. Kash, Purdue University to Marshall W. Nirenberg", "101584910X398", null, "1969", "18 March 1969", "This letter from Purdue University's Department of Political Science asks Nirenberg for his input on federal science policy and the nature of scientific advice.  Kash states that Nirenberg was selected as a leader in science, telling him that \"your scientific reputation coupled with your participation in science policy decision-making makes you a valuable source of information.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Don E. Kash.", "Copyright may apply", null, null, "March 18, 1969 Dear Professor Nirenberg: Federal science policy and the nature of scientific advice is a significant topic of concern.  Science policy is becoming a major area of emphasis in research at several major universities.  Purdue University is one of these and our own Program in Science and Public Policy is receiving substantial support from the National Science Foundation.  A major goal of the Program is to develop an archive of reliable information of the sources of advice used by the Federal government in making science policy. We hope you will assist us in this phase of the project. The attached questionnaire has been sent to you because you are a leader in American science.  Only members of the National Academy of Sciences and an equal number of the most-cited scientists in the Science Citation Index will receive this questionnaire.  Your scientific reputation coupled with your participation in science policy decision-making makes you a valuable source of information. We are asking you to include your name on the questionnaire. This is done to enable us to achieve as high a return as possible.  Rest assured that individual responses will remain absolutely confidential and any data reported will be in aggregate form only. We hope this questionnaire covers the most significant questions concerning this important subject area.  The questionnaire has been carefully pre-tested; but, regardless of how sensitively constructed, all questionnaires seem simplistic and confining. Your additions, deletions, and comments are, therefore, respectfully solicited. Completion of the questionnaire will take about 10 minutes of your time. For your convenience a self-addressed, stamped return envelope is enclosed. Sincerely yours, Don E. Kash Director, Program in Science and Public Policy", "Kash, Don E. ; Purdue University", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-y4ix-77k2.8fhs", "00000000-0000-0000-390A-C0D269CBC0CA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Alfred Kastler to Marshall W. Nirenberg", "101584910X399", null, "1976", "14 June 1976", "Kastler, who won the Nobel Prize in Physics in 1966, insists that laureates have a responsibility to use their public prestige to prevent misuse of science.  He recommends that they \"meditate upon the Nobel lecture delivered in 1974 by Gunner Myrdal,\" in which he showed that only a radical change of attitude can head off catastrophe initiated by food shortages and population explosion.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Daniel Kastler.", "Copyright may apply", null, null, "June 1976 Jun 14 1976 To the Nobel-laureates in Science Dear Colleagues, The problems with which humanity is faced today are of such a gravity, and governmental decisions on the national or international scale are often so bizarre that -as was stated by a journalist at a press conference on Human Rights held by Nobel-laureates in Stockholm, 12 December 1975- Nobel-laureates have the responsibility to use their public prestige to help make things go in the right direction and to prevent the misuse of science for purposes leading in the short or in the long run to degeneracy and to destruction of mankind. Therefore I would strongly recommend to all Nobel-laureates who receive every year the Nobel-yearbook of the preceding year, to read and to meditate upon the Nobel lecture delivered in 1974 (see page 263) by Gunnar Myrdal who was awarded the prize in Economics in 1974. Myrdal has devoted his long life to the problems of the desherited people of the earth. He is an expert in the fields of economics and sociology. His lecture highlights the gravity of the present world situation. He shows that only a radical change of attitude of the citizens of the industrialized countries towards the people of the developing areas can protect humanity from the coming catastrophe initiated by the population explosion and food shortages, leading to hunger for billions of people and inevitably to violence. In the present situation, the aid given to underdeveloped areas being very limited, the cruel but rational doctrine of \"triage\" has to be considered, condemning millions of people to death by starvation. If, however, as Myrdal proposes, this aid could be substantially increased, if in our countries the citizens could be educated to consciousness on these problems and to a more frugal mode of alimentation, and if agriculture could be developed in retarded countries, all people on the globe could be saved from undernutrition and starvation. Such an increase in aid is difficult to achieve at the present time when even the industrial countries are severely touched by the economic crisis and by the unemployment problem. But it could be easily done, as emphasized by Prime Minister Olof Palme of Sweden in his speech at the Nobel banquet 1975, if \"the enormous potential of human creativity and material resources at present invested in the arms race, could instead be channeled into the fight against poverty and underdevelopment in the poor countries of the world. Thus, the tremendous resources would be used in the service of peace and reason\". At this point, may I draw your attention to another Nobel lecture published in the same yearbook 1974, page 208, that of Sean Mac Bride, laureate of peace, entitled: \"The imperatives of survival\". Mac Bride strongly supports the view of Gunnar Myrdal on the gravity of the present world situation: \"Never before has humanity been presented with so many or such grave problems\". At present the competition of the nuclear arms race between USA and USSR goes on at a rate which -- as our colleague George Wald has said -- may be characterized as MAD. It is not only an offence to ethical principles, as emphazised by Albert Schweitzer; it becomes a defiance to human intelligence. When some years ago the governments of both nations proposed to other nations the non-proliferation treaty of nuclear arms, they promised to engage themselves in the task of nuclear disarmament. This promise has not been fulfilled. If the present trend goes on, before the end of this century, a dozen more nations will possess nuclear arms. I strongly request all Nobel laureates to join our Japanese colleagues Yukawa and Tomonaga in their Pugwash appeal published in the Bulletin of Atomic Scientists, December 1975. I may also inform you that the \"Stockholm declaration of twelve Nobel laureates on disarmament, help to poor countries and the aim of the Paris conference\" remains open for signatures. Your signature would be welcome. With kind regards. Yours sincerely, Alfred Kastler", "Kastler, Alfred", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-s7nd_cwtr_p3sw", "00000000-0000-0000-05B2-D89A32F8E3D7", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Gerald Grunwald, University of Wisconsin to Marshall W. Nirenberg", "101584910X400", null, "1980", "1 December 1980", "Grunwald thanks Nirenberg for his help in putting grant proposals together, requests a letter of support, and mentions meeting a group of Nirenberg's present and former postdoctoral students.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Gerald Grunwald.", "Copyright may apply", null, null, "12/1/80 [stamped, DEC 3 1980] Dear Dr. Nirenberg: Thanks again for your help in putting my grant proposals together. I hope your recent travels have been pleasant. Please find enclosed: (1) A complete copy of the NIH proposal for your files. (The NIH now has a complete package. I sent a copy of the same grant to NSF. With my application to Dr. O'Reilly, that makes three applications out now.) (2) A cover sheet for the Damon Runyon grant which must be signed and returned to me in the S.A.S.E. provided. This I need A.S.A.P., please. (3) Instructions for you for the material I need to complete my applications to [END PAGE ONE] [BEGIN PAGE TWO] Damon Runyon and Muscular Dystrophy. If you find it simpler, you can send me the originals for each and I can run off the copies myself and forward them to the agencies. These letters (and C.V., publications, etc) are less urgent than the cover letter (item number 2) which I need right away. With these other two grants in, I'll have five possibilities riding. I'm confident enough to stop there, unless you come up with some other idea. Thank you once again for your help Sincerely, Jerry Grunwald P.S. -- I met many of your present and former post docs in Cincinnatti [sic]. Had a great time -- they're a fine group of people, and I look forward to joining them.", "University of Wisconsin ; Grunwald, Gerald", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bxpm.psdx~67ay", "00000000-0000-0000-DDDF-AF47DEF19C15", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Claude D. Stout to Marshall W. Nirenberg", "101584910X402", null, "1970", "15 March 1970", "Stout, an attorney from Wisconsin, conveys his interest in Nirenberg's work on the genetic code.  He writes that \"there must be a scientific reason why the descendents of the ancient Roman-Norman stock who fled to England brought such contribution to culture.\"  He explains that he remains confident that \"further research by the geneticists will reveal the impact of dominant genes of heredity.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "March 15, 1970 Dear Dr. Nirenberg: I am intrigued at your achievement in \"Cracking the genetic code.\" My investigation in the area of genealogy has brought a conviction that there must be a scientific reason why the descendants of the ancient Roman-Norman stock who fled England brought such contribution to culture. I enclose a copy of the concluding chapter of my manuscript along with other copies having a pertinent connection. Incidentally, by grandson graduated with honors from the University of California and is pursuing advanced studies at the University of Wisconsin in biochemistry. I am confident that further research by the geneticists will reveal the impact of dominant genes of heredity. Cordially, Claude D. Stout", "Stout, Claude D.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-up9h.e2wz_j555", "00000000-0000-0000-DFDD-5AC008F7161A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Cyril Clemens to Marshall W. Nirenberg", "101584910X403", null, "1977", "8 November 1977", "Clemens, editor of the Mark Twain Journal, informs Nirenberg that \"In recognition of your outstanding contribution to Modern Science, you have been unanimously elected this day, in succession to the late Sir Alexander Fleming\" an honorary member of the Mark Twain Society.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Joanna Clemens.", "Copyright may apply", null, null, "8th November 1977 Dear Dr Nirenberg In recognition of your outstanding contribution to Modern Science, you have been unanimously elected this day, in succession to the late Sir Alexander Fleming Honorary Member The Mark Twain Society", "Clemens, Cyril", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ssij_93zr_m9p6", "00000000-0000-0000-22FD-3EA74292B92A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Second Annual Norton Levy Memorial Lecture Presents Nobel Laureate Marshall Nirenberg", "101584910X404", null, "1985", "22 March 1985", "This press release announces Nirenberg's lecture on \"the future of genetics and the importance of using this knowledge in an appropriate way.\"", "Press releases", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "FOR IMMEDIATE RELEASE March 22, 1985 SECOND ANNUAL MORTON LEVY MEMORIAL LECTURE PRESENTS NOBEL LAUREATE MARSHALL NIRENBERG The Second Annual Morton Levy Memorial Lecture on Sunday, March 24th at 7:30 p.m. at the Loch Haven Art Center, will present Marshall W. Nirenberg, Ph.D., recipient of the Nobel Prize in 1968 for uncovering the structure of the genetic code. Dr. Nirenberg's discovery has important implications for genetic engineering and may one day provide the cure for certain inherited diseases. He will discuss the future of genetics and the importance of using this knowledge in an appropriate way. The Morton Levy Memorial Lecture Series was created to perpetuate the memory of long-time Orlando physician Morton Levy, who died in 1983. The annual programs are devoted to preserving the ideals by which Dr. Levy lived his life and are presented by his family and colleagues from Orlando Regional Medical Center. His wife, Rita Levy, says the lecture series deals with a part of living that was close to Dr. Levy's heart.  \"It's the philosophy of medicine--the philosophy of humanism--that says the patient, above all, is most important.\" Morton Levy was the grandson of a pioneer Jewish family that settled in Orlando in 1911.  What made Dr. Levy unique in the medical community and in the lives of his patients and friends was the blending of his medical skills with his compassion and love of people.  Dr. Levy felt that preserving the dignity of his patients was essential to their proper medical care. \"Morton Levy made a tremendous contribution toward raising the standard of medical ethics and medical practice in this community,\" says Barry Seiger, M.D., Chairman of the Morton Levy Memorial Lecture Committee. \"It's because of that contribution that we honor Morton Levy with this annual lecturship.\" The first Morton Levy Memorial Lecture was held in 1984 and featured Nobel Laureate Rosalyn Yalow, who discussed radioimmunoassay testing, a technique she developed which revolutionized biomedical research and laboratory diagnosis.  Dr. Yalow's lecture focused on society's fear of radiation and the value of radioactive substances when used constructively in science and medicine.", "Orlando Regional Medical Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9s3d_7hy2_ahui", "00000000-0000-0000-A745-0F8FA6912572", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Georgetown University Department of Biology to Marshall W. Nirenberg", "101584910X405", null, "1986", "24 November 1986", "Indicating Nirenberg's status in the scientific community as a prized advisor, in this letter Neale asks him for help in finding a scientist to fill a faculty position in the Department of Biology at Georgetown University.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Joseph H. Neale.", "Copyright may apply", null, null, "November 24, 1986 Dear Dr. Nirenberg: We are writing to request your help in finding an excellent scientist to fill a faculty position in our Department.  We are interested in hiring an individual with a record of achievement in molecular genetics.  We are enclosing material which describes this position, including a copy of the announcement which will appear in Science and Nature within the next few weeks.   Do you know of a promising young scientist who would flourish in an academic position at Georgetown where the proximity to NIH permits research collaborations to be initiated or maintained as part of an independent research program?  We are interested in interviewing such individuals. Over the past few years we have built a strong core of research programs in the Department of Biology at Georgetown by recruiting productive young investigators.  We anticipate continuing that process with this appointment and additional tenure track positions to be filled in the near future.   Inasmuch as the University and metropolitan area provide an environment which is physically and intellectually stimulating, we are optimistic that these positions at Georgetown will continue to be attractive to well-qualified candidates. We appreciate whatever assistance you may be able to provide us in the task of identifying appropriate applicants. Yours sincerely, Joseph H. Neale, Ph.D. Ellen Henderson, Ph.D. Otto Landman, Ph.D.", "Neale, Joseph H. ; Georgetown University. Department of Biology", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Henderson, Ellen ; Landman, Otto E." ]
, [ "row-9mm5~qp2f_ityg", "00000000-0000-0000-6ECB-0B34104AA726", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from William E. Gordon to Marshall W. Nirenberg", "101584910X406", null, "1988", "31 May 1988", "Gordon, Foreign Secretary for the National Academy of Sciences, alerts Nirenberg to the opportunity for him to participate in three aspects of the NAS program of cooperation with the Academy of Sciences of the USSR: first, to visit the USSR as an Academy Scholar; second, to participate in annual scientific workshops; and third to facilitate a program of exchange of postdoctoral scientists.  Supplemental information in each area is provided and Nirenberg's input is requested.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "6", "pages", "Text", "English", "Reproduced with permission of William E. Gordon.", "Copyright may apply", null, null, "May 31, 1988 Members of the Academy Dear Colleagues: As you probably know, during the past several years we have been gradually expanding our program of scientific cooperation with the Academy of Sciences of the USSR. In January of this year all of the NAS officers and several other NAS members met in Moscow with the leadership of the Soviet Academy to discuss future opportunities for scientific cooperation. The Presidents of the two Academies signed a new five-year agreement for cooperation and developed an ambitious program of joint activities for the next several years. Within the Soviet Academy, a new generation of scientists, well attuned to the international aspects of science and technology, is emerging. For the first time, we have begun exchanging ideas with these new scientific leaders on the reform of their economic and scientific systems. Also, we have had frank exchanges on human rights in the USSR, on free circulation of scientists, and on other topics of great importance to the international scientific community.  The willingness of the Soviet scientific leadership to candidly and openly confront key issues that retard their scientific progress and impede international cooperation has greatly encouraged us as to the possibilities for highly productive collaboration between the scientific communities in the United States and the USSR. The purpose of this letter is to bring to your attention opportunities for your participation in three aspects of our program of cooperation with the Soviet Academy. First, each year we nominate six NAS members to visit the USSR for two to four weeks as Academy Scholars; we invite interested members to apply for the program. Secondly, we sponsor with the Soviet Academy four scientific workshops annually; we seek proposals for workshops from our membership. Finally, we are initiating a program of exchange of postdoctoral scientists; we request your assistance in identifying potential opportunities for placement of Soviet scientists in the United States. Also, as noted in the enclosure, we encourage NAS members to work with Soviet colleagues outside interacademy channels. I am enclosing details about each of these activities. I hope that you will find one or more of them of interest. We are also sponsoring other types of activities such as longer-term visits to the USSR to conduct research. Should you be interested in learning more about our collaborative activities with the Soviet Academy, I suggest that you call Glenn Schweitzer of our staff (202/334-2644) or write to me directly. Sincerely, William E. Gordon Foreign Secretary Exchange Visits by NAS Members to USSR The interacademy agreement signed on January 12, 1988, by the Presidents of the NAS and Academy of Sciences of the USSR continues the Academy Scholars Program begun in 1986. Under this program, each Academy makes arrangements to exchange six Academy members for visits of two to four weeks for the purpose of presenting scientific and public lectures and for scientific consultations. The NAS and the Soviet Academy make all administrative arrangements for the visits. For NAS members who are selected, the NAS provides APEX tickets to Moscow while the Soviet Academy covers living and travel expenses in the USSR. Academy Scholars may be accompanied by a scientific colleague, spouse, or family member, for whom the Soviet Academy will also cover in-country expenses. The NAS members who have participated in the Academy Scholars program during 1987 and 1988 were generally very pleased with the experiences. The NAS members who have completed such exchanges are Gilbert White (University of Colorado), Norman Ness (University of Delaware), Irving Segal (MIT), Saunders Mac Lane (University of Chicago), Michel Boudart (Stanford University), Vernon Hughes (Yale University), Ray Davis, Jr. (University of Pennsylvania), Melvin Green (University of California - Davis), George Herbig (University of California - Santa Cruz), Robert Gomer (University of Chicago), Anton Lang (Michigan State University), Robert Perry (Fox Chase Cancer Center). The NAS is currently soliciting applications from NAS members to participate in the Academy Scholars program during 1989 and 1990. The NAS selection process will be completed by December 1988, with the officers of the NAS making the final selection. Applications should be submitted in the form of a letter of approximately two pages and should include the following information: Name and current affiliation of applicant; a brief description of applicant's current research activities and relevance to the proposed visit; scientific rationale for visiting the USSR and anticipated benefits to be derived from the visit by both the Soviet and US scientific communities; the proposed program in the USSR, including the names of proposed Soviet hosts and Soviet institutions of particular interest; the timing of the proposed visit. Applications should reach the following address no later than August 15, 1988: William E. Gordon Foreign Secretary National Academy of Sciences 2101 Constitution Ave. Washington, DC 20418 Attn: Soviet and East European Affairs - Academy Scholars Further inquiries about the details of the program should be directed to Ms. Cassandra Turczak (202/334-3652). Interested NAS members who have previously applied for the program but did not participate are requested to update their applications with the information described above. The Academy officers will be fully informed about previous applications which, due to administrative constraints within the Soviet Academy of Sciences, did not result in visits. In view of the very limited number of NAS members who can participate in the Academy Scholars Program, members interested in visiting the USSR may wish to arrange such visits on a personal basis directly with Soviet colleagues and Soviet institutions outside interacademy channels. In the recent past, a number of NAS members have visited or have been visited by Soviet colleagues in response to private invitations, and this method of cooperation is likely to increase substantially. Scientific Workshops Sponsored by the NAS and the Soviet Academy The NAS and the Academy of Sciences of the USSR co-sponsor four scientific workshops per year, with one-half held in the United States and one-half in the USSR. Workshops that have been held in 1987 and 1988 (*) or are scheduled in 1988 and 1989 are: 1987: *Condensed Matter Theory, in US 1988: *Use of Lasers in Linear and Nonlinear Photochemistry, in US *Creation of New Vaccines, in USSR *Nonlinear Processes in Dense Plasmas, in US Earthquake Prediction, in USSR Planetary Exploration, in USSR 1989: Dynamical Symmetries & Supersymmetries, in US Biotechnology and Its Applications to Agriculture, in US Structure of Eucaryotic Genome & Regulation of Its Expression, in USSR High Energy Astrophysics, in USSR We currently are soliciting proposals for workshop topics that might be considered for the latter half of 1989 and 1990. Selection of workshop topics will be made at the meeting of Officers of the NAS and Soviet Academy in December 1988. It is therefore necessary that all proposals by NAS members be received at the following address by August 15, 1988: William E. Gordon Foreign Secretary National Academy of Sciences 2101 Constitution Ave. Washington, DC 20418 Attn: Soviet and East European Affairs - Scientific Workshops Each workshop lasts three to four days with an additional ten days devoted to visits to research facilities in the host country. About ten American and ten Soviet scientists participate in each workshop together with additional observers from the host country. The participants, agenda, and technical aspects of the workshops are determined jointly by the American and Soviet co-chairs who are selected by the respective Academies. The NAS and the Soviet Academy assume responsibility for the direct costs associated with the workshops, but the American co-chairs are expected to work with the NAS staff in raising the required funding from Government and private sources.  NAS staff will also assume considerable responsibility for the administrative aspects of the workshops and will provide advice to the American co-chairs on the substantive aspects. Each proposal should include the theme for the proposed workshop, subthemes which might be considered as specific agenda topics, a discussion of the scientific importance of the theme and subthemes, and an assessment of the particular strengths of the Soviet scientific community in the field. The proposal should include the name and affiliation of the American co-chair and the possible co-chair from the Soviet side. Names and affiliations of appropriate American and Soviet participants should also be included. Themes that are closely tied to military applications should be avoided. The proposal should be in the form of a letter of about two pages in length. Questions concerning the proposal or other aspects of the workshop program can be directed to Ms. Virginia Martin on 202/334-3655. Topics that have already been covered in previous workshops will not be repeated for workshops in 1989 or 1990. Proposals previously submitted to the NAS for themes that have not yet been adopted by the two Academies should be updated in accordance with the above suggestions. Postdoctoral Opportunities for Soviet Scientists In an effort to encourage greater interactions between young scientists in the United States and the USSR, the NAS and the Academy of Sciences of the USSR have agreed to exchange information about opportunities for postdoctoral scientists from each country to spend one or more years at appropriate laboratories in the other country. We are asking for your help in identifying opportunities for Soviet postdoctoral scientists to work in laboratories in the United States in fields that would not be considered sensitive from the point of view of military applications of research results. Specifically, we would appreciate your consulting with possibly interested colleagues about such opportunities. If you or your colleagues can identify postdoctoral positions for which highly talented Soviet scientists might compete during the next several years, we would greatly appreciate receiving such information. NAS is acting only as an expediter of information for this program. Therefore, we plan simply to forward to the Soviet Academy of Sciences the names and addresses of US scientists and institutions which would consider Soviet nominees in specific fields, presumably in competition with nominees from the United States and from other countries. The Soviet scientists will then deal directly with the US institutions on all administrative, financial, and other aspects of such arrangements. If you can assist us, we would greatly appreciate your sending information on postdoctoral positions which might be of interest to Soviet scientists to the following address by August 15, 1988: William E. Gordon Foreign Secretary National Academy of Sciences 2101 Constitution Ave. Washington, DC 20418 Attn: Soviet and East European Affairs - Postdoctoral Positions The information should include as much detail as possible on the documentation required from the Soviet scientists and the time table for postdoctoral appointments beginning in 1989. Any questions may be directed to Ms. Kathleen Trivers on 202/334-3654.", "Gordon, William E. ; National Academy of Sciences (U.S.)", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-svzs.izpj-z5ke", "00000000-0000-0000-DD23-079EFBAC449B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Shelia G. Handy to Marshall W. Nirenberg", "101584910X407", null, "1986", "25 February 1986", "Handy thanks Nirenberg for agreeing to serve as keynote speaker for the Region B Elementary Science Fair Awards Program.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "February 25, 1986 Dear Dr. Nirrenberg: Thank you for consenting to serve as the keynote speaker for the Region B Elementary Science Fair Awards Program.  The program will be held on Friday, March 7, 1986, at John Eaton Elementary School, 34th and Lowell Streets, Northwest, at 2:00 P.M. Your presence and active participation at our Awards Program will help to make this a successful event for our students. Sincerely, Shelia G. Handy Assistant Superintendent Region B", "Handy, Shelia G.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7nuv_rybi-aakm", "00000000-0000-0000-8003-1AFE0E1F8F7B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Henry Margenau, Yale University to Marshall W. Nirenberg", "101584910X408", null, "1989", "31 January 1989", "Interested in historical questions and battles between science and religion, Margenau asks Nirenberg to respond to a series of questions for an edited anthology titled Origins: Scientific Perspectives.  Included are such questions as: \"What do you think should be the relationship between religion and science?\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Rolf Margenau.", "Copyright may apply", null, null, "January 31, 1989 Dear Professor Nirenberg: As you are well aware, the history of science has periodically been punctuated by fierce battles between scientists and theologians. Several modern scientists and scientific theories, however, have been surprisingly sympathetic to religious issues. I recall that my late teachers/colleagues/friends, Einstein, Schroedinger and Heisenberg, who were all distinguished scientists, had a passionate interest in religious questions. Theories like the Big Bang, black holes, quantum theory, relativity, and the Anthropic Principle have introduced science to a world of awe and mystery that is not far removed from the Ultimate Mystery that drives the religious impulse. These twentieth century trends seem to call for a new metaphor in describing the relationship of science and religion. Nowhere is the tension between science and religion more pronounced than in the origin issues: the origin of the universe, the origin of life and the origin of homo sapiens. As a scientist and a philosopher of science for over forty years, I have reflected on these questions in my books Foundations Of Physics, The Nature Of Physical Reality and The Miracle Of Existence. These issues have now drawn me to an even more extensive exploration. I would like to map modern scientific perspectives on these issues. To this end, I am working on an origins anthology project with a science journalist. This project entails a compilation of views on the three main origin issues from the most noted scientists of the present day. You would honor me greatly by responding to the questions I have outlined below before April 1.  Your responses will be included in the anthology I will be editing to be titled Origins: Scientific Perspectives. Sir John Eccles, the Nobel Prize winning neurophysiologist who has been described as one of the greatest brain scientists of the century, has kindly agreed to write the foreword to Origins. These are the questions to which I would like you to respond: 1. What do you think should be the relationship between religion and science? 2. What is your view on the origin of the universe: both on the scientific level - and if you see the need - on a metaphysical level? 3. What is your view on the origin of life: both on a scientific level - and if you see the need - on a metaphysical level? 4. What is your view on the origin of home sapiens? 5. How should science - and the scientist - approach origin questions, specifically the origin of the universe and the origin of life? 6. Many prominent scientists - including Darwin, Einstein and Planck - have considered the   concept of God very seriously. What are your thoughts on the concept of God and on the existence of God? I look forward to hearing from you. With many thanks Sincerely, Henry Margenau", "Yale University ; Margenau, Henry, 1901-1997", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-d8xw_4it5.shkn", "00000000-0000-0000-8B63-B8C3CAF2ABCA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Shail K. Sharma, All India Institute of Medical Sciences to Marshall W. Nirenberg", "101584910X409", null, "1990", "6 March 1990", "Sharma informs Nirenberg that he has formal approval for the Indo-US project involving scientific exchange.  He provides Nirenberg with an update on his scientific work, including details on his planned stay at NIH, and submits some research proposals.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Shail K. Sharma.", "Copyright may apply", null, null, "6th March, 1990. Dear Dr. Nirenberg, I have now formal approval of the Indo-US project.  If it is convenient to you may I come to your lab. from 14th May to 16th June, 1990, for about one month. I had written to American Type Culture Collection for my participation in a 5 day programme on \"Recombinant DNA -Techniques and Applications\" from June 4-8, 1990.  If it is all-right with you, I will spend five days (4th to 8th June, 1990) at ATCC in the said workshop.  In your lab, it is possible I would like to purify the growth factor from the C6 glioma conditioned medium.  You may remember that this factor was shown to stimulate the growth of murine hybridomas.  If we could grow the C6 cells in the synthetic medium (devoid of FCS) then it will be very easy to purify this factor.  If this is a new factor, then we can do the cDNA cloning for this. Looking forward to come to the Lab at NIH if it is convenient to you. With kind regards to you and Perola, Yours sincerely, (Shail K. Sharma)", "All-India Institute of Medical Sciences ; Sharma, S. K. (Shail K.)", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rgp6_8cdg~fa8j", "00000000-0000-0000-A7CB-B62457D49BFF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Gerald C. Goeringer to Marshall W. Nirenberg", "101584910X410", null, "1990", "5 March 1990", "Goeringer thanks Nirenberg for volunteering to come to the Department of Anatomy and Cell Biology at the Georgetown University Medical Center to talk with students about homeoboxes.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Gerald C. Goeringer.", "Copyright may apply", null, null, "March 5, 1990 Dear Dr. Nirenberg: Just a brief note to thank you for taking the time to come and talk with our students about homeoboxes. Many of the students have come to me to ask that I express their appreciation also. I have had any number of comments about how interesting your talk was. Many thanks. It of course remains to be seen how effective this series of presentations will be in encouraging students to get into the research laboratory. Sincerely, Gerry Goeringer G. C. Goeringer, Ph.D.", "Georgetown University. Medical Center ; Goeringer, Gerald C.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ckvr_8tmq-4vz3", "00000000-0000-0000-BEA2-B11D00AB540F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Hiroshi Matsuzawa to Marshall W. Nirenberg", "101584910X411", "101584910X366", "1990", "24 July 1990", "Matsuzawa, a scientist from the Department of Agricultural Chemistry at the University of Tokyo, thanks Nirenberg for his hospitality while he stayed in Bethesda.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Hiroshi Matsuzawa.", "Copyright may apply", null, null, "July 24, 1990 Dear Marshall: I have enjoyed my travel of this time, and I have been pleased to see you are fine. I would like to extend my thanks for nice dinner at Bethesda. I have enclosed a picture, and please remind Perola of me. Sincerely yours, Hiroshi Matsuzawa Department of Agricultural Chemistry The University of Tokyo Bunkyo-ku, Toyko 113 Japan", "Matsuzawa, Hiroshi", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xe5v-as2z-29qh", "00000000-0000-0000-09A1-DF3A6C238863", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Israel Halperin to Marshall W. Nirenberg", "101584910X412", null, "1987", "24 August 1987", "Halperin addresses two main issues in this letter to Nirenberg: the International Campaign for Human Rights; and an edited book project he is coordinating on the actual experience of working as a scientist and a scholar.  He requests Nirenberg's involvement in each.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "9", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "UNIVERSITY OF TORONTO TORONTO. CANADA MSS tA! DEPARTMENT OF MATHEMATICS August 24, 1987 Dear Dn. Miri heorg , Some time ago I wrote to about one hundred Nobel Laureates, most of whom had supported the International Campaigns for Human Rights which I have been directing, about a purely historical project. I said that I contemplated publishing a book about the work of Nobel Laureates and asked for whatever documentation could be spared, by those to whom I was writing. My intention was not to publish a biographical account of the usual style. I was thinking of a story about the actual work of the scientists and scholars, the emphasis being on the problems, the disappointments, the difficulties , the final success, but above all, emphasizing the role of hard work. It was my intention to put out a work that would be inspiring to young workers. My own contact with John von Neumann demonstrated to me how much it means to witness, and work with,a great scientist or scholar. It was not my plan to do the actual writing. That was to be done by someone competent in the area under discussion,and it would be my task to find such writers. Undoubtedly more than one book would be involved. Of course this entire project would be unnecessary if such books had been al- ready written or were in process of being written. I therefore asked for such information as was known about such books. I am most grateful to many of you who have sent me material, especially many splendid photographs. I will see to it that one way or another, all the material I collect, is disposed of in a way that will be useful to future historians. Some of you have sent me copies of the pamphlets written by you and published by the Nobel Foundation. These are most welcome. I have enquired from the Nobel Foundation and these pamphlets are available in bound volumes but rather expensive. With respect to my request that you support the International Campaigns for Human Rights that I direct, of course I appreciate that in some sense I am intruding on your privacy. But it is a realistic fact that famous persons enjoy popular respect and Nobel Laureates are surely among the most respected persons. By speaking out against torture and oppression you strengthen enormouslythe action of thousands, perhaps millions,of your fellow persons. The International Campaigns have this charateristic: they are separated away from partisan political action,they pursue doggedly one issue at a time, and the Campaign sticks with the issue until the Campaign is successful. There are 94 Nobel Laureates who support the present Campaignfor all victims in Chile(to the list on page 4 of enclosed Bulletin add: Robert Schrieffer (USA)*), If your name is not in the list, it would be welcomed. SE, ae le ts Let ott OA Yours sincerely, ~p Der ae LL, sya mad Sone) Weise rie 8 Xp» orb te Yew at- OT 5 hex pgs Israel Halperin anh untial tA ard rclion t fora copy) ae Thank for: 1981-1984 : INTERNATIONAL CAMPAIGN - PROFESSOR J.L. MASSERA (URUGUAY) 1984-1986 : INTERNATIONAL CAMPAIGN - ORLOV AND SHCHARANSKY (USSR) 1986-1986 : INTERNATIONAL CAMPAIGN - ORLOV (USSR) 1986- : INTERNATIONAL CAMPAIGN - ALL VICTIMS (CHILE) c/o Department of Mathematics, University of Toronto Toronto, Ontario, Canada, M5S 1A1 Telephone (416) 978-4156   BULLETIN of March-April 1987 Revised August 1987   The objective of this Campaign is to bring to an end all torture, abduction and oppression by agents of the Pinochet regime in Chile.   This International Campaign is directed by: Israel Halperin, Fellow of The Royal Society of Canada Secretary of the Canadian Committee of Scientists and Scholars Former Vice-President of The Canadian Mathematical Society with the help of a world-wide network of colleagues, who copy and distribute the Bulletins; to mention just one (as an example), Professor Kenichi Shiraiwa and his group, who translate the Bulletin into Japanese and distri- bute it throughout Japan. We thank the many thousands of supporters in many countries whose contributions pay our only expenses (very high): printing and postage. The work for these Campaigns, the enormous (and growing) volume of correspondence, are now impossible to handle without a secretary. We appeal to persons and organizations that value these Campaigns, to contribute funds that will pay for a secretary: payable to INTERNATIONAL CAMPAIGNS.   Persons and Organizations listed as supporting the Campaign, are not responsible for the material printed in the Bulletin: they are simply stating their strong support for the objective of the Campaign. Our previous Campaigns have been for individual victims: Prof. J.L. Massera in Uruguay, and Dr. Anatoly Shcharansky and Dr. Yuri Orlov in the USSR. So strong has become our international support (at the end of the Orlov Campaign the number of Nobel Laureate supporters was 98) that we take on now the problem of all victims in Chile. -2- 13 years ago, General Pinochet’s men murdered the legally elected President of Chile and the General seized control of the country, control enforced with the tactics of torture, abduction, and murder of opponents or even critics. Rick Jackson of the Canadian Labor Congress (Globe and Mail, August 13, 1986) called Pinochet one of the world’s bloodiest dictators.   From the Globe and Mail, Feb. 11, 1987: A Chilean army officer gave evidence implicating military officials in the 1976 murder in Washington of a lead- ing Pinochet opponent, former Ambassador from Chile Orlando Letelier. General Pinochet brushed off this event by calling the officer a deserter. From Newsletter Numbers 1 and 2, 1986, Canadian Inter-Church Committee on Human Rights in Latin America: In December, 1984, A.A.V. Morales of the Chilean Air Force Intelligence Service confirmed the existence of an ‘legal’ organization whose job it was to eliminate suspected or known opponents of the regime. The Horrible Practice of Disappeared-Misstng: Agents of the Pinochet regime, sometimes in uniform, sometimes not, seize a victim, take the victim to a secret camp (the location is changed frequently), torture the victim and then murder him or her. The Government and other authorities deny all knowledge of the event and refuse to investigate. Relatives are left in a state of unending despair and agony and the entire population is terrorized (those who complain are themselves often targets of this inhuman practice); sometimes the dead body is found, discarded. From Amnesty International pamphlet: Chile, September, 1986: Juan Aguirre Ballesteros went missing on 4 September 1984. On 22 October his headless and mutilated body was found in a river near Santiago. In July, 1986, Rodrigo Rojas and Carmen Quintana Aranciaba, university students, were seized by a military control, beaten, doused with petrol and set on fire. Rojas died in hospital on 5 July, Carmen Quintana came to Canada, and is in the Hospital Dieu in Montreal, with 80% of her body burned, fighting for her life. Pinochet commented that they had probably burned themselves accidentally. Maximo Baex Jaramillo was beaten by the police, kicked and thrown into a water canal. Fuente Galdames was forced to walk over burning tyres in his bare feet. Luis Caucao Munoz was taken from his home, beaten and burned. One 13-year old girl was put on a police bus and beaten and kicked, later put in a barrel of very cold water. The Archbishop of Santiago protested “methods used to instill fear and insecurity in the whole commun- ity, especially the children”. The threat of arrest, abduction, torture and even death is ever present for thousands of Chileans, from church workers, human right activists, and the urban poor, to members of opposition organizations. Chilean law contains ample provisions for the protection of the rights to life, freedom from arbitrary detention, freedom from torture and ill-treatment, and the right to justice. However, in practice, legal safeguards have been rendered ineffective by emergency laws and outright flouting of procedures. Delegations Vistting Chile: 4 The National Academy of Sciences (U.S.A.) sent a delegation, including two Nobei Laureates, to Chile, March 17, 1985. The delegation reported that the Vicaria de la Solidaridad (under the protection of the Archbishop) documented, in 1983: 60 cases of people who were tortured while detained by the security forces; in 1984: 156 such cases. The most common forms of torture used in Chile, the delegates were told, are beatings on the soles of the feet, use of an electric prod, and electric torture involving strapping the victim to a metal bed, often with strips of wet cloth. The American Committee for Human Rights, the American Psychiatric Association and the American Psycho- logical Association sent a delegation to Chile on November 25, 1985. They reported “One case involved several school children who watched the seizure and shooting in their schoolyard of adults, including a parent and a teacher; those seized were found the next day with their throats slit”. This delegation stated that, based on their own findings, they believed that torture was used by Chile’s military regime. Harassment of Doctors in Chile: On 11 July, 1986, Chilean police arrested and jailed Dr. Juan Luis Gonzalez and Dr. Francisco Rivas, president and secretary general, respectively, of the Chilean Medical Association. Six weeks before, Gonzalez and Rivas had accepted the American Association for the Advancement of Science Award for the efforts of the Chilean Medical Association to prevent torture in Chile and to halt the complicity of physicians in such torture. From Frederick H. Lowy, M.D., Dean of Medicine, University of Toronto, to the American Association For The Advancement of Science, Jan. 26, 1987: ...[ have learned that during the past year the President and Secretary of the Chilean Medical Association have been temporarily imprisoned and harassed .... Canadian academic and medical communities vigorously oppose such abuses of human rights... Are these colleagues still being harassed?   This International Campaign-Chile will escalate publicity by regular Bulletins in several languages, including Spanish and Portuguese, and distribute these Bulletins widely in many countries, including Chile. There are other countries where human rights are abused, but by focusing world-wide public opinion on this notorious case, we will make an effective contribution to the struggle to eliminate torture, as slavery and canni- balism were eliminated long ago from civilized society. -4- Who supports this International Campaign? Thousands, in many countries. We can name only a few. Included are 93 Nobel Laureates (those marked * cosigned the message to the Human Rights Commission of the United Nations printed on the next page): Philip Anderson (USA) Christian Anfinsen (USA)* Kenneth Arrow (USA)* Julius Axelrod (USA)* David Baltimore (USA)* Sir Derek Barton (USA)* Baruj Benacerraf (USA)* Paul Berg (USA)* Hans Bethe (USA)* Gerd Binnig (Germany)* Konrad Bloch (USA)* Daniel Bovet (Italy)* Willy Brandt (Germany) Adolph Butenandt (Germany )* S. Chandrasekhar (USA)* Stanley Cohen (USA)* Leon Cooper (USA)* Sir John Cornforth (U.K.)* James Cronin (USA) Jean Dausset (France)* Gerard Debreu (USA)* Renato Dulbecco (USA)* Manfred Eigen (Germany )* Adolfo Perez Esquivel (Argentina)* E.O. Fischer (Germany)* William Fowler (USA)* Murray Gell-Mann (USA) Donald Glaser (USA)* Sheldon Glashow (USA)* Ragnar Granit (Sweden)* Herbert Hauptman (USA) Dudley Herschbach (USA)* Gerhard Herzberg (Canada)* Robert Hofstadter (USA)* Dorothy Hodgkin (U.K. )* Robert Holley (USA)* David Hubel (USA)* Francois Jacob (France)* B.D. Josephson (U.K.)* Jerome Karle (USA)* Sir Bernard Katz (U.K.)* Aaron Klug (U.K. )* Arthur Kornberg (USA)* Polycarp Kusch (USA)* Yuan T. Lee (USA)* Baruch Blumberg (USA)* Sir George Porter (U.K.)* Andre Lwoff (France)* Sean MacBride (Ireland)* A.J.P. Martin (U.K.)* Sir Peter Medawar (U.K. )* J.E. Meade (U.K. )* Simon van der Meer (Switzerland)* Czeslaw Milosz (USA)* Cesar Milstein (U.K. )* Peter Mitchell (U.K. )* Francisco Modigliani (USA)* Sir Nevill Mott (U.K. )* Daniel Nathans (USA)* George Palade (USA)* Linus Pauling (USA)* Arno Penzias (USA)* Max Perutz (U.K. )* John Polanyi (Canada)* Tadeus Reichstein (Switzerland)* Carlo Rubbia (USA)* Ernst Ruska (Germany)* Abdus Salam (Italy)* Frederick Sanger (U.K. )* Arthur Schawlow (USA)* Julian Schwinger (USA)* Glenn Seaborg (USA)* Kai Siegbahn (Sweden)* Herbert Simon (USA)* George Snell (USA)* Roger Sperry (USA)* Henry Taube (USA)* Howard Temin (USA)* Jan Tinbergen (Netherlands )* Nicolaas Tinbergen (U.K. )* James Tobin (USA)* Lord Todd (U.K)* Charles Townes (USA)* Desmond Tutu (South Africa)* George Wald (USA)* Patrick White (Australia)* Elie Wiesel (USA)* Torsten Wiesel (USA)* Robert Wilson tise Antony Hewish (U.K. )* Andre Cournand (USA)* Paul Samuelson rae Louis Neel (France)* -5- Message to every member nation of the Human Rights Commission of the United Nations: The inhuman practice of abduction, torture and oppression in Chile has been condemned by world-wide opinion. The Human Rights Commission has rebuked the Government of Chile but has failed to take effective measures to stop the practice. This failure undermines the credibility of the Commission and weakens the United Nations. The undersigned Nobel Laureates call on the Commission to take action that will be effective in stopping torture and oppression in Chile. This Campaign has the support of (also): The Academia Nazionale dei Lincei (Italy) Sir Yehudi Menuhin Jack Lemmon, actor (star of the movie ‘Missing’) Ephriam Katzir, former President of Israel The Federation of Australian University Staff Associations Dr. Yuri Orlov and of (also): (i) (ii) Organizations The InterChurch Committee on Hyman Ri I ti j The national Mathematical Societies of Canada, Norway, Orgel Galette Sap Anerice (Canada ) The United Steelworkers of America, The Canadian Labor Congress The Ontario Federation of Labor National Academies of Science of Bolivia, India The Chemical Institute of Canada The New York Academy of Sciences The Social Responsibility and Justice Committee of the United Church of Australia International Playwrights, Essayists and Novelists (Canadian Centre) Faculty Associations of Canadian Universities: Toronto, Brock, Mount Allison, Western Ontario, Moncton, Faculty of Science of the University of Witwatersrand, South Africa SFU, UBC. The Committee of Concerned Scientists (CCS), The Law Union of Ontario The Alliance of Canadian Cinema, Television and Radio Artists (ACTRA) Actors Equity of New Zealand The German Physical Society The Jesuits of Canada, The Dominican Friars of Toronto Presidents, Vice-Chancellors, Principals, Rectors, Masters of Universities P.J. Boyce (Murdoch, Australia) John Mallea (Brandon, Canada) Aldee Cabana (Sherbrooke, Canada) John Marburger (Stony Brook, USA) J.E. Chamberlin (New College, Toronto) B.C. Matthews (Guelph, Canada) Jill Conway (formerly Smith, USA) Jean-Guy Paquet (Laval, Canada) Margaret Fulton (formerly Mt. St. Vincent, Canada) George Pedersen (Western Ontario, Canada) John Godfrey (King’s College, Halifax) Jose Pinotti (formerly Campainas, Brazil) R.D. Guthrie (NSW Institute of Technology, Australia) | Peter Richardson (University College, Toronto) Keith Hancock (Flinders, Australia) William Saywell (Simon Fraser, Canada) Evelyn Handler (Brandeis, USA) David Smith (Queen’s, Canada) Leslie Harris (formerly Memorial, Newfoundland) Robert Smith (Western Australia) Theodore Hesburgh (Notre Dame, USA) David Strangway (British Columbia, Canada) Ron Ianni (Windsor, Canada) Ron Watts (formerly Queen’s, Canada) Paulo Renato Costa Jonga (Unicamp, Brazil) Brian Wilson (Queensland, Australia) Alvin A. Lee (McMaster, Canada) Douglas Wright (Waterloo, Canada) M.I. Logan (Monash, Australia) Di Yerbury (Macquarrie, Australia) Lord Flowers(University of London, U.K.) Walter Pitman (OISE, Canada) Rodolfo J. Pinto de Luz(Santa Catarina, Brazil) A. LeRoy Greason(Bowdoin, USA) Klaus Ring(J. W. Goethe, Frankfurt ) Brian Segal(Ryerson, Canada Jerome Wiesner(formerly M.I -T.) Jurgen Timm( Bremen, German Patrick Kenniff(Concordia, Canada) Donald Wells(Me . Allison, tdnada) Richard Cyert(Carnegie-Mellon, USA) J. Lyons(Trinity Hall, Cambridge, U.K.) L.P. Blanchard(Moncton) (continued on next page) ~6- (ii) Continued: Rectors of Universities in Brazil: Fund. Univ. Estad. Maringa Univ. Fed. Fluminense Univ. Fed. Paraiba Fund. Univ. Blumenau Fund. Univ. Fed. Mato Grosso Univ. Estad. Paulista “Julio de Mesquita\" Univ. Estad. Campinas Univ. Fed. Uberlandia Univ. Catolica Goias Univ. Fed. Rio de Janeiro Pontif. Univ. Catolica Campinas (iii) Academicians in many countries, we list those who are Members of The Royal Netherlands Academy of Arts and Sciences: Prof.Mr. J.A. Ankum Prof.Dr. E.H. Kossmann Prof.Dr. J. Ariéns Kappers Prof.Dr. D.J. Kuenen Prof.Dr. G. Blasse Prof.Dr. C.J. Lammers Prof.Dr. D.P. Blok Mr. G.E. Langemeijer Prof.Dr. J. de Boer Prof.Ir. D.G.H. Latzko Prof.Dr. D. Bootsma Prof.Dr. W.J.M. Levelt Prof.Dr. P. Borst Prof.Dr. H. Linnemann Prof.Dr. N.G. de Bruijn Prof.Dr. J.H. van Lint Prof.Dr. J. Bruyn Prof.Dr.Ir. J.P. Mazure Prof.Dr. H.M. Buck Prof.Dr.Ir. R. Meischke Prof.Dr. W.H. van Dobben Prof.Dr. G.R.F.M. Nuchelmans Prof.Dr. P.J.D. Drenth Prof.Dr. J.H. Oort Mr. H. Drion Prof.Dr. J.Th.G. Overbeek Prof.Dr. C.W. Drooger Prof.Dr. S.L. Radt Prof.Dr. H.W. von der Dunk Dr. A.R. Ritsema Prof.Mr. P. van Dijk Prof.Dr. J.J. van Rood Prof.Dr. W.T. van Est Prof.Dr. C.J. Ruijgh Prof.Dr. N.H. Frijda Prof.Dr. A. Teeuw Prof.Dr. I. Gadourek Prof.Dr. E. den Tex Prof.Dr. P.J. Gaillard Prof.Dr. J. Tinbergen Prof.Dr. H. van Genderen Prof.Dr. G.J. de Vries Prof.Dr. A.D. de Groot Prof.Dr. J.H. van der Waals Prof.Mr. P. de Haan Prof.Dr. V. Westhoff Prof.Dr. P. Hennipman Prof.Dr. A.A. Weijnen Prof.Dr. W.K.B. Hofstee Mr. G.J. Wiarda Dr. M.W. Holtrop Prof.Dr. J.T. Wiebes Prof.Dr. F.J. van Ingen Prof.,Dr. D. de Wied Prof.Dr. L. de Jong Prof.Dr. J.C.M. van Winden Prof.Dr. J. Joosse Prof.Jhr.Dr. P.J. van Winter Prof.Dr. P. van de Kamp Prof.Dr. P. de Wolff Prof.Dr. P.W. Klein Prof.Dr.Ir. P.M. de Wolff Prof.Dr. A.J.F. Kébben Prof.Dr. A.S. van der Woude Prof.Dr.Ir. W.T. Koiter Prof.Dr. E. Ziircher Prof.Dr. J. Kommandeur -7- (iv) Members of L'Académie des Sciences(Frar c} and Members Associés R. D' Aubigné Y. Choquet-Bruhat W. Koeiter J.C. Pecker P. Auger CG. Cohen-Tannoudgi H. Lacombe R. Peierls E. Baulieu J.» Coulomb P,. Lacombe J. Roche J. Blamont P. Deligne , Y. Laporte E. Schatzman B. Bleaney J. Dieudonne BE. Lederer L. Schwartz J. Brachet J. Doob J. Lehn P. Slonimski E. Boureau C. Dubost P. Lepine kK. Thimann H. Cartan F. Gros G. Millot A. Thomas H. Casimir A. Guinier T. Monod J. Tits R. Castaing P. Jacquinet A. Moyse V. Weisskopf P. Chambon A. Jost G. Ourisson R. Wurmser G. Choquet P. Karli (v) Fellows of the Royal Society of Canada, 94, among them: Northrop Frye Margaret Atwood Marc-Adelard Tremblay Guy Rocher Robert Finch Robert Haynes L.S.Wolfe Bryce Kendrick Edith Fowke Hon Eugene Forsey John Meisel Pierre Bois George Connell Ermest Sirluck Sydney Friedman Louis Siminovitch Leslie Green Digby McLaren (Pres. RSC) (vi) Musicians Maurice Lebel (former Pres. RSC) John: Weanzweig, Harry Freedman, Anton Kuerti, Louis Applebaum, Maureen Forrester, John Beckwith, Oscar Peterson, Oskar Morawetz, Leon Pommers (vii) Writers Per Wastberg, Arthur Miller, Timothy Findley, Susan Sontag, June Callwood, Marion Andre, Robert Fulford, Robertson Davies (viii) Physicists in many countries, among them: Kurt Gottfried, Elliot Lieb, John Charap, Herman Feshbach, Daniel Kastler, Rudolph Haag, William McGowan, J. Trumper Myer Bloom, Paul Kessler, J.P.Mathieu, Ephriam Katzir(former President of Ispael)s. Eduardo Amaldi, Rafael Sorkin, Fritz Rohrlich (ix) Mathematicians Fields Medalists: Sir Michael Atiyah, David Mumford, Stephen Smale, Charles Fefferman, Michael Freedman Laurent Schwartz Jacques Dixmier, Dennis Sullivan, Edward Nelson, Robert Edwards, Hale Trotter, Lipman Bers, Leon Kushner, Ezio Stagnaro, William Parry, Miles Reid, Jean Louis Verdier, Jean Francois Mela, Robert Anderssen, Marcos Sebastiani, Udo Simon, Gyorgy Targonski, L. Arnold, Dieter Kohnlein, Kurt Leichtweiss, Shokichi Iyanaga, Kenichi Shiraiwa and 23 other senior mathematicians in Japan, and many others in other. countries, e.g. M. Kreck, W. Ambrose, B. Monteiro, J. Seade (x) Fellows of the Australian Academy of Science: C.A. Hurst, S.J. Redman, B.G. Hyde, Jacques Miller, Allen Kerr, G.M. Kelly, Sir Gustav Nossal. (xi) Religious Leaders Rt. Rev. Paul Moore Jr., Bishop of New York Most Rev. Michael J. Peers, Primate of the Anglican Church of Canada Very Rev. Lois Wilson, President of the World Council of Churches Very Rev. Edward Scott, President of the Canadian Council of Churches Moderators of the United Church of Canada, present and former: Dr. Anne Squires, Clarke MacDonald, Bruce McLeod, Robert Smith Rabbi Dow Marmur, Holy Blossom Temple, Toronto Father William M. Addley, S.J., Provincial Superior, Jesuit Order Henri Goudreault, O.M.I., Provincial Superior, St. Joseph's Province Marilyn Matz, F.C.J., Provincial Superior, Faithful Companions of Jesus RF. Wooton, Associate Secretary, United Church of Australia (xii) Mayors of cities Ron Wallace(Halifax), Robert Morrow(Hamilton, Sylvia Sutherland(Peterborough) Gordon Campbell(Vancouver), Arthur Eggleton(Toronto), Manning MacDonald(Sydney) _ (xiii) Senators(Canada) Earl Hastings, John Godfrey, L. Norbert Theriault, David Croll, Royce Frith, Joan Neiman (xiv) Members of Parliament(Canada) Neil Young, Steven Langdon, Hon Warren Allmand, Michael Cassidy, Bill Blaikie, Hon Charles Caccia, Dan Heap, Sheila Copps, Ernie Epp, Dr. Maurice Foster, Svend Robinson, Lynn McDonald, John Parry, Robert Kaplan (xv) Members of the Ontario Legislature Susan Fish, Jim Henderson, Ian Scott, Karl Morin-Strom, Richard Allen, Dave Cooke, Mike Breaugh, Elinor Caplan, William Wrye, James Foulds, Jim McGuigan, Bob Rae, Marion Bryden, C. J. Wildman (xvi) Many, many other persons and groups, among them: Hon J.D. Arnup, Justice(retired), s. . FRS: Charles Hanes, Michael Smith, L. Mestel, John S. Bell, Robert Bell, (Court, ont Harold Copp, David Tabor, Donald Ramsay, H.S.M. Coxeter, Frank MackIntosh, C.T.C. Wall, Anthony Leggett : Asociacion Universitaria y Cultural Andres Bello(Chile) The German Commission For Rights of Chilean Women The Criminal Lawyers’ Association(Canada), Fellowship of Reconciliation Noam Chomsky and many other linguists in many countries To Help This Campaign, which will escalate until torture stops in Chile: You can distribute copies of this Bulletin, you can contribute funds so that a secretary can be employed, you can write a letter to the Ambassador of Chile to your country (sufficient address: Ambassador, Embassy of Chile, Capital City of your country) and express your opinion of the situation in Chile.", "Halperin, Israel", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-siaq-rucs-z342", "00000000-0000-0000-7ED8-C29813D3CC2F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Ralph Simon to Marshall W. Nirenberg", "101584910X413", null, "1987", "23 May 1987", "Simon conveys his \"strong impression that we must now begin an active campaign to counteract popular belief in supernatural power.\"  He suggests that Nirenberg, as a Nobel laureate, make some kind of public statement regarding the \"incompatibility between natural law as discovered by application of the scientific method, and the experience of supernatural power.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Ralph Simon.", "Copyright may apply", null, null, "23 May 1987 Dear Dr. Nirenberg: I, a retired physicist, am writing this letter to all American Nobel Laureates in physics, chemistry and physiology or medicine whose addresses I could find, to convey my strong conviction that we must now begin an active campaign to counteract popular belief in supernatural power. Fundamentalist religions may have served some useful purposes in the past, and still continue to do so in certain limited areas. On balance, however, I believe they are now becoming a major impediment to the progress of the human species toward a world that is relatively free from military conflicts and in which political, social, economic and environmental problems can be investigated and solutions proposed and tested in a rational manner. We find today in the United States, in a society that is scientifically and economically advanced, a strong resurgence of religious fundamentalism.  The advocates of this movement are becoming ever more powerful politically and their voices reach a friendly ear in the White House.  They cry out for such measures as the legalization of prayers in the public schools and for the teaching of \"creation science*' on an equal basis with evolution.  Recently a federal judge in Alabama ruled that textbooks which cover rational approaches to social or moral problems were advocating the particular \"religion\" of secular humanism, and are therefore unconstitutional. Fundamentalists claim that the \"absolute truths\" regarding such matters are revealed in religious dogma. What all this is leading up to is very frightening.  Why bother to make arms limitation agreements with the Soviets, to protect the environment, or to pursue vigorously physical, chemical or biomedical research? Since the apocalyptic destruction of the world is coming soon anyway, the only truly meaningful activity is the seeking of personal salvation. My suggestion for an initial step to counteract this growing mania is for you, as a Nobel Laureate, to make some kind of public statement regarding the incompatibility between natural laws as discovered by application of the scientific method, and the existence of supernatural power.  Since the general public understandably has a great respect for Nobel Laureates, such a statement should help considerably to halt the current spread of religious fanaticism. I would appreciate receiving your comments regarding the above suggestion, or any other ideas for a course of action that you feel might be more appropriate and effective. We must do something; we cannot let human civilization, which has shown such great strides in the last two centuries mainly because of scientific progress, go down by default. Sincerely, Dr. Ralph Simon", "Simon, Ralph", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dq7f.8m9s_jms3", "00000000-0000-0000-EFD8-4D04BB699DFA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Nicolas G. Bazan to Marshall W. Nirenberg", "101584910X414", null, "1993", "27 May 1993", "Bazen, Professor of Ophthalmology, Biochemistry, and Molecular Biology and Director of the Neuroscience Center at the School of Medicine in New Orleans, updates Nirenberg on the neuroscience company he and Lloyd Horrocks founded.  Nirenberg agreed to be a scientific advisor to the company as the main theme of the company was to apply signal transduction technology to develop drugs to treat neurological diseases.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Nicolas G. Bazan.", "Copyright may apply", null, null, "27 May, 1993 Dear Marshall: I would like to bring you up-to-date on the neuroscience company established by Lloyd Horrocks and myself originally called BrainChemTech. Recently, we have decided to rename it as B&H Pharmaceuticals, Inc. It was very kind of you to agree to be a scientific advisor to this company when I contacted you a few months ago.  I would like to reaffirm this relationship with you. As I mentioned at that time, Lloyd will be full time in the company and I will Chair the Scientific Advisory Board. I plan to continue to devote most of my time to my current university research laboratory. The company has recently picked up momentum and seems ready to be launched.  We have enlisted the services of Dr. Alex To in putting the start-up activities together.  Dr. To is a very experienced management consultant and was previously vice president of a large New York venture capital firm which specialized in medical product companies. We have interviewed a series of CEO candidates as Chief Executive Officer and will make the final selection shortly. The company is also under negotiation with several institutions in order to obtain licenses to patents. Most importantly, we are under discussion with venture capital firms and investors, and it looks very likely that the funding will be in place in the near future. Some of these investors may contact you in their due diligence process. I am wondering if you would be so kind as to take time from your busy schedule and answer their inquires, please give your honest opinion to any question the investors may have. To reiterate my previous communication, the main theme too of the company is to apply signal transduction technology to develop drugs to treat neurological diseases. Products include neuroprotective agents, CDP-ethanolamine, and a slow release dopamine microcapsule, among others. Thanks again for your kind support.  I hope that I will be in the position to discuss specific terms of your advisorship in the near future. Best regards, Nicolas G. Bazan, M.D., Ph.D. Professor of Ophthalmology, Biochemistry and Molecular Biology, and Neurology Director, Neuroscience Center", "Bazan, Nicolas G.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-vqpc-vksj.q7mt", "00000000-0000-0000-4126-073E834D099B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Stephen Lanham Tarason to Marshall W. Nirenberg", "101584910X415", null, "1997", "10 February 1997", "Tarason thanks Nirenberg for agreeing to be a judge for the Silver Spring High School Science Fair in Maryland.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Stephen Lanham Tarason.", "Copyright may apply", null, null, "February 10, 1997 Dear Judge: Thank you for agreeing to be a judge for the Silver Spring Community High School Science Fair on Wednesday, March 26, 1997. I will be forwarding a packet to you in the near future. The packet will contain information needed for the day of the Science Fair. I look forward to meeting you. If you have any questions or concerns, please call me at 301-230-5463. Sincerely, Stephen Lanham Tarason Director of Innovative High School Programs", "Tarason, Stephen Lanham ; Montgomery County Public Schools (Md.)", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-imu5.iwwt_63u7", "00000000-0000-0000-EB30-72B55D5E21BE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Robert Ehrlich to Marshall W. Nirenberg", "101584910X416", null, "1986", "13 March 1986", "Ehrlich thanks Nirenberg for agreeing to speak at George Mason University on \"Frontiers of Genetic Engineering\" as a part of their fall lecture series \"Six Nobel Laureates Look at the Next Fifty Years.\"  The letter suggests that nearly twenty years after he won the Nobel Prize for work in genetics, Nirenberg was still seen as a leading authority in the field.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Robert Ehrlich.", "Copyright may apply", null, null, "March 13, 1986 Dear Dr. Nierenberg: Thank you for agreeing to speak at George Mason University on October 21 at 7:30 pm on \"Frontiers of Genetic Engineering\". This talk will be one of six in our Fall Lecture Series: \"Six Nobel Laureates Look At The Next Fifty Years\". As I mentioned on the phone, the series is being supported by ten area companies, and it will be attended by engineers, scientists and business people, most of whom know little about genetic engineering. A large number of students and faculty should also be present. At this moment, the other five topics and speakers have not yet been finalized, however, I am thinking of the topics listed on the enclosed outline. In the past the format of these lectures has allowed for an extensive question and answer session, so that with a coffee break in the middle and Q&A the session has typically gone until 9:30. I hope, incidentally, it would be possible for you to join a group of us before your talk for dinner at 6:00 pm. As I mentioned on the phone the stipend for this lecture would be $1,000. Sincerely yours, Robert Ehrlich Six Nobel Laureates Look at the Next 50 Years Possible lecture topics include: The Shape of Tomorrow's Technoloqy extrapolations from today's discoveries in pure science communications, transportation, new materials The Future in Health and Medicine life prolongation extended mental and physical capabilities transplants - \"bionic\" humans? The Future of Space Exploration and Utilization commercial utilization space habitation and colonization scientific exploration The Future of Artificial Intelligence Computers, and Neuroscience understanding the brain brain - computer interfaces tomorrow's computers Frontiers of Physical Science (alternative: Frontiers of Genetic Engineering) subatomic particle physics cosmology grand unification Beyond Physical Science holistic views of the universe nature and origin of life science, philosophy, and religion", "Ehrlich, Robert", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mf4u-4mam-6b3p", "00000000-0000-0000-1DD6-5C7DC9E96DD0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Mark C. Fishman to Marshall W. Nirenberg", "101584910X417", null, "1990", "10 October 1990", "Fishman, Chief of the Developmental Biology Laboratory for Massachusetts General Hospital, Harvard Medical School, asks Nirenberg to continue a chain letter supporting the demand to abolish apartheid in South Africa.  Fishman states, \"I am very willing to express my commitment as a scientist in the promotion of human rights and civil liberty.  I am accordingly asking if you would do likewise.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Mark C. Fishman.", "Copyright may apply", null, null, "October 10, 1990 Dear Doctor Nirenberg: I have received the attached letter from Dr. Norbert Perrimon asking me to join in the continuation of a chain letter supporting the demand to abolish apartheid in South Africa. This letter has been initiated by Dr. M. Mboya, Department of Education, University of South Africa, Private Bag, Rondebosch 7700, South Africa. I am very willing to express my commitment as a scientist in the promotion of human rights and civil liberty. I am accordingly asking if you would do likewise. If so, please write ten other scholars whom you know personally and ask them for a similar letter of support with a copy to Dr. Mboya. Sincerely, Mark C. Fishman, M.D. Enclosures: CC: Dr. M. Mboya, U. of S. Africa Dr. Norbert Perrimon, Harvard Medical School, Boston, MA 02115 Circulation: Dr. John Potts, Jr., Massachusetts General Hospital, Boston, MA 02114, USA Dr. Anne Flamand, Centre National de la Recherche Scientifique, 91190 Gif sur Yvette, France Dr. Judah Folkman, Children's Hospital, Boston, MA 02115, USA Dr. Willem Gispen, University of Utrecht, 3584 CH Utrecht, The Netherlands Dr. Jerome Gross, Massachusetts General Hospital, Boston, MA 02114, USA Dr. Robert Horvitz, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Dr. Roger Keynes, University of Cambridge, Cambridge CB2 3DY, England Dr. Richard Klausner, National Institutes of Health, Bethesda, MD 20205, USA Dr. Eva Neer, Brigham & Women's Hospital, Boston, MA 02115, USA", "Fishman, Mark C.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-62pc_s659~w49m", "00000000-0000-0000-CA6A-10848201BD7A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from William A. Shurcliff to Marshall W. Nirenberg", "101584910X418", null, "1986", "15 March 1986", "Retired Harvard physicist Shurcliff urges members of the National Academy of Sciences to join him in denouncing President Ronald Reagan's Strategic Defense Initiative, or \"Star Wars,\" plan.  Questions regarding the potential success of the program are included along with a return envelope.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of William A. Shurcliff.", "Copyright may apply", null, null, "March 15, 1986 Dear Member of the National Academy of Sciences: It seems to me and many of my physicist colleagues that the President's SDI (Star Wars) program is doomed to failure. If most of the members of the National Academy of Sciences likewise regard it as doomed, the President, Congress, and the public should be so informed. No other advisory group is more broadly competent or more impartial than the NAS. I am now making a personal effort to sound out the NAS membership on its views on Star Wars by means of the following questionnaire.  I intend to compile the results promptly and to publicize them appropriately. (Seventeen years ago, when the threat of the sonic boom of the proposed supersonic transport (SST) plane was at issue, I wrote -- as Director of the Citizens League Against the Sonic Boom -- to all of the NAS members, received strong confirmation that the sonic boom would do much damage to buildings, and was able to bring additional pressure to bear on Congress to halt the program. It soon did so.) I hope you will indicate your views on the following questions and will return the sheet to me in the enclosed stamped envelope. Is it likely that the proposed Star Wars program would succeed in providing an effective shield against a surprise attack by a flight of several thousand rocket-launched, high-altitude atomic-bomb-carrying warheads? Yes; No (The attack could, I suppose, be launched on a stormy night and could be accompanied by tens of thousands of decoys of new and sophisticated type.) Would the Star Wars program, even if capable of defeating a high-altitude attack, prevent atomic-bomb delivery by various other methods? Yes; No (My impression is that an enemy could introduce dozens of city-destroying atomic bombs by cruder methods such as smuggling them ashore in innocent-looking lobster boats and yachts, then transporting the bombs by truck to our biggest cities, ready for detonation by clockwork.  Or they could be smuggled in across the Mexican or Canadian borders with the aid of a bribed customs officer. Or the bombs could be delivered by short-range low-altitude rockets launched from close-offshore freighters and submarines.) Would the Star Wars, program prevent ten-minute short-range delivery of atomic bombs to England, France, and West Germany? Yes; No What is your overall attitude toward the Star Wars program? Strongly for; For; Neutral; Against; Strongly against. Your signature (Comments welcomed. Use reverse side?) Sincerely, William A Shurcliff Physics Dept., Harvard Univ. Retired. Senior Editor of \"Smyth Report\" (\"Atomic Energy for Military Purposes: The Official Report on the Development of the Atomic Bomb, 1940 - 1945\") Senior Author of Top Secret Report on the First Atomic Bomb Tests at Bikini. Recipient of the 1980 Forum Award of the American Physical Society.", "Shurcliff, William A.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bgkt~37k6_jvn2", "00000000-0000-0000-A776-815CE27AACB7", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Eliot Stellar to Marshall W. Nirenberg", "101584910X419", null, "1986", "20 March 1986", "Stellar, chairman of the Committee of Human Rights for the National Academy of Sciences, provides Nirenberg with background on the important work of the committee, founded in 1976 at the request of NAS membership.  Stellar provides examples of some of the cases with favorable outcomes including Pedro Castillo in Chile, Ismail Mohamed in South Africa, and Anatoly Scharansky in the Soviet Union.  Nirenberg is invited to be a correspondent to the committee.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "4", "pages", "Text", "English", "Reproduced with permission of the University of Pennsylvania University Archives and Records Center.,Reproduced with permission of James R. Stellar.", "Copyright may apply", null, null, "March 20, 1986 Dear Dr. Nirenberg: At the last meeting of the Committee on Human Rights, it was decided that I should write to all NAS members who are not correspondents of the Committee.  Thus, as the Committee's chairman, I am writing to tell you about its important work and to invite you to actively support our efforts by becoming a \"correspondent.\" The Committee on Human Rights was created in 1976, at the request of the NAS membership.  Over the years the Committee has worked in behalf of more than 200 scientists, engineers, and medical professionals who have been, or still are, prisoners of conscience. Every year since its creation, the Committee has approached newly elected NAS members to invite them to become correspondents; more than 750 members of the NAS, NAE, and IOM have accepted our invitation. The Committee takes on cases of individuals from the scientific community, anywhere in the world, who are believed to be victims of severe repression, that is, people who are imprisoned, have been sent into internal exile, or have \"disappeared.\" We write letters of inquiry or appeals to government officials, prison directors, heads of scientific academies and societies, human rights organizations, and people who may have personal knowledge of or influence on a particular case. In a few instances, the Committee makes public statements or appeals.  These public statements, which must be approved by the Council of the Academy, usually follow numerous unsuccessful private attempts by the Committee to obtain satisfactory results from the government of a country in which a colleague is being held. While our work is often discouraging and we cannot claim sole responsibility for the release from prison or the amelioration of the plight of any one individual, we do believe that our activities have played an important part in the many efforts--occasionally successful efforts--made by groups and individuals in behalf of persecuted scientists. To give you an example of some of the cases that the Committee has worked on and which had favorable outcomes within the past year, I would like to describe those of Pedro Castillo in Chile, Ismail Mohamed in South Africa, and Anatoly Shcharansky in the Soviet Union. Pedro Castillo is a surgeon, a member of the independent and highly respected Colegio Medico de Chile, and president of the unofficial National Commission against Torture. Dr. Castillo met with many representatives of human rights groups who had visited Chile, including Committee members Gerard Debreu and Robert Perry, NAS/IOM member Baruch Blumberg, and IOM member Robert Lawrence.  He was arrested at his home in Santiago on August 4, 1985, and subsequently banished without charges or trial to a tiny and practically inaccessible island off the southern coast of Chile.  The Committee immediately sent delegates to discuss Dr. Castillo's case with the Chilean Ambassador to Washington, sent telegrams to the Chilean authorities, and asked its correspondents to write individual appeals.  In an unprecedented move, the government granted Dr. Castillo an unconditional release on August 22. Ismail Mohamed is one of South Africa's leading algebraists. He is a professor at the University of Witwatersrand in Johannesburg and a leader of the United Democratic Front.  He was arrested on February 19, 1985, and charged with high treason, a capital offense. The Committee took numerous private actions in Professor Mohamed's behalf, including letters to South African government officials, letters and telephone calls of moral support to Professor Mohamed and his wife, a request to the Committee's correspondents to write individual letters of concern, and requests for information to officials of the American Embassy in Pretoria.  In October I received a letter from Professor Mohamed in which he wrote:  \"I am very grateful for the concern and support the National Academy of Sciences as well as yourself and Professor Lipman Bers have shown for the trial we have to face.\"  He went on to say, \"We are encouraged and strengthened by the continuing interest of the American people in the struggles we face.\"  All charges against Professor Mohamed were dropped on December 10. Soviet computer scientist Anatoly Shcharansky has been one of the Committee's most important and long-standing cases. He was arrested in March 1977, charged with anti-Soviet propaganda and espionage, and sentenced to 3 years in prison and 10 years' hard labor. Mr. Shcharansky was one of the founding members of the Moscow Helsinki Watch Group and a leading figure in the Jewish movement for emigration to Israel. Over the years the Committee made many efforts in Mr. Shcharansky's behalf, including three public statements.  In 1981 his sentence was changed to a tougher one of 6 years in prison and 7 years' hard labor.  In 1982, in response to the Committee's efforts, Mr. Shcharansky's wife, Avital, wrote to thank the Committee for its continued efforts and support. Mr. Shcharansky's release last month was a happy end to a long, difficult, and often seemingly hopeless journey. Several times a year, for cases in which colleagues are in need of immediate and extensive appeals, we ask our Committee's correspondents to write politely worded letters, as individuals, to the authorities in a country in which a particular scientist is being held. Occasionally we also ask correspondents to write letters of support and to send scientific literature to a prisoner and to his or her family. These requests provide background on the individual and the case, information on the type of inquiry or appeal that should be written, and the names and addresses of people to whom letters should be written.  Our correspondents also receive the Committee's in-house bulletin, Correspondence, which describes the status of cases in various countries and provides the names and addresses of officials to whom individual appeals can be sent. We would welcome your assistance in our work on the problems of human rights abuses.  Needless to say, becoming a correspondent does not obligate individuals to any specific action, nor does it imply blanket approval of past or future actions of the Committee.  If you complete and return the enclosed card, your name will be added to our list of correspondents. Yours, Eliot Stellar Chairman Enclosures:  List of Committee Members Membership Card 1985-1986 Committee On Human Rights National Academy Of Sciences Eliot Stellar, Chairman ('87)* Institute of Neurological Sciences, University of Pennsylvania Gerard Debreu ('87) Departments of Economics and Mathematics, University of California, Berkeley Daniel C. Drucker ('88) Engineering Sciences Department, University of Florida Gertrude S. Goldhaber ('87) Department of Physics, Brookhaven National Laboratory, Upton, New York Francis E. Low ('88) Department of Physics, Massachusetts Institute of Technology Daniel Nathans ('88) Department of Molecular Biology and Genetics, Johns Hopkins University Donald S. Ornstein ('88) Department of Mathematics, Stanford University Robert P. Perry ('86) Institute of Cancer Research, Philadelphia, Pennsylvania Helen M. Ranney ('86) Department of Medicine, University of California, San Diego Peter H. Raven ('87) Missouri Botanical Garden, St. Louis, Missouri William P. Slichter ('88) Materials Science & Engineering, AT&T Bell Laboratories Murray Hill, New Jersey Albert J. Solnit ('86) Child Study Center, Yale University Gilbert F. White ('88) Institute of Behavioral Science, University of Colorado Adam Yarmolinsky (Adviser) Kominers, Fort, Schlefer & Boyer, Washington, D.C. Carol Corillon, Committee Director *Date indicates end of term.", "Stellar, Eliot, 1919-1993 ; National Academy of Sciences (U.S.)", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-eeth_tx4r~has9", "00000000-0000-0000-E494-A4F3C56F2664", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Mark Kac, Joel Lebowitz, and Paul Plotz to Marshall W. Nirenberg", "101584910X420", null, "1983", "29 June 1983", "In this letter from the Committee of Concerned Scientists, Inc., the co-chairmen asks Nirenberg for his sponsorship of an international scientific conference addressing the worsening situation for colleagues who wish to emigrate from the USSR.  Updates on the plight of specific scientists are included.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Paul Plotz.", "Copyright may apply", null, null, "June 29, 1983 Dear Prof. Nirenberg: We are writing to ask for your sponsorship of an international scientific conference, designed to call attention to the worsening situation of colleagues who wish to emigrate from the USSR. Timed to turn to advantage the presence of numerous scientists and a sizable press contingent in Stockholm for events surrounding the Nobel awards, the International Refusenik Seminar-in-Exile on Collective Phenomena will be convened there December 1 and 2, 1983. Chairing an organizing committee of distinguished scientists is Prof. Inga Fischer-Hjalmars, Vice President of the Royal Swedish Academy of Sciences. The conference will consist of scientific sessions devoted to the fields of interest of many of the refuseniks. As you will recall, the Fifth International Conference on Collective Phenomena, scheduled for Moscow in September 1981, was squashed as ten Soviet refusenik would-be participants were threatened with serious reprisals and ten Americans were denied entry visas to the USSR. Since that time the possibility of emigration has become even more remote and the refusenik scientists are experiencing increasing isolation and despair. Prof. Lerner, whose decade-old biweekly seminar on mathematical biology has been unable to meet since September 1981, discontinued -- under threat of criminal prosecution -- his meetings with foreign colleagues visiting Moscow.  Prof. Naum Meiman, who recently endured a house search and interrogation, has reason to believe he will be tried on contrived charges in retaliation for his human rights activities.  And Dr. Viktor Brailovsky, former host of the Moscow Seminar is languishing in internal exile, apprehensive that as the time for his scheduled release approaches, authorities will try him again and extend his sentence.  More and more refusenik scientists are being demoted or dismissed from professional employment, called in for interrogations, and issued \"final refusals\" of emigration. An alarming number of them are facing trials and imprisonment. Against this backdrop of gross violations of the human rights of our Soviet colleagues, scientists from a number of countries conceived of the Stockholm conference as a concerted demonstration of solidarity. By focusing on the latest developments in their fields, sessions in honor of individual members of the Moscow Seminar will not only dramatize their plight but also will point to the loss sustained by science as a result of their repression. We look forward to your early response that you will sign on as a sponsor of this event, lending your good name and prestige to its hoped for success. Sincerely yours, Mark Kac Cochairman Joel Lebowitz Chairman Paul Plotz Cochairman", "Lebowitz, Joel ;Plotz, Paul ; Kac, Mark ; Committee of Concerned Scientists, Inc.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bvep-czrm.8nn9", "00000000-0000-0000-80C7-5857EB162C4A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from James A. Pittman, Jr., University of Alabama School of Medicine to Marshall W. Nirenberg", "101584910X401", null, "1990", "21 February 1990", "Pittman thanks Nirenberg for serving as a member of the Visiting Committee for the University of Alabama School of Medicine.  He suggests that Nirenberg's presence \"added a substantial measure of dignity and stature to the group.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of James A. Pittman Jr.", "Copyright may apply", null, null, "February 21, 1990 Dear Marshall: Many thanks indeed for taking the time and effort to come to Birmingham as a member of the Visiting Committee to the University of Alabama School of Medicine, UAB.  I know how valuable time becomes, particularly if you have personal interests that you would like to pursue, and particularly as we grow older and the time available ahead becomes less and less.  Your presence, however, added a substantial measure of dignity and stature to the group, and I very much appreciate your having been here. Apologies for burdening you with so many books on your departure, but I love them, and I hope you will find them interesting too. Many thanks again for the trip. Best regards, James A. Pittman, Jr., M.D., Dean University of Alabama School of Medicine, UAB", "The University of Alabama School of Medicine ; Pittman, James A., Jr. (James Allen), 1927-2014", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-vxa9.44qv-f4vf", "00000000-0000-0000-F73A-383BDC16385E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Christian B. Anfinsen to Halfdan Mahler", "101584910X421", null, "1979", "11 May 1979", "This copy of a letter from Anfinsen to Mahler was included with Nirenberg's own correspondence.  Anfinsen addresses the intention of Iraq to introduce a resolution to the General Assembly of the United Nations that would request the suspension of Israel for membership in the World Health Organization.  Anfinsen assures Mahler that Nirenberg is \"fully in accord\" with his point of view that the actions of Iraq should be denounced.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "May 11, 1979 Dear Dr. Mahler: I have just heard about the reckless and rather frightening decision by the Republic of Iraq and the bloc of nations with which it is associated.  As you know, they intend to introduce a resolution in the General Assembly of the United Nations that would request the suspension of Israel from membership in the World Health Organization.  Having visited Israel frequently over the past years, I have had the opportunity to observe the excellent handling of general medical care for all the inhabitants of this nation, Arab and Jew alike.  This is certainly true in the West Bank area as well as the rest of Israel and the construction and operation of hospitals, clinics and medical supply centers in the West Bank area is particularly impressive. It is well documented that the levels of health, life expectancy and infant mortality are almost exactly the same for the Arab and non-Arab inhabitants and, furthermore, that these levels are an order of magnitude higher than the situation that exists in the neighboring Arab nations.  The intended resolution is clearly of a purely political nature, very reminiscent of a similar proposal made with regard to UNESCO several years ago.  My colleagues here at the NIH, Dr. Julius Axelrod and Dr. Marshall Nirenberg, both of whom are Nobel Laureates, are fully in accord with my own point of view and our position is completely adhered to by all those associates with whom we have discussed this matter. We strongly urge you to take into consideration the extremely negative effects that such an action would have, not only on the population within Israel but also on the usefulness, worldwide, of the highly successful World Health Organization. Expulsion of Israel from WHO would make it impossible for many of us to consider cooperation with those nations that go along with the senseless stand that has been taken by Iraq.  We would be extremely grateful to you if you could take into consideration, and publicize, the attitude that I am conveying to you which I am sure would be taken by everyone truly interested in the maintenance and improvement of health throughout the world. Sincerely yours, C. B. Anfinsen Chief, Laboratory of Chemical Biology, NIAMDD Nobel Laureate in Chemistry, 1972", "Anfinsen, Christian B. (Christian Boehmer), 1916-1995", null, null, null, "Mahler, Halfdan T.,The World Health Organization", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bycc.5nz3-639b", "00000000-0000-0000-CE71-B1CA3BC7CE08", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from David H. Hubel to Marshall W. Nirenberg", "101584910X422", null, "1989", "24 February 1989", "Hubel informs Nirenberg that as President of the Society for Neuroscience he has decided to \"concentrate on offensive tactics, rather than to limit myself to fighting brush fires\" caused by animal activists.  He contrasts the boldness with which the Surgeon General Koop addressed smoking and the reluctance with which he dealt with the animal rights question.  Hubel asks for Nirenberg's opinion on the idea of writing a letter to Koop signed by Nobel Prize winners urging him to take a more active stance on the issue.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of David H. Hubel.", "Copyright may apply", null, null, "February 24, 1989 Dear Marshall: This year I find myself President of the Society for Neuroscience. As you might guess, one of my main preoccupations is with the animal activists. I've decided to concentrate on offensive tactics, rather than to limit myself to fighting brush fires. When I heard Koop's (the Surgeon General's) comments on smoking a few weeks ago I was struck by the contrast between his boldness on that issue and his reluctance -- and also that of the administration of the NIH and the NSF -- to deal with the animal rights question. I want to get your reaction to the idea of writing the Surgeon General a letter, signed by as many Nobel Prize winners as we can get, urging him to take a more active stance on this issue. Would you be willing to sign such a letter, and perhaps similar ones to the top people at NIH and NSF? If enough of us are enthusiastic I will prepare drafts of a few such letters for your approval. Sincerely, David H. Hubel, M.D. Professor of Neurobiology, Harvard Medical School President, Society for Neuroscience P.S.: Too bad we don't see more of each other. I well remember our conversation on the porch at Cold Spring Harbor.", "Hubel, David H.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8wt8_i4yk-3d9k", "00000000-0000-0000-6188-984DFD04107C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Howard H. Hiatt, Harvard University to Marshall W. Nirenberg", "101584910X423", "101584910X424", "1982", "27 September 1982", "Hiatt, Dean at the Harvard School of Public Health, encloses the statement presented to Pope John Paul II at the recent meeting of Presidents of the National Scientific Academies.  Carlos Chagas and Viki Weisskopf ask that Nirenberg sign the statement, \"Declaration of Prevention on Nuclear War,\" as a member of the Pontifical Academy of Sciences.  Hiatt informs Nirenberg that there was virtual unanimity among the scientists regarding the statement.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Howard H. Hiatt.", "Copyright may apply", null, null, "September 27, 1982 Dear Marshall: I enclose the statement presented to the Pope at the recent meeting of Presidents of the National Scientific Academies (and others) at the Vatican.  Carlos Chagas and Viki Weisskopf ask that you sign the statement as a member of the Pontifical Academy, if you are willing, and then that you telex the Academy to inform them that you have done so.  The signed documents should be sent to Viki's office, 6-303, MIT, Cambridge 02139. It was a very moving meeting.  The statement represents a virtual unamity among the scientists.  It was received with gratitude by the Pope, who said that he considers no other issue more important. With best wishes, Sincerely, Howard H. Hiatt", "Hiatt, Howard H. ; Harvard University", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fyrb-d5gj_f3i6", "00000000-0000-0000-6E3D-A13D146A9D19", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Declaration on Prevention of Nuclear War", "101584910X424", "101584910X423", "1982", "24 September 1982", "A copy of this statement was presented to Pope John Paul II at the meeting of Presidents of the National Scientific Academies.", "Manifestoes", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "6", "pages", "Text", "English", "Reproduced with permission of Howard H. Hiatt.", "Copyright may apply", null, null, "September 24, 1982 DECLARATION ON PREVENTION OF NUCLEAR WAR Presented to His Holiness the Pope by an assembly of Presidents of Scientific Academies and other scientists from all over the world convened by the Pontifical Academy of Sciences I. PREAMBLE Throughout its history, humankind has been confronted with war, but since 1945 the nature of warfare has changed so profoundly that the future of the human race, of generations yet unborn, is imperilled.  At the same time, mutual contacts and means of understanding between peoples of the world have been increasing.  This is why the yearning for peace is now stronger than ever. Humankind is confronted today with a threat unprecedented in history, arising from the massive and competitive accumulation of nuclear weapons.  The existing arsenals, if employed in a major war, could result in the immediate deaths of many hundreds of millions of people, and of untold millions more later through a variety of after-effects.  For the first time, it is possible to cause damage on such a catastrophic scale as to wipe out a large part of civilization and to endanger its very survival.  The large-scale use of such weapons could trigger major and irreversible ecological and genetic changes, whose limits cannot be predicted. Science can offer the world no real defense against the consequences of nuclear war.  There is no prospect of making defenses sufficiently effective to protect cities since even a single penetrating nuclear weapon can cause massive destruction.  There is no prospect that the mass of the population could be protected against a major nuclear attack or that devastation of the cultural, economic and industrial base of society could be prevented. The breakdown of social organization, and the magnitude of casualties, will be so large that no medical system can be expected to cope with more than a minute fraction of the victims. There are now some 50,000 nuclear weapons, some of which have yields a thousand times greater than the bomb that destroyed Hiroshima. The total explosive contant of these weapons is equivalent to a million Hiroshima bombs, which corresponds to a yield of some three tons of TNT for every person on earth. Yet these stockpiles continue to grow. Moreover, we face the increasing danger that many additional countries will acquire nuclear weapons or develop the capability of producing them. There is today an almost continuous range of explosive power from the smallest battlefield nuclear weapons to the most destructive megaton warhead. Nuclear weapons are regarded not only as a deterrent, but there are plans for their tactical use and use in a general war under so-called controlled conditions. The immense and increasing stockpiles of nuclear weapons, and their broad dispersal in the armed forces, increase the probability of their being used through accident or miscalculation in times of heightened political or military tension. The risk is very great that any utilization of nuclear weapons, however limited, would escalate to general nuclear war. The world situation has deteriorated. Mistrust and suspicion between nations have grown. There is a breakdown of serious dialogue between the East and West and between North and South. Serious inequities among nations and within nations, shortsighted national or partisan ambitions, and lust for power are the seeds of conflict which may lead to general and nuclear warfare. The scandal of poverty,    hunger, and degradation is in itself becoming an increasing threat to peace. There appears to be a growing fatalistic acceptance that war is inevitable and that wars will be fought with nuclear weapons. In any such war there will be no winners. Not only the potentialities of nuclear weapons, but also those of chemical, biological and even conventional weapons are increasing by the steady accumulation of new knowledge. It is therefore to be expected that also the means of non-nuclear war, as horrible as they already are, will become more destructive if nothing is done to prevent it. Human wisdom, however, remains comparatively limited, in dramatic contrast with the apparently inexorable growth of the power of destruction.  It is the duty of scientists to help prevent the perversion of their achievements and to stress that the future of mankind depends upon the acceptance by all nations of moral principles transcending all other considerations. Recognizing the natural rights of humanity to survive and to live in dignity, science must be used to assist humankind towards a life of fulfilment and peace. Considering these overwhelming dangers that confront all of us, it is the duty of every person of good will to face this threat.  All disputes that we are concerned with today, including political, economic, ideological and religious ones, are small compared to the hazards of nuclear war. It is imperative to reduce distrust and to increase hope and confidence through a succession of steps to curb the development, production, testing, and deployment of nuclear weapons systems, and to reduce them to substantially lower levels with the ultimate hope of their complete elimination. To avoid wars and achieve a meaningful peace, the powers of intelligence are needed, the powers of ethics, morality and conviction. The catastrophe of nuclear war can and must be prevented. Leaders and governments have a grave responsibility to fulfill in this regard.  But it is humankind as a whole which must act for its survival. This is the greatest moral issue that humanity has ever faced, and there is no time to be lost. II. In view of these threats of global nuclear catastrophe, we declare: --Nuclear weapons are fundamentally different from conventional weapons. They must not be regarded as acceptable instruments of warfare. Nuclear warfare would be a crime against humanity. --It is of utmost importance that there be no armed conflict between nuclear powers because of the danger that nuclear weapons would be used. --The use of force anywhere as a method of settling international conflicts entails the risk of military confrontation of nuclear powers. --The proliferation of nuclear weapons to additional countries seriously increases the risk of nuclear war and could lead to nuclear terrorism. --The current arms race increases the risk of nuclear war. The race must be stopped, the development of new more destructive weapons must be curbed, and nuclear forces must be reduced, with the ultimate goal of complete nuclear disarmament. The sole purpose of nuclear weapons, as long as they exist, must be to deter nuclear war. III. Recognizing that excessive conventional forces that increase mistrust and could lead to confrontation with the risk of nuclear war, and that all differences and territorial disputes should be resolved by negotiation, arbitration or other peaceful means, we call upon all nations: --Never to be the first to use nuclear weapons; --To abide by the principle that force or the threat of force will not be used against the territorial integrity or political independence of another state; --To seek termination of hostilities immediately in the appalling event that nuclear weapons are ever used; --To renew and increase efforts to reach verifiable agreements curbing the arms race and reducing the numbers of nuclear weapons and delivery systems.  These agreements should be monitored by the most effective technical means. Political differences or territorial disputes must not be allowed to interfere with this objective; --To find more effective ways and means to prevent the further proliferation of nuclear weapons.  The nuclear powers, and in particular the superpowers, have a special obligation to set an example in reducing armaments and to create a climate conducive to non-proliferation.  Moreover, all nations have the duty to prevent the diversion of peaceful uses of nuclear energy to the proliferation of nuclear weapons; --To take all practical measures that reduce the possiblity of nuclear war by accident, miscalculation or irrational action; --To continue to observe existing arms limitation agreements while seeking to negotiate broader and more effective ones; IV. Finally, we appeal: 1. To national leaders, to take the initiative in seeking steps to reduce the risk of nuclear war, looking beyond narrow concerns for national advantage; and to eschew military conflict as a means of resolving disputes. 2. To scientists, to use their creativity for the betterment of human life and to apply their ingenuity in exploring means of avoiding nuclear war and developing practical methods of arms control. 3. To religious leaders and other custodians of moral principles, to proclaim forcefully and persistently the grave human issues at stake so that these are fully understood and appreciated by society. 4. To people everywhere, to reaffirm their faith in the destiny of humankind, to insist that the avoidance of war is a common responsibility,  to combat the belief that nuclear conflict is unavoidable, and to labor unceasingly towards insuring the future of generations to come.", "Hiatt, Howard H.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j3vz.aztp_tuub", "00000000-0000-0000-4279-0910614131B3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Richard E. Marcus to Marshall W. Nirenberg", "101584910X425", null, "1982", "4 October 1982", "Marcus, a teacher living in western New York, discusses the idea for a book from the greatest minds in the world on the future of mankind and the solutions to the problems that have been created.  Norman Cousins and Jonathon Schell are quoted and the offer for Nirenberg to contribute an essay on any subject related to how we might \"live together on this planet in relative harmony\" is extended.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Richard E. Marcus.", "Copyright may apply", null, null, "October 4, 1982 Dear Dr. Nirenberg: I am a teacher living in a small community in western New York.  I have no expertise in world affairs, nor any idea how to solve the problems of survival that we human beings face in this century. But I am troubled by the same thoughts Norman Cousins speaks of in his book Human Options.  He asks, \"What basis is there for hope when the human future is increasingly in the hands of men who do not comprehend the meaning of the new [nuclear] power and who are, some of them, puny and fretful and prone to act out of frustration or false pride or mistaken notions of grandeur?\" Since the world seems governed by second-rate minds, my thesis is that the only hope for mankind is to reach the finest minds in the world today as a sauce of solutions to the direction the world has taken.  Unless new and original thinking is done on the relationship of man to the rest of his own species, we are on the road to our own extinction. Thus, this letter to you.  I have discussed with a major New York publisher the idea of a book bringing together the thinking of the finest minds in many fields, worldwide, in a series of essays on the subject of practical ways that we might live together on this planet in relative harmony. We would be honored if you would contribute your ideas on ways in which individuals and world leaders can begin to turn away from this dark path we now follow.  We are not setting any guidelines for you, since we believe that new paths can come only from original thinking.  Nothing is too absurd or unorthodox.  We are looking to exceptional minds for unique solutions, and feel confident that fresh directions will come from the ideas and thoughts expressed by yourself and others in this book. To paraphrase the flyleaf to Jonathan Schell's Fate Of The Earth, \"This must mark the moment at which man wrenches himself free from the psychological habits of pre-nuclear times, permits the nuclear predicament to take hold in his consciousness, rouses himself to confront the stark fact that his species is in imminent danger of extinguishing itself and begins to act to avert a final, absurd, irreversible, boundless calamity.\" Two practical matters:  (1) This invitation is now being sent to Nobel Prize winners and those suggested by a few of you to whom I have spoken.  We want to be sure to reach a broad range of fine thinkers from all disciplines, around the world.  Would you be kind enough to send us the names of four or five people whose thinking you feel should be represented in this book, along with their addresses, if easily available, and an indication of your inclination to contribute, which will help our planning. (2) We hope to begin the editorial process early in the coming year, since the topic is of some urgency.  Naturally, contributions will be accepted after that time, but if a tentative deadline of year-end 1982 could be met you would have our heartfelt gratitude. In short, the future of the world is at stake.  I feel the only hope for survival is if people with a gift like yours are willing to put some thinking into possible solutions. An essay from many like you would be a powerful statement to which the world's leaders and all of us can look for rational direction - the next stage in providing a future for our children and their children, instead of leaving another lifeless planet whirling endlessly through space. In closing, from Human Options: \"All sorts of magnificent notions are at large in the human mind today, and the most revolutionary notion of all is that the problem of human survival is not beyond human intelligence.\" I look forward to hearing from you. Sincerely, Richard E. Marcus", "Marcus, Richard E.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-evy4.hbrp-5en3", "00000000-0000-0000-9BCA-AFBCF1CAA3D7", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Joshua Sussman to Marshall W. Nirenberg", "101584910X426", null, "1989", "[March 1989]", "Nirenberg often received letters on subjects that were not directly related to his scientific expertise.  In this letter, Joshua Sussman, a 17 year old cadet at the Missouri Military Academy asks Nirenberg for his opinions on the necessity of war and whether or not it is preventable.  Sussman hopes that Nirenberg will be able to settle a dispute between he and his father.  He asks, \"Is Dad right by saying there will always be wars or am I right by my conviction that animosities between countries can be negotiated?\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Joshua Sussman.", "Copyright may apply", null, null, "Marshall W. Nirenberg I am a 17 year old cadet at the Missouri Military Academy in Mexico, Missouri. I'm writing to you as a matter of great importance to both my dad and myself. My dad is an ardent student of history.  His being an invalid has created the need for other outlets.  History is his primary interest.  Many evenings the two of us sit and discuss historical events.  At times we disagree. I know of Dad's admiration for you and I thought you might help settle an argument that we have pursued over a year now. I am of the belief that wars have failed as an extension of national politics.  I believe that differences between countries can be solved by diplomacy.  I believe that massive killings of armies and civilians has created a dangerous precedent that may ultimately lead to the destruction of mankind!  What is the answer when wars no longer work?  How can we solve conflict without violence? My dad feels that there will ALWAYS be wars because of historical differences in language, boundaries, religious beliefs, population explosions, etc.  He believes that strength comes from military preparedness.   Yet, General Douglas McArthur, one of our nation's greatest generals and strategists, in a speech given to the American Legion in St. Louis in 1955 said, \"The next great advance in the evolution of civilization cannot take place until war is abolished.\" What are your thoughts?  Is Dad right by saying there will always be wars or am I right by my conviction that animosities between countries can be negotiated? We both would value your opinion and would be most grateful for your kindest help! Sincerely, Joshua Sussman Mar 20 1989", "Sussman, Joshua", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-72b3.dccj_sku8", "00000000-0000-0000-9E28-C4BA2F92CFD9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from William S. Sly to Marshall W. Nirenberg", "101584910X427", null, "1987", "18 April 1987", "Sly, chairman of the Department of Biochemistry at St. Louis University Medical Center and a former postdoctoral student in Nirenberg's laboratory, thanks Nirenberg for the opportunity he provided to him.  Having recently attended a symposium in Nirenberg's honor where another great scientist and former Nirenberg postdoc, Phil Leder, also attended, Sly writes, \"I will always be grateful to you for that opportunity to work in your laboratory, to learn a new direction, and a new way of thinking about problems.\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of William S. Sly.", "Copyright may apply", null, null, "April 18, 1987 Dear Marshall, I'm really grateful to Sidney for inviting me to attend and to participate in the symposium in your honor at Rutgers.  It was wonderful joining with all of those whose lives you've influenced so much in this opportunity to pause, share some science, and say thanks.  Phil Leder probably expressed the sense of appreciation that we all feel toward you better than any of us in his statements at the banquet.  Although I spent less time in your laboratory than most of the other people there, I can honestly say that it was an extremely influential part of my development. I will always be grateful to you for that opportunity to work in your laboratory, to learn a new direction, and a new way of thinking about problems. I've been at St. Louis University long enough to have most of the renovations complete.  I hope we can find a time in the next year or two when you can visit and give a seminar. All the best. Sincerely, William S. Sly, M.D. Chairman, E. A. Doisy Department of Biochemistry", "Sly, William S.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-84kq-invi.3rg8", "00000000-0000-0000-69F4-8327F2CB7B68", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Vulimiri Ramalingaswami, Harvard University to Marshall W. Nirenberg", "101584910X428", null, "1987", "30 July 1987", "Nirenberg continued to provide advice to scientists in the field of genetics throughout his career, long after he switched to work in neurobiology.  In this follow-up to an earlier letter, Ramalingaswami asks for Nirenberg's opinion on a paper written on codons for publication in the Proceedings of the National Academy of Sciences.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "July 30, 1987 Dear Dr Nirenberg I should be most gratified if you would be so kind as to let me have your most valuable opinion on the paper entitled: \"Possible evolution of sphere Janetion [?] Signals from stop Codons\" written by P. Sinepathy for possible publication in PNAS. I know how busy you are and I apologize for this intrusion on your time. With kind regards Sincerely O. Ramalingaswami PS I had written to you on 26th May with a copy of the paper. [stamped AUG 06 [?] 1987] [written below in different handwriting, (M, I have a copy of the paper, but not Ramalingaswami's letter -- I believe Peri forwarded the paper to us) -- S]", "Ramalingaswami, Vulimiri, 1921-2001 ; Harvard University", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mzkj.b96x_ry9a", "00000000-0000-0000-067E-402CFE455E7E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Andrew Blake to Marshall W. Nirenberg", "101584910X429", null, "1996", "19 April 1996", "Blake, director of Seriously Ill for Medical Research (a patients group supporting the humane and necessary use of animals in medical research), asks for Nirenberg's view on medical research.  He informs Nirenberg that he has written all Nobel laureates in physiology or medicine and asks a common question of each, \"Do we really need animal experiments in medical research?\"", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Seriously Ill for Medical Research.", "Copyright may apply", null, null, "19 April 1996 Dear Dr Nirenberg I am the founder and director of Seriously Ill for Medical Research (SIMR), a patients group supporting the humane and necessary use of animals in medical research.  SIMR was formed in 1991 by patients who were outraged by animal rights actions that threatened medical research. SIMR's main objective is to promote a greater public understanding of the methods, aims and benefits of medical research and the role of animal research. I am writing to all Nobel Laureates in Physiology or Medicine as your views on medical research are highly respected.  I wanted to address the common question, \"Do we really need animal experiments in medical research?\" Your answers will be compiled and then presented to the media in a special report.  Your name will only be used if you have added any comment at the end of the questionnaire. I would like a reply by no later than 31 May, if possible. I look forward to hearing from you. Yours sincerely Andrew Blake Director", "Seriously Ill for Medical Research ; Blake, Andrew", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9g6a_yxi4~87dr", "00000000-0000-0000-DE4D-D286511DDBEB", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Bernd Lindemann, Saarland University to Marshall W. Nirenberg", "101584910X430", null, "1971", "9 November 1971", "Nirenberg received requests for advice from all over the world throughout his career.  Here, Lindmann, from Saarland University in Germany, informs Nirenberg that he is part of a group of professors interested in electrical membrane properties of neuroepithelial cells, nueroblasts, and developing nerve cells.  He asks for advice on which review articles and original articles would provide the best summary of the state of the field.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Reproduced with permission of Bernd Lindemann.", "Copyright may apply", null, null, "Nov. 9, 1971 Dear Professor Nirenberg, Recently our group got interested in electrical membrane properties of neuroepithelial cells, neuroblasts and developing nerve cells. We were informed that you are working in this field since many years. This is why we should like to ask you to help us with a first orientation and survey of the literature. In other words: which review articles and original articles would give us an idea about what is known and what is missing? We are aware of some work on cultured cells but could not so far find papers on developing cells in situ. I hope you will find some time to help us in this matter and remain with best personal wishes Yours very sincerely Berrnd Lindemann", "Lindemann, Bernd ; Universität des Saarlandes", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-388h.94xk.ndbz", "00000000-0000-0000-866C-A6295128C038", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Saul Kent, Life Extension Foundation to Marshall W. Nirenberg", "101584910X431", null, "1988", "12 September 1988", "Kent, president of the Life Extension Foundation (a non-profit organization that provides financial assistance to scientists striving to extend the human life span), asks for Nirenberg's assistance in supporting cryonic suspension (freezing of the human body after legal death for the purpose of reanimation).  A lengthy discussion of the scientific, social, and ethical implications of cryonic suspension is included.  Kent asks Nirenberg to send him a letter of support in hope of fighting a ruling by the State of California Health Department making it illegal.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Sep. 12, 1988 Dear Dr Nirenberg: My name is Saul Kent. I am President of the Life Extension Foundation -- a non-profit organization that provides financial assistance to scientists striving to extend the human lifespan. I'd very much appreciate your assistance in helping to preserve the constitutionally-guaranteed right of all Americans to take advantage of advances in science and technology. This right is now being threatened by an arbitrary and unlawful ruling by the State of California Heath Department that the practice of cryonic suspension is \"illegal\". Cryonic suspension is the freezing of the human body after legal death for the purpose of future reanimation. Cryonic suspension is based upon the fact that storage of the human body at the temperature of liquid nitrogen (-196 deg. C.) preserves biologic systems indefinitely; and the premise that, at some future time, advanced medical technologies may be able to repair damage caused by injury, disease, and aging that is clearly irreparable today. Cryonic suspension is controversial because its value lies in the prospect of future medical advances, rather than in the capabilities of today's medicine.  As a result, many scientists have questioned its value. On the other hand, there are a significant number of established scientists who clearly recognize the value of this pioneering new technology, including: James B. Lewis, Ph.D, Fred Hutchinson Cancer Research Center, Seattle, WA; Peter Gouras, M.D., Columbia University Medical Center, New York; K. Eric Drexler & Ralph C. Merkle, Ph.D, Stanford University, Palo Alto, CA; Gerald Feinberg, Ph.D, Columbia University, New York; Gregory M. Fahy, Ph.D, American Red Cross, Rockville, MD; Daniel Alkon, Ph.D., National Institute Of Mental Health, Bethesda, MD: Paul Segall, Ph.D, Hal Sternberg, Ph.D & Harry Waitz, Ph.D, University of California at Berkeley, CA; and Hans Moravec, Ph.D, Carnegie Mellon University, Pittsburgh, PA. I invite you to read the enclosed Question-And-Answer discussion of the scientific, social, and ethical implications of cryonic suspension. The actions of the California Health Department have been challenged by the Alcor Life Extension Foundation of Riverside, California -- a non-profit organization that provides the service of cryonic suspension to its members. An Alcor member afflicted with AIDS has joined Alcor in a lawsuit to obtain a Court Order prohibiting the Health Department from interfering with his (and anyone else's) cryonic suspension. (See enclosed press release and newspaper story) I'd greatly appreciate it if you sent a letter addressed to our attorney indicating that, in your opinion, cryonic suspension is a \"rational gamble\" (or words to that effect) that is of value to the individual who chooses it, and that any individual who wishes to be frozen after legal death has the right to do so. This letter will then be the basis for a legal declaration submitted to the court on behalf of the AIDS patient, the members of Alcor, and the American people. Please send your letter and a copy of your Curriculum Vitae (CV) to: David B. Epstein, Attorney, Garfield, Tepper & Ashworth, 1925 Century Park East, Suite 1250, Los Angeles, California 90067, Telephone: 213/277-1981. If you have any legal questions, please call David Epstein. If you have any other questions, please call Saul Kent at: 714/780-3252. Sincerely, Saul Kent", "Life Extension Foundation ; Kent, Saul", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xm3u.a95s.fjzw", "00000000-0000-0000-AE80-CF40DD35C14A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Walter W. Stewart to Marshall W. Nirenberg", "101584910X432", null, "1988", "24 February 1988", "Stewart asks for Nirenberg's response to an article in the Scientist containing first-hand accounts by scientists who had made allegations of scientific misconduct.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "24 February 1988 Dear Dr. Nirenberg: Several months ago The Scientist carried a special section containing first-hand accounts by scientists who had made allegations of scientific misconduct.   In each case, their allegations were subsequently found to be justified.  Disturbingly, however, all three scientists experienced difficulties in getting their point across. The Scientist also carried a companion article by Ned Feder and myself discussing some of the questions. I think the articles raise disturbing and important issues, which deserve to be as widely considered as possible.  For this reason, I am enclosing a Xerox copy of the articles.  I would be most interested in learning your reaction to the points made there. Sincerely, Walter W. Stewart", "Stewart, Walter W. ; United States. Public Health Service", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-edic~rf6u_2544", "00000000-0000-0000-3124-93BF5260D097", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Dennis Flanagan, Scientific American to Marshall W. Nirenberg", "101584910X433", "101584910X445", "1972", "5 May 1972", "Flanagan thanks Nirenberg for an article written in 1963 and asks him to write a short book on the genetic code for Scientific American.  He suggests there is a need for a more extended account of an area in which Nirenberg's work has played a significant part.  Details for publication are included.", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "2", "pages", "Text", "English", "Reproduced with permission of Dennis Flanagan.", "Copyright may apply", null, null, "May 5, 1972 Dear Dr. Nirenberg: We have pleasant memories of the article you were kind enough to write for us in 1963, and we should now like to make another proposal. We here at Scientific American have always been somewhat frustrated by the fact that the articles we publish do not have more of a life beyond their initial creation. It is true that large numbers of reprints are distributed, but we fell that even more is needed. It has lately seemed to us that a logical extension of each of the major articles we publish would be a small illustrated book of perhaps 50,000 words (roughly 100 pages). Such a book could be written at the level of a Scientific American article. It could of course take advantage of the fact that some of the illustrations had already been prepared for the magazine. Other illustrations could of course be added. The book would be published by our partners, W. H. Freeman and Company of San Francisco. One of the editors at Scientific American, however, would work with the author on the production of the book and its illustrations. Our colleagues at the Freeman company estimate that a book of this kind would have a minimum first edition of 5,000 cloth-bound copies and 7,500 paper-bound copies. These editions would probably be priced respectively at $6 and $3. The usual royalty arrangements would obtain. Would you consider writing such a book that might simply be titled \"The Genetic Code\"? If so, one of us would welcome an opportunity to call on you and discuss that matter further. It would be a privilege for Scientific American and the Freeman company to present a more extended account of this area in which your own work has played such a significant part. I look forward to hearing from you at your convenience. Cordially, Dennis Flanagan", "Flanagan, Dennis, 1919-2005 ; Scientific American", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fszx_xamb.m8ni", "00000000-0000-0000-966E-35F1CCF0ED48", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Steven Kelsen to Marshall W. Nirenberg", "101584910X434", null, "1967", "15 September 1967", "Kelsen invites Nirenberg to be an honored guest and principal speaker at the 22nd Annual Research Day at Hahnemann Medical College.", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of Steven Kelsen.", "Copyright may apply", null, null, "September 15, 1967 Dear Dr. Nirenberg: The Undergraduate Research Society of the Hahnemann Medical College invites you to be our honored guest and principal speaker on the occasion of the Twenty-second Annual Undergraduate Research Day, March 13, 1968. At this time students who have engaged in research programs present brief resumes of their work to an audience of students and faculty. The presentation of student research begins at 2:30 PM with the principal address given at 5:00 PM. An informal dinner and social hour in honor of the essayists and guest speaker completes the evening. A morning program of informal seminars with small groups of faculty, graduate students, and interested medical students also will be given and completes the day. We hope that you will be able to attend the entire day's events. However, if this is not possible, we would still appreciate your participation in the program of the afternoon. The choice of topics is yours, but we hope that it will in some way relate to your interests in the genetic code and regulation of protein synthesis. This invitation carries with it an honorarium of $150 plus expenses for you and your wife. We are looking forward to meeting and speaking with you in March. Sincerely yours, Steven Kelsen, President Undergraduate Research Society", "Kelsen, Steven", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tsu5_9z7z-jvjy", "00000000-0000-0000-7418-8A1F55EAB3C0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Draft of Joshua Lederberg's editorial to Science magazine", "101584910X435", "101584910X121", "1967", "1967", "This is a direct reponse to Nirenberg's August 1967 Science editorial, \"Will Society be Prepared?\"", "Articles, Drafts (documents)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Joshua Lederberg.", "Copyright may apply", null, null, "te Deru In an editorial in the August 11 issue, \"Will Society Be Prepared\", Dr. Marshall W. Nirenberg wrote about the prospects of molecular genetics: \"Cells will be programmed with synthetic messages within 25 years, and when man.becomes capable of programming his own cells, he must refrain fron doing so until he has sufficient wisdom to use this knowledge for the benefit of mankind.\" No subject of policy is more important than this, and it deserves the most critical debate. There is some danger that, whether so intended or not, Nirenberg's language could generate public misunderstandings that might undercut the very research needed to reach sufficient wisdom. His underlying concern, which I share, is for the use of biological control by a malevolent government to the peril of individual freedom. As Hitler's race policy illustrated only too well the States access to forcéable compulsion already gives it the power of genoside. Presumably we have to be even more concerned about subtler mistakes. A well-intentioned government might impose rash commitments for the sake of short-run advantages. Plainly we must be very sensitive about innova- tions that, once introduced, constituted irreversible evolutionary deviations. However, in reading Nirenberg’s editorial, we should emphasize the distinction between evolutionary deviation and euphenics, i.e., the repropraiming of somatic cells and the modification of development. \"Message\" does carry a strong connotation of RNA messengers with somatic effects. To interdict such personal uses of messages would be hard to justify without a prohibition on all new medicine, especially such inter- ventions as the use of hormones. If only germinal messages are meant, we   nes — have other prospects to worry about toe, The manipulation of germ cells for genetic surgery would almost certainly be preceded by techniques for clonal propagation and for chromosome manipulations in human beings,which would already have the most cogent evolutionary implications. Human culture - as Mug/Ller has pointed out - is already a major commitment of individual development to formative influences decided by the community. Our educational systems are certainly a form of psychological engineering scareély different in fundamental principle from the biological interventions that our knowledge of nucleic acids is likely to bring about. eal In point of fact, we already practige biological engineering on a rather large scale by use of live viruses in mass immunization campaigns. While these are of indubitable value for preventing serious diseases, their-pkobal impact on the development of human beings of a wide range of genotypes is hard to aggess at our present stage of wisdom. Crude virus preparations such as are in common use at the present time are also vulnerable to frightful mishaps of contamination and misidentification. Live viruses are themselves genetic messages used for the purpose of programming human cells for the synthesis of immunizing virus antigens. Dr. Nirenberg's cautions are just as relevant to considerations of contemporary policy as they are for the ever-widening applications of molecular biology in the near future. Joshua Lederberg Professor of Genetics Stanford University School of Medicine Our main concern must be to maximize the role of individual decision. This could be defeated by overenthusiastic policing against personal initiative and experimentation es well as by premature positive measures imposed by the State.", "Lederberg, Joshua", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2r2e_fsz5_xrs7", "00000000-0000-0000-0540-28B051BAD5CE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Louis G. Chacos, Montgomery Junior College to Marshall W. Nirenberg", "101584910X436", null, "1967", "14 February 1967", "Chacos asks Nirenberg if any of his experiments \"suggest whether or not any human characteristics, generally referred to as personality, may be genetically determined.\"  Chacos is skeptical regarding the position of social scientists who maintain that all characteristics are acquired.", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of Louis G. Chacos.", "Copyright may apply", null, null, "February 14, 1967 Dear Dr. Nirenberg: I read with great interest the report in the newspaper of your receiving the 31st annual Research Corporation Award for \"outstanding achievements in science\" at the dinner held in New York on January 19 and wish to add our congratulations.  We are the most interested in your experiments pertaining to \"protein synthesis which led to a partial 'cracking' of the genetic code and stimulation of further research activity all over the world.\" In the teaching of health education to our many students, we are covering this area of genetics and the inherited physical aspects of man.  I am interested in finding out if your experiments suggest whether of not any human characteristics, generally referred to as personality, may be genetically determined.  I for one am not ready to concede that all characteristics are acquired as irrefutably stated by associates who are social scientists. If time will permit, we shall be most appreciative of any response you may give to the question posed. Sincerely yours, Louis G. Chacos Chairmen of Health and Physical Education", "Chacos, Louis G. ; Montgomery Junior College", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-r682_b685_txgv", "00000000-0000-0000-EE19-65A836FBE272", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from A. Clark Griffin, University of Texas to Marshall W. Nirenberg", "101584910X437", null, "1963", "22 March 1963", "A. Clark Griffin, professor of biochemistry at the University of Texas M. D. Anderson Hospital and Tumor Institute, thanks Nirenberg for reprints of his articles and commends him for his recent article \"The Genetic Code: II\" published in Scientific American 208, no. 3 (1963): 80-94.  He mentions research progress made in the manipulation of tumor ribosome's and promises a subsequent letter with detailed findings.", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of the University of Texas. M. D. Anderson Cancer Center.", "Copyright may apply", null, null, "March 22, 1963 Dear Marshall: Many thanks for the reprints and your note. We have had an unusually good winter, a little on the cold side for the native Texans, but I thought it was just right. Next year you must come down when the weather is at its worst in Bethesda. Just read your article in the latest issue of Scientific American, an excellent presentation. We are gradually learning the ways of manipulation of tumor ribosomes and have some interesting studies in progress.  I'll bring you up to date in a subsequent letter, we are in the process of evaluating and writing up some of our findings. Wendell Stanley is going to present this year's Mike Hogg Lecture, he will have to perform very well to meet the high standards you set last year.  Incidentally, Marshall, I would greatly appreciate if you would send me an autographed photograph for my office, I have them from all of those who have participated in this lecture series. We all send our best wishes for continued success and progress in your exciting research program. Sincerely, A. Clark Griffin, Ph.D. American Cancer Society Professor of Biochemistry", "Griffin, A. Clark ; The University of Texas", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2p6f.wpdf_y8vb", "00000000-0000-0000-9D45-C60A98D220F4", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Gordon Tomkins to Marshall W. Nirenberg", "101584910X438", null, "1961", "25 October 1961", "This letter from Tomkins, includes a brief discussion of personal matters during a particularly difficult period in Nirenberg's  life, when both his mother and father passed away.  Tomkins, writing from France, mentions briefly his work at the Pasteur Institute and his opportunity to discuss his work with world-famous scientists Jacques Monod and Francois Jacob.", "Letters (correspondence)", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of Millicent Tomkins.", "Copyright may apply", null, null, "25 Oct Dear Marshall: I just learned that you lost your father quite suddenly and I wanted to tell you how sorry I feel about it. It must be especially difficult after your mother's death too. Funny thing about couples: that seems to happen fairly often. There are now things too which, of course, helps, and I'm sure being married makes it somewhat easier to bear. How is Perla? I guess things in the lab must be fantastically interesting now. It's funny to experience them at so great a distance, but spiritually, I'm there in some way. We're well settled and enjoying France immensely. The lab is new, full well equipped and the people are very nice. I haven't quite decided on a problem and am finishing up those expts that Arnie did with some interesting results. I may try the same thing in Misman's particle. He's out here at Gif and almost every day Monod, Cohen, and Lennox come out -- so people now believe in him. [END PAGE ONE] [BEGIN PAGE TWO] His system is one in which people want to do in vitro genetics which apparently can be done. I'll try to learn it and bring it back. Also may work on transformation. Not sure yet but I'll let you know. Millient is inspired by the scenery and has finished 3 or 4 paintings. Leslie is in French school but not too happy yet since she can't speak with the kids. I get in to the Pasteur family often but not often enough although I've had a couple of discussions with Monod but not yet with Jacob. Anyway, my best wishes to you and Perla Give my regards to all thereabouts. as ever Gordon", "Tomkins, Gordon", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mwzg_cgqu_ztkt", "00000000-0000-0000-98CD-C13EADB6741D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on the code, taken at an art exhibit", "101584910X439", null, "1963", "March 1963", "In the middle of the race to discover the genetic code, Nirenberg worked steadily toward its completion.  This program from a neo-abstract art exhibit at the Obelisk Gallery includes handwritten codon combination possibilities on the outside cover, which suggest that Nirenberg did not confine his work to the laboratory.", "Notes,Brochures", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "MARINA NUNEZ DELPRADO “A Aaa a | Awe ABE Guar 4 “OU Uhh AYU     Cu4 a Ccly g © 6 @® eee Cc. GA Cue usy Ga—- = 6G cAc hAad A uot She S94 cucu oud \\\\ cs d seo KSA 6cC 6C g Ace we Bed AWS. ug pPA oh BWA UR ane up at 4 1 un Af CAA ¢ eu? CAC Ay UW v + uC pew Ave & ar UK 6 U4 CARY wae at ih Cun Aho CCA oe TAY CAC MARCH 6-30, 1963 PREVIEW, MARCH 5, 5-7 P.M.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8c6s_vkhy.s35v", "00000000-0000-0000-E402-05BE4418A172", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from R. Lee Clark, University of Texas to Marshall W. Nirenberg", "101584910X440", null, "1967", "3 July 1967", "Clark informs Nirenberg that he has won the Bertner Foundation Award for \"an outstanding contribution to research of significance in the field of cancer.\"  An invitation to the 22nd Annual Symposium on Fundamental Cancer Research is included.", "Letters (correspondence)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "2", "pages", "Text", "English", "Reproduced with permission of the University of Texas. M. D. Anderson Cancer Center.", "Copyright may apply", null, null, "July 3, 1967 Dear Doctor Nirenberg: Doctor Robert B. Hurlbert has been in correspondence with you, I believe, concerning our Twenty-second Annual Symposium on Fundamental Cancer Research, to be held in 1968. A principal feature of this Annual Symposium is the Bertner Foundation Lecture and Award.  The nominee for this honor is selected for an outstanding contribution to research of significance in the field of cancer.  Enclosed for your information is the Bertner Foundation Award brochure for the 1967 Symposium. An honorarium of $500 and reimbursement for travel expense are provided by the Bertner Foundation. It is my great pleasure to inform you that the Symposium Committee has recommended you as our 1968 Bertner Foundation Lecturer in recognition of your many contributions to our knowledge concerning the nucleotide code for amino acids in protein synthesis. A cordial invitation is hereby extended to you to accept this nomination and to participate in the Symposium. With your acceptance, our Symposium Program Committee Chairman, Doctor Hurlbert, will write you in more detail about the Lecture. It is scheduled for 5:30 p.m. on March 7, 1968, and should be a presentation of approximately 45 minutes in time of delivery. The Committee would hope your topic would be one in keeping with the theme of the Symposium which, as you know, is \"Exploitable Molecular Mechanisms and Neoplasia,\" and would like to have your specific title as soon as you have made the decision.   The manuscript of the Bertner Lecture will be published in the Symposium monograph and should be submitted at the time of the Lecture. We realize that you may be working with a tight schedule next spring, but in our quest for the top contributors to cancer research as candidates for the Bertner Award, we gave your name our first consideration. We look forward to the prospect of a favorable reply. Sincerely yours, R. Lee Clark", "The University of Texas ; Clark, R. Lee", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9zb5-kw9s_nthu", "00000000-0000-0000-D361-7DAFBF842427", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Memorandum from Mike Fuortes, United States Department of Health, Education, and Welfare to Marshall W. Nirenberg", "101584910X441", null, "1968", "13 November 1968", "Fuortes informs Nirenberg that they can begin to make plans for the utilization of laboratory and office space at NIH reserved for neurobiology.  During the early period of Nirenberg's transition from genetics to neurobiology, this allotment of space supported the direction of his research.", "Memorandums", null, "Transition to Neurobiology, 1965-1969", "2", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "DATE: November 13, 1968 TO: M. Nirenberg FROM: M. Fuortes SUBJECT: I think that our negotiations have come to a conclusion and that we can now start making some plans on how to utilize the space reserved for Neurobiology. As I wrote to you yesterday, Wagner has decided that we can have four modules in the D wing of the second floor of Bldg. 10 as soon as the rooms are vacated.  Phil told me that l/2 module will remain available for him in your wing on the sixth floor, and I should like to reserve three modules for Neurobiology on the second floor of Bldg. 36. Phil and I thought that the following layout might be reasonable. 2nd floor, Bldg. 10 Biochemistry - one module Electrophysiology - one module Darkroom - l/2 module Shop - l/2 module Two offices - 1 module (Phil Nelson, Jerry Fischbach, Eric Schwartz) 6th floor, Bldg. 10 Electrophysiology - l/2 module (Phil Nelson) 2nd floor, Bldg. 36 Electrophysiology - 2 modules Tissue Cultures & Nematodes - 1 module (Mike Fuortes, Phil Nelson, Jerry Fischbach, Paul O'Bryan) This means that Phil and Jerry would have space in both Buildings and would try to carry on some research in both. If you wish to move some of your activities and detach some of the people to the second floor of Bldg. 10 or to Bldg. 36, we should be very happy to cooperate.  Also, we should be glad to change the plans outlined above in accordance with your suggestions. At the moment we do not have the equipment required for the work to be performed in Bldg. 36 (we do not have inverted microscopes and micromanipulators), but things are on order and we shall be ready to start as soon as the equipment arrives. Please let me know your comments on our plans. Sincerely, Mike", "United States Department of Health, Education, and Welfare ; Fuortes, Mike", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qys8-7ggi.k2bc", "00000000-0000-0000-C3D7-470C9E5E4B43", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Man's Power to Shape His Own Biologic Destiny--Will Society Be Prepared to Use It Wisely?", "101584910X442", "101584910X121", "1967", "[Spring 1967]", "This appeared shortly before Nirenberg's more famous editorial in Science in the fall of 1967.  Nirenberg confronts the same issues of knowledge and power and the future of genetic research.  Anticipating the time that \"man may be able to program his own cells with synthetic information.\"  Nirenberg notes that this will happen long before man can \"assess adequately the long-term consequences of such alterations\" and resolve the ethical and moral problems it will raise.", "Articles", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "2", "pages", "Text", "English", "Reproduced with permission of the Research Corporation.", "Copyright may apply", null, null, "[od Bo AT RE Movs TRE AE Ao QR ATTHON, BX PR OU WORT as tr Oat BUR ING t967 UATE: Ane NDAGete A VARSNS HN indie ici ella: GLAM IMe: Ate SOEI] CLHARae ht SIAU AMAL OF PEE. egy tomas coi dy if sant . . “ . eb ale ok Man’s Power to Shape iis O<wn Biologic Destiny — Wall Society be Prepared to Use it Wisely? By Marshall W. Nirenberg, National Heart Institute New information is being obtained in the field of biochemical genetics at an extremely rapid rate which will undoubtedly continue to increase within the foreseeable future. Thus far, this knowledge has had relatively little effect upon man, basically, because more information must be obtained before practical application will be possible. The tech- nical problems that must be overcome are for- midable, to say the least. However, when these. obstacles have been removed this knowledge will greatly influence man’s future, for man then will have the power to shape his own biologic destiny. Such power can be used wisely or unwisely, for the betterment or detriment of mankind. I think it is fair to say that most biochemical geneticists work because the search for knowledge is an exciting creative adventure—much as an artist’s or poet’s exploration is creative. His pri- mary aim certainly is not that of creating powerful tools for society. However, the cumulative accre- tion of knowledge, bit by bit, does create such tools; thus the scientist has a responsibility which an eminent virologist, Salvadore Luria, has stated Clearly and eloquently. Let me quote part of . Luria’s statement: . “The impact of science on human affairs im- poses on its practitioners an inescapable responsi- bility. On the onc hand, it creates the urge to seck useful applications and to foster their gencral ac- ceptance. On the other hand, it may restrain the scientist from pursuing a line of research that is clearly leading to evil applications. Yet the prog- ress of science is so rapid, almost catastrophic, that it creates an imbalance between the power it places in the hands of man and the social condi- tions in which this power is exerted. Then neither warnings of scientists, nor breadth of public in- formation, nor wisdom of citizens may compen- sate for inadequacies of the institutional framework to cope with the new situations.   This paper was presented by Dr. Nirenberg, Chief, Lab- oratory of Biochemical Genetics, National Heart Insti- tute, National Institutes of Health, at the Research Corporation Award Dinner in New York, January 19, 1967. Dr. Nirenberg received the foundation's 1966 Award for his pioneering experiments on protein synthe- sis which led to a partial “cracking” of the genetic code. “The scientist should cultivate his own alertness to prospective scientific developments that may suddenly add new powers to man. The scientific habits of skepticism and restraint, of curbing fan- tasy, and distrusting fancy, inhibit the scientist's effort to speculate on what the future may bring. He must, however, within the limited means at his disposal, prepare the public to cope with the fore- seeable consequences of advances he anticipates.” During the 1930s physicists realized that the release of nuclear energy was theoretically certain, but they doubted whether it would be of practical interest. Because of this uncertainty and the notion that scientists should mind their own business, physicists did little to inform society of what might come. When this power became reality society was unprepared, both intellectually and institutionally, to deal with it. The public understands to some extent the “recent developments in biochemical genetics, but has only a vague notion of what may be expected in the future, in spite of the efforts of many scien- tists to inform the public about probable future developments. Where do we stand today? The genctic language now is known, and it seems clear that most, if not all, forms of life on this planet use the same lan- guage with minor variations. Simple genetic mes sages now can be synthesized chemically. Genetic surgery, applied to microorganisms, is a reality. Genes can be prepared from one strain of bacteria and inserted into another which is then changed genetically. Such changes are inheritable. Thus far, it has not been possible to program mam- malian cells in this way. What may be expected in the future? Short but meaningful genctic messages will be synthesized chemically. Since the instructions will be written in the language which cells understand, the messages will be used to program cells. Cells will carry out the instructions and the program may even be inherited. I don’t know how long it will take before it will be possible to program cells with chemically synthesized messages. Certainly the experimental obstacles are formidable. However, I have little doubt that the obstacles eventually - -will be overcome. The only -question is WHEN? (Continued on page 4) Man’s Power to Shape His Own Biologie Destiny... (Continued from page 1) If a poll were taken of a dozen knowledgeable investigators a dozen different answers probably would be obtained. My guess is that cells will be programmed with synthetic messages within twenty-five years. If effort along these lines were intensified, bacteria might be programmed within five years. The point which deserves special emphasis is that man may be able to program his own cells with synthetic information long before he will be able to assess adequately the long-term conse~ ~ uences of such alterations, long Before he will Be hs td Iaulute goals, and: long before he cam resolve the ethical and moral problems which will be raised. When man becomes capable of instruct- ing his own eells, he must refrain from doing so until he has sufficient wisdom to use this knowk edge for the benefit of mankind. The zm I tite this antihiem weulliin adhance of the need' tr do sa; is because the decisions concerning the application of this knowledge ultimately must be made by society, and only an informed society can make", "Nirenberg, Marshall W.", null, "Research Corporation Quarterly Bulletin", "The Research Corporation", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-is4k_dhay.h7q6", "00000000-0000-0000-2309-75A93E1F9774", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Takehiko Amano to Marshall W. Nirenberg", "101584910X443", null, "1972", "12 May 1972", "Amano thanks Nirenberg for his kindness and support while working as a postdoctoral student in his laboratory.  He laments the lack of a neurobiology program in Japan and suggests that Nirenberg would be a welcome guest for all molecular biologists in his country.", "Letters (correspondence)", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Text", "English", "Reproduced with permission of the Mitsubishi Chemical Corporation.", "Copyright may apply", null, null, "May 12, 1972 Dear Dr. Nirenberg: Almost two months have passed since I left your laboratory. I believe you have already settled down in your new laboratory. When I was leaving Bethesda, you were so busy I could not find a time to express my thanks to you and Mrs. Nirenberg for your kindness and discussions in my stay at your laboratory. I stopped by in Europe, spent fourteen days in several places and returned to my home in Japan. I am now beginning to set up a new, but small laboratory oriented to molecular ? neurobiology. I hope we could establish a basis in neurobiology in Japan. Whenever you have a chance to visit Japan, (actually we wish to invite you for the opening ceremony of our new institute being held this coming fall.) please contact with me without hesitation.  You are the most welcomed person for me and also for molecular biologists in Japan seeking for new field. With best regards Takehiko Amano", "Amano, Takehiko ; Mitsubishi Kasei Seimei Kagaku Kenkyu-jo", null, null, null, "National Heart and Lung Institute. Laboratory of Biochemical Genetics ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ttr5-pje5.huci", "00000000-0000-0000-D4FA-8996785656A7", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Jane H. Hu, National Naval Medical Center, Naval Medical Research Institute to Marshall W. Nirenberg", "101584910X444", null, "1972", "22 February 1972", "Hu discusses the work done by Nirenberg and his laboratory in the area of electrophysiology.  She offers some of her own experimental findings, suggests some implications of Nirenberg's work, and offers to provide any experiments or techniques that would be helpful.", "Letters (correspondence)", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Text", "English", "Reproduced with permission of Jane H. Hu.", "Copyright may apply", null, null, "Feb. 22, 1972 Dear Dr. Nirenberg, I attended your recent seminar at our Institute. Your experimental data and their implications are most interesting. If I understand correctly, you found that B-type cells which contain Acetylcholine Esterase and respond to electrical stimulations with local potentials. A-type cells can respond to electrical stimulation with action potentials. Your findings remind me of the basic differences between the so-called synaptic membrane which has acetylcholine receptors or chemoreceptors and the nonsynaptic membrane which has no chemosensitivity. I wonder whether the differentiation between the A-type cells and B-type cells also depends upon the presence of chemoreceptors. The synaptic membrane has the following properties: (1) it is chemically excitable, (2) it only generates local and graded potential responses, (3) it contains chemoreceptors. The nonsynaptic membrane has the following characteristics: (1) it is electrically excitable, (2) it is not chemically excitable, (3) it can generate action potentials which are regenerative and propagated events. It seems to me that the B-type cell membrane may be chemically excitable, which indicates the presence of chemoreceptors. To test this, one may use intracellular and extracellular iontophoresis to eject acetylcholine or other transmitters to the surface of the cells and record cell membrane potential changes. If you are interested in these experiments and techniques, I would be glad to be helpful. I am sure the electrophysiologists in your lab can do these experiments easily. Your lecture was very stimulating and I enjoyed listening to you. Cordially yours, Jane H. Hu, Ph.D. Research Neurophysiologist", "Naval Medical Research Institute. National Naval Medical Center ; Hu, Jane H.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p8gc_ak8y.w69f", "00000000-0000-0000-49C7-308297F6F85A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to Dennis Flanagan, Scientific American", "101584910X445", "101584910X433", "1972", "26 June 1972", "Nirenberg turns down an offer to write a small book on the genetic code for Scientific American.  He informs Flanagan that for the previous five years he had \"been working on a different kind of coding problem--namely, how neurons are assembled into functional circuits during the development of the nervous system and how neurons encode and decode information.\"", "Letters (correspondence)", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "June 26, 1972 Dear Mr. Flanagan: Thank you for your kind invitation to write a small book on the genetic code. For the last five years I have been working on a different kind of coding problem--namely, how neurons are assembled into functional circuits during the development of the nervous system and how neurons encode and decode information. So at the present time I am not interested in writing a book on the genetic code but might in a year or so consider writing an article for the Scientific American on neural coding. Sincerely yours, Marshall Nirenberg Laboratory of Biochemical Genetics National Heart and Lung Institute", "Nirenberg, Marshall W.", null, null, null, "Scientific American ; Flanagan, Dennis, 1919-2005", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3xn4-wkf8-uf9m", "00000000-0000-0000-30B7-C888214089A5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Lloyd H. Elliott, George Washington University to Marshall W. Nirenberg", "101584910X446", null, "1971", "28 October 1971", "Elliot informs Nirenberg that the trustees of the George Washington University have voted to confer upon him the honorary degree of Doctor of Science due to his outstanding contributions in the field of biochemistry.", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of Lloyd H. Elliott.", "Copyright may apply", null, null, "October 28, 1971 Dear Dr. Nirenberg: It is my great pleasure to inform you that the trustees of The George Washington University have voted to confer upon you the honorary degree of Doctor of Science in recognition of your outstanding leadership in the field of biochemistry. On behalf of the trustees, as well as myself, may I congratulate you upon this recognition, and express our hope that it will be possible for you to accept this honor. Our convocation is scheduled for February 21, 1972 at 8 p.m. in Constitution Hall, and we look forward to your participation. Other details will be sent along by the University Marshal, Dr. Robert G. Jones, as soon as plans are completed. In the meantime, it would be helpful if your secretary could supply me with an up-to-date biographical sheet and a glossy print of yourself by late December for publicity purposes. May I ask further that this information be kept confidential until such time as our news office makes appropriate public announcements. We look forward to having you with us on February 21. With every good wish, Sincerely, Lloyd H. Elliott", "George Washington University ; Elliott, Lloyd H.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ipjn_e65c_948h", "00000000-0000-0000-DEEF-29BDE1D3595E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Edward C. Rosenow, Jr., American College of Physicians to Marshall W. Nirenberg", "101584910X447", null, "1966", "17 November 1966", "Rosenow informs Nirenberg that he has won the American College of Physicians Award for distinguished contributions in science as related to medicine.  Details for black tie receptions and academic processions are included.", "Letters (correspondence)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "2", "pages", "Text", "English", "Reproduced with permission of Susan J. Vig.", "Copyright may apply", null, null, "17 November 1966 Dear Doctor Nirenberg: You will be pleased to know that the Board of Regents of the American College of Physicians has elected to present you with the American College of Physicians Award for distinguished contributions in science as related to medicine.  The award includes a bronze medal bearing the name of the recipient and a certificate, plus an honorarium of $250.00.  Travel and other expenses of the recipient and one member of the family will be paid for by the College.  The award will be presented during the 48th Annual Session of the College in San Francisco, April 10-14, 1967. It is expected that the recipient of the award will give a lecture at one of the plenary sessions.  Dr. Irving S. Wright, our President, has asked me to tell you that this can be on any phase of medicine in which you are particularly interested.  Please send us a title and an informative abstract of about 150 words in length so that we can include it in the printed program.  This should be sent in by December 20th.  The award will be presented at the time of this lecture. There is no requirement that the recipient of the award prepare a manuscript for submission to the editors for publication in the Annals Of Internal Medicine. However, I am sure that the editors would be pleased to consider publication of such a manuscript. Please let me know if you can accept this award.  If you can, there are certain functions to which you and a member of your family are invited.  There is a reception and dinner for Officers, Regents, Governors and special guests of the College, and their wives, on Sunday evening, April 9 at the Fairmont Hotel.  The President's reception and dinner-dance will follow the annual convocation on Thursday, April 13. This also will be at the Fairmont Hotel.  Both of these affairs are black tie. If you can be with us on Thursday, you are invited to march in the academic procession and you would be seated on the dais and introduced at the convocation.  This is always in full academic regalia and it would be appropriate for you to bring your own cap, gown and hood. If you do not have your own, please let us know your hat size and height so that we can order regalia for you. You are also invited to attend a concert by the San Francisco Symphony, as a guest of the College, on Tuesday, April 11. Please send us a recent autographed photograph of yourself for the permanent College archives.  We would also like you to let us know, on the enclosed card, the exact way in which you want your name engrossed on the certificate. Finally, please send us a curriculum vitae or biographical material which will be needed in the preparation of a suitable citation.  It is important that we have this by December 20th. With this letter, I am happy to send you my congratulations and best wishes. Cordially, Edward C. Rosenow, Jr., M.D., F.A.C.P. Executive Director", "American College of Physicians ; Rosenow, Edward C.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6zqe.mff8.t57p", "00000000-0000-0000-6F35-4859FBD3B654", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Nirenberg to Receive 1966 Research Corporation Award", "101584910X448", null, "1966", "[Fall 1966]", "This article reports on the $10,000 award granted annually for outstanding achievements in science.  Nirenberg's pioneering work is showcased, a short biography provided, and previous winners mentioned.  Candidates were nominated and elected by Nobelists and winners of other major awards.  Nirenberg was the first scientist in a government organization to earn it.", "Articles", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Reproduced with permission of the Research Corporation.", "Copyright may apply", null, null, "Nirenberg to Receive 1966 Research Corporation Miward Marshall W. Nirenberg, National Heart Institute; Research Corporation Awardee for 1966.   Marshall W. Nirenberg, Chief of the Laboratory of Biochemical Genetics at the National Heart Institute, National Institutes of Health, has been chosen to receive the Research Corporation Award for 1966. The $10,000 award, given annually for outstanding achievements in science, will be pre- sented to Dr. Nirenberg at a dinner in his honor to be given in New York on January 19. Dr. Nirenberg is being honored for his pioncer- ing experiments on protein synthesis which led to a partial “cracking” of the genetic code and stimu- lation of further research activity all over the . world. The result of :this activity is a major ad- vance in the understanding, on a molecular basis, “of the mechanism by which genetic information is translated into the various types of proteins that determine the nature of all living matter. The continuing studies of Dr. Nirenberg have , contributed importantly to comprehension of much ‘of the mysterious way information is coded into the nucleic acids and used to direct the incorpora- tion of specific amino acids into proteins. While it is difficult to conjecture the ultimate practical utility of this work, it has been a major contribu- tion toward understanding on a molecular basis how the chemicals of the cell nucleus carry the hereditary message from one generation to the next. A native New Yorker, Dr. Nirenberg holds B.S. and M.S. degrees from the University of Florida and a Ph.D. in biological chemistry from the Uni- versity of Michigan. He joined the National Insti- tutes of Health in 1957 as a postdoctoral fellow of the American Cancer Society, remaining at the NIH National Institute of Arthritis and Metabolic Diseases until 1962 when he went to the NIH National Heart Institute. Among previous honors received by Dr. Niren- berg are National Medal of Science, President Lyndon B. Johnson, 1965; Paul Lewis Award in Enzyme Chemistry, American Chemical Society, 1964; Award for Research in Molecular Biology, National Academy of Sciences, 1962, He has been awarded honorary D.Sc. degrees by the University of Michigan, Yale University, the Uni- versity of Chicago and Windsor University, Ontario. THIRTY-FIRST AWARD The Research Corporation Award to be pre- sented to Dr. Nirenberg is the thirty-first given since the award was established in 1925, and the first ever to be given to a scientist in a government organization. Among past awardees who have later received Nobel Prizes for the work recog- nized by the foundation’s award are Ernest O. Lawrence, Edwin M. McMillan, Willard F. Libby. Melvin Calvin, James D. Watson, Francis H. C. Crick, and Charles H. Townes, Candidates for the Research Corporation Award are nominated and elected by Award Juries com- posed of Nobelists and winners of other major science prizes. Members of the, 1966 Jury are Harold S. Black, General Precision, Inc.; Melvin Calvin, University of California; Edward C. Ken- dali, Princeton University; Polykarp Kusch, Co- lumbia University; Willis E. Lamb, Jr. Yale University, James D. Watson and Robert B. Woodward, Harvard University; and Chien-Shiung Wu, Columbia University.   that occur within the living organism.   RESEARCH CORPORATION AWARD FOR 1966 FOR OUTSTANDING CONTRIBUTIONS TO SCIENCE IS PRESENTED TO MARSHALL W. NIRENBERG For his pioneering work in the discovery of the mechanism through which the code in genetic material determines the proteins synthesized by a cell. His investigations have given a new and productive stimulus to the quest for an understanding, on a molecular basis, of the processes", "The Research Corporation", null, "Research Corporation Quarterly Bulletin", "The Research Corporation", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2vcg-n4rw~5uec", "00000000-0000-0000-D1CC-79271D8920DA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Paul Berg, Stanford University Medical Center to Marshall W. Nirenberg", "101584910X449", null, "1969", "15 April 1969", "Berg acknowledges that Nirenberg is no longer doing work on the genetic code and proceeds to explain to Nirenberg the genetic research he is engaged in and the nature of their discoveries.  Admitting that he is not a skilled chemist, he asks Nirenberg for two triplets--UAG and CAG--so that they can complete their work.", "Letters (correspondence)", null, "Transition to Neurobiology, 1965-1969", "1", "pages", "Text", "English", "Reproduced with permission of Paul Berg.", "Copyright may apply", null, null, "April 15, 1969 Dear Marshall, I know that you are no longer doing work on the Genetic Code, but I would like to get a favor from you if it is possible.  We are in the midst of trying to identify a new suppressor of UAG which we have isolated.  We are almost sure that it inserts glutamine, but in contrast to the usual su2 suppressor, this mutation is lethal for the organism.  We can carry the mutation in diploids and therefore have been able to study the mechanism of its suppression.  In trying to extend this work to in vitro suppression, we have used the f2 sus3 mutant and looking for tRNA-dependent suppression in vitro.  We would also like, if we can, to show the suppression by triplet binding, but we are not such good chemists that we can quickly make appropriate triplets.  I wonder, therefore, if I can prevail upon you to get some.  What we would like to have is some UAG and CAG.  We would like to be able to fractionate the suppressor species and monitor that fractionation by the coding response.  If you could let us have some of each of the two triplets, it would very much help our effort. Hope to see you soon, Sincerely, Paul Berg", "Berg, Paul ; Stanford University. Medical Center", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bwdd_m5ia.hvz4", "00000000-0000-0000-6FF9-E7E1FDFEA662", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Characteristics and Composition of RNA Coding Units", "101584910X450", null, "1962", "April 1962", "The authors report on further results concerning the poly-U in directing polyphenylalanine synthesis and explain the nature of much of the early code-breaking work.", "Articles", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "12", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Reprinted from the Proceedings of the NaTionaL ACADEMY OF SCIENCES Vol, 48, No. 4, pp. 666-677. April, 1962. CHARACTERISTICS AND COMPOSITION OF RNA CODING UNITS* By J. Hernricn Marruast,t OLIveR W. JonEs, Ropert G. Marrin, AND MarsHauu W. NiRENBERG NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, BETHESDA Communicated by Richard Roberts, February 27, 1962 The translation of a four-letter nucleotide code into a twenty-“word” amino acid dictionary has been the subject of much speculation. Although DNA and RNA polymers were inferentially involved in determining amino acid sequence, a cell- free protein synthesizing system dependent upon these polymers was not available. We have recently described such a system which is dependent upon the addition of template RNA.!—* This system affords a sensitive assay for both naturally occur- ring and synthetic template RNA. Polyuridylic acid directed the synthesis of polyphenylalanine; thus, one or more uridylie acid residues in poly U appeared to be the coding unit corresponding to phenylalanine.!: 2 Phenylalanine linked to soluble RNA is an intermediate in this process.‘ The finding that synthetic polyribonucleotides of known composition could be used to direct cell-free protein synthesis, suggested a reasonable experi- mental approach for establishing the characteristics of the genetic code, an ap- proach which has been utilized by ourselves® and by others.®: 7 The purpose of this communication is to report further results concerning the: influence of poly U in directing polyphenylalanine synthesis and the effects of randomly ordered copolymers upon the incorporation of other amino acids into protein. Some of these results have been reported in a preliminary communication.® Methods and Materials —The preparation of stable H. coli enzyme extracts (DN Aase-treated, preincubated 8-30 fractions) has been described.* Such extracts were dialyzed and stored under liquid nitrogen after preincubation. Reaction mixtures used in determining C!*amino acid incorporation into protein contained the following components: 0.1 M tris(hydroxymethyl)- VoL. 48, 1962 BIOCHEMISTRY: MATTHAEI ET AL. 667 aminomethane, pH 7.8; 0.01 4 magnesium acetate; 0.05 M KCI; 6 x 10~* M mercaptoethanol; 1 X 10-3 M ATP; 5 X 10-*M potassium phosphoenolpyruvate; 20 ug/ml of crystalline phospho- enolpyruvate kinase (California Biochem. Corp.); 0.8 — 1.6 x 10-4 M C'-amino acids; 2 x 10-* M each of 19 L-amino acids minus the C1*-amino acid; 3 X 10-5 M each of GTP, CTP, and UTP (except where stated); and FE. colt extracts. Total volume was 0.50 ml except where specified. All assays were performed in duplicate. Techniques used in. washing, plating, and counting protein precipitates have been reported.? Protein analyses were performed by a micro modi- fication of the method of Lowry et al. The lithium and sodium salts of nucleotide diphosphates were obtained from Schwarz Biochemical Corp. and Sigma Chemical Co., respectively. U-C1- amino acids were obtained from Nuclear-Chicago Corp. The purity of each C+-amino acid was checked by high voltage electrophoresis followed by radioautography.* C1-isoleucine was contaminated with C'-leucine; therefore, C'-isoleucine was purified electrophoretically before use. C14methionine was contaminated with C!-methio- nine sulfoxide. All of the other C'-amino acids were found to be free of C'4-contaminants. S*- cysteine was reduced before use either electrolytically or with mercaptoethanol. The radioactive amino acids used, their source, and their respective specific radioactivities are as follows: U-C14- glycine, U-C\"4-L-isoleucine, U-C'*-L-tyrosine, U-C!“L-leucine, U-C1!-L-proline, L-histidine- 2(ring)-C™, U-C14L-phenylalanine, U-C1*-L-threonine, L-methionine (methyl-C), U-C'4-L- arginine, and U-C14-L-lysine obtained from Nuclear-Chicago Corporation, 5.8, 6.2, 5.95, 6.25, 10.5, 3.96, 10.3, 3.9, 6.5, 5.8, 83 mC/mM, respectively; C1*-L-aspartic acid, C'4-L-glutamic acid, C'4-L-alanine, obtained from Volk, 1.04, 1.18, 0.75 mC/mM, respectively; D-L-tryptophan- C4, obtained from New England Nuclear Corporation, 2.5 mC/mM; 8*®-L-cystine obtained from the Abbott Laboratories, 2.4mC/mM; U-C™-L-serine obtained from the Nuclear-Chicago Corporation, 0.2mC/mM. The specific radioactivities of C'-phenylalanine, valine, and leucine reported by Nuclear-Chicago Corp. were validated by assay. Amino acid concentration was determined by the ninhydrin method.° These assays agreed well with the commercially reported data. In a few instances the concentration of C4-phenylalanine also was assayed by measure- ment of the absorption of the enol-borate complex of phenylpyruvic acid generated enzymatically from phenylalanine by L-amino acid oxidase.\" Polyribonucleotides were synthesized enzymatically with Micrococcus lysodeikticus poly- nucleotide phosphorylase purified by the method of Singer and Guss.!? Reaction mixtures contained 0.15 M tris(hydroxymethyl)aminomethane pH 9.0; 0.01 M MgCh; 4 x 10-4 M ethylenediaminetetraacetate; 0.06 M nucleotide diphosphate; and polynucleotide phosphorylase purified through the acid ammonium sulfate fractionation step.1* Approximately 1.4 units of polynucleotide phosphorylase. were added per ml of reaction mixture. Reaction mixtures were incubated at 37° and the formation of polynucleotide was followed by determining phosphate liberation by the Fiske-SubbaRow method.'* After the reaction had proceeded to 20-35% completion, the polynucleotides were precipitated by the addition of 3 volumes of cold absolute ethanol plus a few drops of IM NaCl and the precipitates were collected by centrifugation. The polynucleotides were redissolved in H.O and were reprecipitated as before. The pellet was dissolved in H,O and the solution was deproteinized three times by the method of Sevag.* The aqueous phases obtained after deproteinization were combined and solid KCl. was added to a final concentration of 0.025 M. The solution was dialyzed against 0.025 M KCl for 24 hr and against H,O for an additional 48 hr. The polynyrleotide solutions were then lyophilized and were stored at —15°. Some polynucleotides were t!. generous gifts of Drs. Leon Heppel, Maxine Singer, Daniel Bradley, David Davies, and Robert Steiner. The base-ratio of each polynucleotide was determined by acid hydrolysis.5 The bases were separated by descending chromatography on Whatman No. 1 paper for 18 hr using isopropanol- concentrated HCI-H.0 (130:33:37). The base-ratio analyses obtained by acid hydrolysis were verified in some cases by comparison with base-ratio data obtained by alkaline hydrolysis* and by determination of phosphate.” Good agreement was found between base-ratio analyses obtained by the three methods. Results.—Stoichiometry: In Table 1 the mymoles of C1-phenylalanine incor- porated into protein are compared with the mymoles of uridylic acid residues (pU) in poly U added to a reaction mixture. Limiting concentrations of poly U were 668 BIOCHEMISTRY: MATTHAEI ET AL. Proc. N. A. 8. TABLE 1 RELATIONSHIP BETWEEN C-L-PHENYLALANINE INCORPORATED AND Potyvuripriic Acip PRESENT i in = ILE 7 mgmole pU Experiment mumoie ny eR Toe Phen! mumole C1phenylalanine 1 11.35 . 9.12 1.24 2 17.7 ” 10.25 1.78 3 25.7 16.9 1.52 The components of the reaction mixtures are presented under Methods and Materials. -In addition, each reaction mixture contained 80 mymole of C'4“L-phenylalanine with a specific activity of 1 mC per mM; 2.6 mg preincubated 8-30 protein and the amounts of polyuridylic acid specified. In each experiment the amount of polyuridylic acid present was limiting. Final volume was 0.5 ml. eaction mixtures were incubated for 60 minutes at 37°. .. added and incubations were continued until phenylalanine incorporation had ceased. Approximately 1 mymole of C'«-phenylalanine was incorporated into protein per 1.5 mymole of uridylic acid residue in poly U. These data represent the lowest ratios obtained. The results of many similar experiments demonstrated that widely different stoichiometry ratios could be obtained depending upon the enzyme and poly U preparations used. In some experiments more than 50 mymoles of uridylic acid residues in poly U were required to direct the incorporation of 1 mpmole of C'-phenylalanine into protein. It should be noted that the presence of trace amounts of RNAase in the system, the molecular weight of poly U, etc., are vari- ables which affect the incorporation of phenylalanine. For these reasons, it is likely that smaller ratios than those presented in Table 1 will be obtained. Since it is not known whether one molecule of poly U directs the synthesis of one or many molecules of polyphenylalanine, these data alone cannot be used to determine the number of uridylic acid residues in one phenylalanine coding unit (coding ratio). Effect of Molecular Weight upon Template Activity of Poly U.—Poly U was sepa- rated into fractions of different molecular weights by sucrose density-gradient cen- trifugation.% Sixteen fractions were collected and both the absorbancy at 260 mil- limicrons of each fraction and its ability to stimulate C14-phenylalanine incorpora-   T          600 8 b 500 50 8 & 400 140 5 = = 2 = 2300 30 8 ut ! in —l i . m Q 200: 420 Sc, é 100 - 10 b o¢ > L L L 2 1 4 0 7 8 9 10 Tt 2 13 14 15 6 FRACTION NUMBER Fig. 1.—The relationship between the molecular weight of polyuridylic acid and its activity in stimulating C'-phenylalanine into protein. Polyuridylic acid was separated into fractions of dif- ferent sizes by sucrose density-gradient centrifugation. A linear gradient of sucrose concentration ranging from 16% at the bottom to 5% at the top of the tube was prepared. The sucrose solutions (4.4 ml total volume) contained 5 X 10-8 M imidazole, pH 6.8 and 0.1 M@ NaCl. 0.5 mg poly- uridylic acid in 0.2 ml was layered on top of the tube which was centrifuged at 39,000  g for 4.0 hours at 3°C in a swinging bucket rotor, Spinco type SW-39, using a Spinco Model L ultracentri- fuge. Sixteen fractions containing 0.29 ml each were collected after piercing the bottom of the tube. Aliquots containing 25 mumoles of uridylic acid residue in polyuridylic acid were used for C'4-phenylalanine incorporation assays. This amount of polyuridylic acid was limiting under the conditions of the assay. The components of each reaction mixture is presented under Methods and Me ee Total volume of each reaction mixture was 0.5 ml and incubations continued for 60 min at 37°. , Von. 48, 1962 BIOCHEMISTRY: MATTHAEI ET AL. 669 tion into protein were determined (Fig. 1). Equal amounts of poly U were added to each reaction mixture and incubations were continued until the reactions had stopped. The concentration of poly U in reaction mixtures limited the rate of the reactions. The total activity of each fraction of poly U thus was determined. Poly U molecules of higher molecular weight were distributed toward the bottom of the tube; the lower molecular weight molecules were nearer the top of the tube. It can be seen that fractions of poly U of higher molecular weight were more active in directing polyphenylalanine into protein than lower molecular weight fractions. Although the biologic activity of poly U is related to its molecular weight, the                     10 4 3S 150 mumoles [pU] & + J s Ss | ~~ 4 5 wy 4 3S ey . 5 Sor 7 g +15 r eco gq ug fo _ est & Vm g / 5S ’ RS - +15 / a NY A 0 10 20 863006 400 50 0 Bs HO min. S lo- t T T t = be <x = . Oo a a [o] oO < li z 5 | a a =z 5b 4 — > = us =x a + “o wn ut + ° = a Eo J i i 1 1 30 60 90 120 150 B my MOLES [pU] IN POLYURIDYLIC ACID Fic. 2.—Kinetics of C'«-L-phenylalanine incorporation. (A) C1phenylalanine incorporation plotted as a function of time. The components of reaction mixtures are described under Methods and Materials. In (A) the mumoles of uridylic acid residues (pU) in polyuridylic acid added to 0.5 ml of reaction mixture is shown. Each point represents a 0.5 ml reaction mixture containing 2.6 mg’of preincubated S-30 protein. In (B) total C'~-phenylalanine incorporation (each final 90- minute analysis shown in (A )) is plotted as a function of polyuridylic acid concentration. 670 BIOCHEMISTRY: MATTHAEI ET AL. Proc. N. A. 8. minimum molecular weight of poly U active as informational RNA is not known and experiments designed to answer this question are in progress. Kinetics of Phenylalanine Incorporation —In Figure 2 the rate of phenylalanine incorporation into protein is shown. From 7.5 to 150 mymoles uridylic acid resi- due (pU) in poly U were added per 0.5 ml reaction mixture. C1‘-phenylalanine incorporation ceased within 30 minutes after addition of poly U. Further addition of poly U after incorporation had stopped resulted in an additional stimulation of incorporation. After the reactions had again stopped, addition of more poly U did not appreciably stimulate C'*-phenylalanine incorporation. When the mixtures were incubated for 30 minutes in the absence of poly U, and at that time 15 mymoles of pU were added, a somewhat greater incorporation of C14-phenylalanine was ob- served compared with incorporation resulting from the addition of poly U at the onset of incubation. These experiments demonstrate that even though C'- phenylalanine incorporation stops after 20-30 minutes of incubation, the enzyme extracts are fully active. The fact that C!4-phenylalanine incorporation stops after 30 minutes even though a large excess of poly U is present (150 mumoles pU) shows that poly U is being inactivated. Such inactivation of excess poly U does not ap- pear to be dependent upon protein synthesis. The data of Figure 2 B show that phenylalanine incorporation is proportional to poly U concentration in the range of 10-50 mumoles of pU in poly U per 0.5 ml reaction mixture. In the linear part of the curve about 6 mumoles of pU were re- quired to direct the incorporation of 1 mumole of C'*-phenylalanine. Stimulation of Amino Acid Incorporation by Polynucleotides Containing One Base.—The effect of “homopolynucleotides” upon amino acid incorporation into protein is presented in Table 2, As has been reported previously’? poly U specifi- cally stimulated phenylalanine incorporation into protein. Poly U also stimulated the incorporation of small amounts of C’*leucine and C'-valine into protein. TABLE 2 ; Speciriciry oF AMINO Acip INCORPORATION STIMULATED BY HOMOPOLYNUCLEOTIDES AND . Yeast RNA Minus poly- Yeast C.L-amino acid nucleotide poly A poly C poly U RNA Alanine 27 18 21 26 82 Arginine 19 19 18 25 204 Aspartic acid 23 23 18 28 177 Cysteine 109 111 114 113 133 Glutamic acid 53 53 50 53 204 Glycine 32 31 28 31 284 Histidine 5 4 5 3 312 Isoleucine 50 22 55 74 229 Leucine 17 71 43 263 178 Lysine 14 22 11 26 129 Methionine 29 35 31. 40 83 Phenylalanine 24 24 22 5424 128 Proline 22 20 336 27 118 Serine 250 250 250 281 687 Threonine 26 23 28 28 110 Tryptophan 245 . 239 238 262 §14 Tyrosine : 38 38 37 42 83 Valine 11 11 11 47 67 The figures represent the incorporation of C!-amino acids in kumoles. The components of reaction mixtures are presented under Methods and Materials. GTP, CTP, and UTP were omitted. ach 0.6 ml reaction mixture contained 80 mymole of the appropriate C!-amino acid (in addition to 19 C!2-amino acids); 2.6 mg preincubated 8-30 protein; and either 100, 20, 10, or 500 yg of poly A, poly C; poly U or yeast RNA (prepared by the method of Crestifield et al.‘4) respectively. Samples were incubated for 60 minutes at 37°. Vou. 48, 1962 BIOCHEMISTRY: MATTHAE!I ET AL. : 671 Base-ratio analyses (see Methods and Materials) showed that this polymer contained 2.5% guanylic acid present as an impurity. Thus, the small stimulation of leucine and valine incorporation by poly U probably is due to the presence of guanylic acid (see next section). Poly C stimulated the incorporation of proline.? However, the effectiveness of poly C in stimulating proline incorporation varied with different preparations of poly C; for example, many preparations stimulated 5- to 10-fold and three prepara- tions stimulated 75- to 100-fold. Since the effectiveness of poly C in stimulating proline incorporation was not fully reproducible, the purity of several preparations was examined (see Methods and Materials section). These preparations contained 4-14 per cent uridylic acid. Commerical preparations of CDP were analyzed and were found to contain no UDP. As will be seen in the next section, randomly-mixed poly UC markedly stimulated proline incorporation, therefore, the observed activity of poly C may result from the presence of small amounts of U in the polynucleotide preparations. Poly A did not stimulate the incorporation of any amino acid into protein. The addition of smaller concentrations of poly A also gave similar results. The mo- lecular weight of the poly A was approximately 30,000. In contrast, poly U of similar molecular weight markedly stimulated phenylalanine incorporation. The ineffectiveness of poly A in coding for any amino acid could be due to either its double-stranded structure in solution or to the possibility that a sequence of A does not specify any amino acid. Previously, it was found that yeast ribosomal RNA prepared by the method of Crestfield et al.,48 stimulated amino acid incorporation in this system.? Yeast RNA was used as a control to show that the system was active with respect to - every amino acid. ‘These experiments also demonstrate that the addition of natu- rally occurring template RNA stimulated the incorporation of every amino acid in contrast to the specificity displayed by poly U. Since poly G is difficult to prepare enzymatically, it was not available to us in sufficient quantities to test with each individual amino acid. However, when poly G was tested with a C'-algal protein hydrolysate which contained 16 C1-amino acids, it did not stimulate the incorporation of any C14-amino acid. An oligonu- cleotide primer (tetraadenylic acid) was required for the enzymic synthesis of poly G and primarily low molecular weight polynucleotides having an average chain length of 15 nucleotides were obtained. Therefore, the fact that poly G was unable to stimulate amino acid incorporation should be interpreted with cau- tion. The Effects of Randomly Ordered Polynucleotides upon Amino Acid Incorporation.— The data of Table 3 demonstrate that randomly ordered polyribonucleotides direct the incorporation of amino acids into protein in a highly specific manner. Since each preparation of polynucleotide will differ in molecular weight, it is difficult to compare directly the incorporation of activity of one polymer with another. There- fore, the figures in Table 3 represent the per cent of any amino acid incorporated compared to phenylalanine incorporation stimulated by the same polynucleotide. The counts/minute of C'“-L-phenylalanine incorporated due to the addition of each polynucleotide is presented in the legend accompanying Table 3; therefore, the percentage figures in the table readily may be converted into counts/minute. The 672 . BIOCHEMISTRY: MATTHAEI ET AL. Proc. N. A. S. base-ratio of each polynucleotide, determined experimentally, is given also. If a polynucleotide containing two bases stimulated incorporation of an amino acid, inclusion of a third base,in the polynucleotide did not prevent this stimulation. Poly UG and poly UGC previously were found to direct the incorporation of small amounts of methionine and glutamic acid respectively.5 Further experiments have shown that poly UGA codes for these amino acids more effectively; thus, the coding units for methionine and glutamic acid contain U, Gand A. Lysine incorporation was stimulated by the addition of poly UA (*/; ratio) but not by randomly mixed poly UA containing lower proportions of A. The coding unit for lysine therefore probably. contains UAA.... The stimulation of phenylalanine incorporation by poly UA (1/4 ratio) was negligible, so these data have not been expressed in Table 3 as the ratio of lysine to phenylalanine incorporation. _A series of polynucleotides each containing a different ratio of U to A were pre- pared. Similar series of polynucleotides containing either U and C or U and G ‘were synthesized also. It was found that stimulation of incorporation of a given amino acid by a polynycleotide varied with the base-ratio of the polymer. Thus the relative amounts of two nucleotides in a coding unit could be estimated. These data are too extensive to be reported here and will be the subject of a future com- munication. The coding ratio is not known definitively. However, assuming a triplet code, the probability of a triplet occurring in a polynucleotide, relative to UUU, may be calculated from the base-ratio data presented in Table 3. For example, if poly UG had a base-ratio of 3U/1G, the probability of obtaining the sequence UUU would be °/, X #/4 & 3/4 = */q4. The probability of obtaining the sequence UUG would be */, XK °/4 X 1/4 = °/e4. Thus, 3 UUU would occur for 1 UUG and, as- suming the frequency of UUU to be 100 per cent, the frequency of UUG would be 33 per cent. The theoretical frequency of each possible triplet, relative to UUU is presented in Table 3. Amino acid incorporation agreed with predictions based upon probability theory in most cases. The nucleotide composition of coding units corresponding to each amino acid may be derived from these data and a sum- mary is presented in the last column of Table 3. Dots after each “word” are used to indicate the possible presence of additional uridylic acid residues. Discussion.—Since the coding units of arginine, alanine, and glutamic acid each contain three different nucleotides, the minimum coding ratio would appear to be three. Poly GC does not code for arginine or alanine and poly AG does not code for glutamic acid.??. Possibly the coding ratio is larger, but these data rule out the possibility of singlet and doublet codes. In this preliminary study we will assume that every amino acid has the same coding ratio” and that in this system, as in cellular systems,?! an overlapping triplet code is improbable. It is important to determine whether one molecule of poly U directs the synthesis of either one or several molecules of polyphenylalanine, i.e., whether poly U func- tions stoichiometrically or catalytically. If, for example, each molecule of poly U directed the synthesis of only one molecule of polyphenylalanine and a nonover- lapping sextuplet code were operative, six uridylic acid residues in poly U would be necessary to mediate the incorporation of one phenylalanine. Instead, the data of Table 1 show that almost one mumole of phenylalanine was incorporated into Polynucleotide Base-ratio Probability of triplet relative to phenylalanine (UUV) = 100% Amino Acid Phenylalanine Arginine Alanine Serine Proline Tyrosine Tsoleucine Valine Leucine Cysteine Tryptophan Glycine Methionine _ Glutamic acid Lysine The figures in the main part of the table re henylalanine incorporated. 100). e components of the reaction mixtures are presented under Methods and Materials. acid, 0.075 umole of the C1amino acid, and approximately 25 ug of each polynucleotide were added to each 0.5 ml reaction mixture. Incorporation of C14-phenylalanine in counts per minute due to the a: incorporated/mpzmoles a@ given amino acid. Uae co UAA— 2.2 a) ae o Hops ee cllBoorno8 AWW Coe I it 7 Fea Dw U Cc eo UUU—100 UUC—157 UCC—244 CCC—382 100 0 ony > oo | bh oO on Seeccddoro TABLE 3 ‘ Amino Acip INcornPoraTION INTO PRorein STIMULATED BY RANDOMLY Mrxep PoLyNUCLEOTIDES tla G U = 0.76 G = 0.24 UUU—100 UUG— 32 UGG— 10.6 GGG— 3.4 ~e Sowors t _ o bo ° o[hlel& 81S 6 respectively. The reproducibility of the above percentage figures was +3. * Sequence of nucleotides in a coding unit is not specified. ROO WeE © oe | roooceone bt i! OO NOD e& UGC 0.341 0.152 0.502 UUU—100 UUG— 46.2 Q toed UGG— 21.0 ° GGG— 1.0 UUC—147 UCC—218 CCC—322 UGC— 68.1 GGC— 31.7 GCC—101 e aonons WwW oO a oO ~ ~T “| ol Sl 83) S| £ a iw] Composition of coding units* UUU... UCG... UCG... UUC.. + UGG... UCC... UUA... UUA... UUG... UUC.. + UUG... UUG... or UGG UGG... UGG... UGA... UGA... UAA... (?) present the incorporation of any amino acid compared to phenylalanine incorporation expressed as percentages (muzmoles amino acid Underlined figures refer to the polynucleotide containing the nucleotides necessary to stimulate the incorporation of 0.1 wmole each of 19 L-amino acids minus the appropriate C!amino Samples were incubated at 37° for 15 minutes. dition of polynucleotides UA, UC, UG, UAC, UGC, and UGA were 731, 2900, 714, 804, 2144, and 2744, 2961 ‘8b “IOA ‘TV Ld IAVALIVA -AYLSINGHOOIT €29 674 BIOCHEMISTRY: MATTHAEI ET AL. Proc. N. A. 8. protein per mymole of uridylic acid residue in poly U. Therefore, each phenyl- alanine coding unit appears to function catalytically for a limited time. A degenerate code is one in which two or more different coding units can direct the incorporation of the same amino acid into protein. In a completely degenerate code, every permutation of nucleotides would code for an amino acid. In a par- tially degenerate code, certain coding units would direct amino acid incorporation, whereas others would not. Coding units which would not direct amino acids into protein will be called “nonsense words.” Up to now it has not been possible to de- termine directly whether the code contains nonsense units. The data of Table 2 demonstrate that poly A does not direct the incorporation of any amino acid into protein. Other polynucleotides such as poly AG also do not direct incorporation of amino acids.22 Such experiments indicate that nonsense words exist and thus would preclude the possibility of a completely degenerate code. Nonsense regions in template RNA may be functionally important, for it is possible that these nucleotide sequences serve as periods, i.e., may specify C- or N-terminal groups in proteins. The data of Table 3 demonstrate that a coding unit corresponding to leucine can contain either U and C or U and G. Since two words containing different nucleo- tides,correspond to leucine, the code is partially degenerate when synthetic poly- nucleotides are used to direct amino acid incorporation. Whereas the U content of RNA viruses is not excessive, a surprisingly high pro- portion of U has been found in coding units thus far (Table 3). This dichotomy’ cannot be explained at the present time. However, it is probable that additional degenerate code words will be found which will not contain U. Trichloracetic acid insoluble peptides only have been measured in this study. Since polyphenylalanine becomes insoluble when four or five phenylalanine residues are linked together, such an insoluble “handle” may ensure the precipitation of a polypeptide which otherwise would be soluble. If a code with much degeneracy is assumed, it is possible that coding units containing U have been selected for by the assay method. However, other explanations are possible and are being considered. The foregoing data demonstrate that the code is partially degenerate, the mini- mum coding ratio is three and that nonsense coding units exist. Crick ef al., on the basis of ingenious genetic experiments employing the rII region in T, phage, have come to similar conclusions.”* It is important to note that such information, obtained by means of both biochemical and genetic approaches, is in complete ac- cord with one another. The treatment of tobacco mosaic virus (TMV) RNA with nitrous acid results in the deamination of nucleotides*‘ and the formation of mutant strains of TMV with altered plaque morphology.* Cytidine is converted to uridine, adenosine to hypo- xanthine and guanosine to xanthosine. The recent striking work of Wittmann” at Tubingen and Tsugita and Fraenkel-Conrat®: ” at Berkeley has shown that the protein isolated from such mutant strains is, in many cases, different from the pro- tein produced by wild type TMV. The amino acid sequences of both wild type and mutant TMV proteins were determined and certain amino acids were found to be replaced by others rather frequently. A comparison has been made in Table 4 between nitrous acid-induced amino acid substitutions and the nucleotide com- positions of corresponding coding units. Only amino acid substitutions which have Vou. 48, 1962 BIOCHEMISTRY: MATTHAEI ET AL. 675 TABLE 4 Comparison oF Nrrrovs Acrtp Inpucep REPLACEMENTS In Topacco Mosaic Virus PROTEIN WITH THE NUCLEOTIDE Composition oF RNA Coping UNits Nucleotide composition Possible No. of mutant strains Amino acid of corresponding coding nucleotide A Bt replacement units changes 1 2 Ser ove: . , Phe UUU... U 1 2 Glu UAG... A { | t Gly UGG... G 1 2 Prol . UCC... C 1 1 } Leu UUC... U 2 Teoleu UUA... ‘ Val vic. .- G 5 Arg UGG... C { | t Gly UGG... G * The mutant amino acid replacement data cited and obtained either by (A) Tsugita and Fraenkel-Conrat*: 27 or (B) Wittmann.” been found more than once have been cited. Thus, the chance of a substitution occurring spontaneously rather than by deamination is lessened. Only those amino acid substitutions are included which correspond to RNA coding units with ex- perimentally determined nucleotide compositions. We are indebted to Drs. Tsugita and Fraenkel-Conrat for allowing us to cite some of their unpublished replacement data. Replacement of serine by phenylalanine, glutamic acid by glycine, proline by leucine, glutamic acid by valine, and arginine by glycine have been observed. Most of the nucleotide conversions corresponding to the amino acid replacements thus would be the conversion of either cytidine to uridine or of adenine to guanine. As has been predicted by Freese,” a fairly high proportion of nitrous acid induced amino acid substitutions should be the result of conversion of cytidine to uridine in a coding unit. Wittmann has also shown that nitrous acid will convert adenine to guanine.* Deamination of guanosine has no mutagenic effect. Comparison of amino acid substitution data with the proposed nucleotide compositions of coding units shown in Table 4 strikingly confirm these predictions. Previously we have shown that sRNA is an intermediate in polyphenylalanine synthesis.‘ It is likely that amino acids lose their identity after being linked to sRNA and that aminoacyl-sRNA molecules recognize template RNA coding units by base-pairing. During the course of evolution, mutations affecting the nucleo- tide sequence of sRNA or the specificity of amino acid activating enzymes might occur and might result in a series of phylogenetically related codes rather than one code universal to all species. From an evolutionary point of view, a mutation resulting in a changed code wherein one amino acid substitutes for another in all proteins probably would be lethal to a highly organized cellular system. The use of synthetic polynucleotides affords a unique opportunity to determine whether the code is universal. The data of Table 4 demonstrate that both TMV coding units directing protein synthesis in tobacco plants, and coding units functioning in EZ. coli have similar nucleotide compositions. It is not improbable that minor changes in the code will be found in 676 BIOCHEMISTRY: MATTHAEI ET Ab. Proc. N. A. S. different species; however, these data strongly suggest", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9fsu-3ega.5ud9", "00000000-0000-0000-1A45-116FF118B3CA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Code of Life Finally Cracked", "101584910X451", null, "1961", "24 December 1961", "The author reports that Nirenberg \"announced his achievement\"--deciphering the first word of the genetic code--at the Moscow International Biochemical Congress in August 1961.  The news, however, did not leak to the press until December when a group of researchers from New York University announced they had \"taken Dr. Nirenberg's code-breaking procedure several steps further and deciphered fourteen out of twenty words of the code of life, and had thus effectively broken it.\"", "Newspaper columns", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of Earl Ubell.", "Copyright may apply", null, null, "December 24, 1961 New Horizons in Science   By Earl Ubell Science Editor Bruecke and I have sworn to make the truth prevail: no forces are effective in the organism other than the purely physical-chemical—-Emil Du Bois-Reymond, 1847. Eventually, physiology must completely dissolve into or- ganic physics and chemistry— Emil Du Bois-Reymond, 1848. * » * When Du Bois-Reymond set those brave words down as a thirty-year-old biologist in .|Berlin.more than a century ago, they seemed as revolutionary as the Communist Manifesto of those same years. His call for a physical-chemical basis for life hammered at the prevailing theory of vitalism:. the “old men” believed an unseen, im- measurable vital force gave the spark to life. In typical revolutionary fashion and in keeping with the times, the young men in science rallied to the young Berliner’s side. Bruecke was Ernst Bruecke, who later became Sigmund Freud’s physiology teacher in Vienna. Hermann von Helmholtz, then twenty- seven and a founder of the law of conservation of energy, and Karl Ludwig, thirty-two, a physiologist, joined the fight. Of course, with their methods they failed) to prove their biological manifesto. Last week another groyp of! Predoninatitly” young ~ men} announced they had come closer than ever to prov- ing the Germans right. They had tapped one of the inner- lmost. secrets of living things. They had found the key to the chemical code of life: they had learned the basic ‘chemical language of heredity and of the living chemistry by which protoplasm regenerates itself. They had given more reason than ever to believe that at bottom the life force is chem- istry and physics. Credit for the fundamental crack in the code goes to Dr. Marshall W. Nirenberg, thirty- two, of the National Institute of Arthritis and Metabolic Diseases, Bethesda, Md. Work- ing with Dr. J. Heinrich Matthaei, Dr. Nirenberg six months ago deciphered the first “word” of that code. The News Leaks Out When Dr. Nirenberg an- nounced his achievement in Moscow at the International Biochemical Congress last August, he set the world of biochemistry abuzz. However, the news did not leak into the newspapers until last week. A group of four researchers from New York University announced they had taken Dr. Nirenberg’s code-breaking procedure several steps further and deciphered fourteen out of twenty words of the code of life, and had thus effectively broken it. After this announcement by the NYU group headed by Nobel Prize winner Dr. Servo Ochoa, Dr. Nirenberg’s insti- tute announced that he and Dr. Matthaei had also de- ciphered as many words. At week’s end, it seemed it would be only a short time before the full code was known, now that its basic secret was revealed. What is this code? What is the “language” involved? What are the “words?” These are but metaphorical ways of ex- pressing the great modern question in biology: how does a living thing transmit its char-     acteristic to its offspring? The Code of Life Finally Cracked In the mood of 1848 trans- ferred to today, biologists and chemists know the transfer is chemical. They know that when sperm ‘unites with . egg, chemicals are joined. When an amoeba or a cell or a germ splits in two, chemicals are distributed. It was only a few generations ago that biologists traced the site of those important chemi- cals to the nucleus, the central core of the cell; and in the nucleus to the chromosomes, microscopic rods. Twenty years ago, they iden- tified the chemical that carries the hereditary. information—it was called - deoxynucleic’ acid (dee-OXY-NOO-KLEE-ik), and known as DNA for short. The past two decades have been spent trying to understand its chemistry. and its role in the hereditary mechanism. Composed of two long strings of four basic molecules, per- muted thousands of times, the DNA holds the information for the‘manufacture of proteins in cells. The type and variety of proteins make an animal the size, shape and color if is. The proteins, composed of twenty different aminoacids, carry on the basic life chemistry.’ | Stull Unknown But the biologists did not know how the information from the DNA moves from the nucleus of the.cell. inte the pro- tein manufacturing region, the | cytoplasm, and into the ribo- somes, the microscopic protein factories. Recently they found the role of the transfer agent played, by. another chemical, ribonucleic acid (RYE-bo- NOO-KLEE-ik), a cousin to DNA and known as RNA. Unknown: What sequence of submolecules in RNA (as trans- ferred from the DNA) produced the proper sequence of amino acids in the proteins? The whole sequence of RNA sub- molecules—identified by letters representing the 4 chemicals, G, C, U, A—is the language of life; eaeh three-letter combina- tion is a “word.” Each word— UUU, UAA, CUA, etc.—controls the addition of a particular amino acid to a particular pro- tein. Dr. Nirenberg fed artificial RNA to the ribosomes. ‘This artificial RNA had but one word —UUU—UUU---UUU—repeated over and over. The ribosomes read the code and produced a protein containing only one amino acid, phenylalanine. This was the news last August in — Moscow. The NYU group tried other RNAs and worked out—with some ambiguities still—the code words for fourteen of the twen- ty amino acids. Dr. Nirenberg has also done so. Future Uses. This work has great practical implications. If biologists know the whole hereditary code and learn how to change it at will, they. may be able to. control. heredity by chemical means. They could raise plants and animals of almost any desired character, and do it in a hurry. In human beings, it could lead to control of hereditary diseases like diabetes and gout, to name but two. Some scien- tists are even worried that it could be applied to control the intelligence of large human populations wholesale; i. e., breed @ super-race. But all this is in the distant future. At the moment, the biological manifesto of 1848 looks very good indeed. ‘", "Ubell, Earl", null, "New York Herald Tribune", "New York Herald Tribune (Firm)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-c6q6~k2wv_ptei", "00000000-0000-0000-73A3-426A633320B1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "NIH Researchers Crack the Genetic Code", "101584910X452", null, "1962", "5 January 1962", "This article details the pioneering work of Nirenberg and Matthaei to decipher the RNA molecules that carry cellular blueprints, and reports \"the enigma of genetic coding, considered a fundamental secret to life, may be on the verge of solution.\"  A photograph of Nirenberg working out genetic coding units is included along with visual representations of RNA sequences.", "Articles", null, "Public Reactions to the Genetic Code, 1961-1968", "2", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "MEDICAL WORLD January 5, 1962 Vol. 3, No, 1 NIH RESEARCHERS CRACK THE GENETIC CODE In pioneering experiments, two young biochemists have begun to decipher the RNA molecules that carry cellular blueprints he enigma of genetic coding, con- sidered a fundamental secret of life, may be on the verge of solution. In just-published and about-to-be- - published papers, several research teams are reporting experimental proof of what has been largely theory: the intricate process by which struc- ture and function of living organisms are shaped. One group has begun to crack the DNA-RNA code—the key to the whole mystery. Soon they expect to decipher the entire set of instruc- tions by which genetic messengers di- rect the manufacture of proteins—the basic stuff of life. The major achievement in RNA research is the work of two young biochemists at the National Institute of Arthritis and Metabolic Diseases, Drs. Marshall W. Nirenberg and J. Hein- rich Matthaei. Behind their work, however, is a whole series of investi- gations which has produced the basic theory and its preliminary experi- mental support. Fundamentally, the theory states that the hereditary “blueprints” of cell structure and function are coded with- in the cell nucleus as long-chain molecules of desoxyribonucleic acid (DNA). These plans are transmitted, in a series of steps, to the cytoplasmic “assembly line” where they direct the synthesis of each cell’s characteristic products. In all species, from bacillus to man, DNA molecules are organized around the same general plan, chains of only four nitrogen-containing units (bases): adenine, guanine, cytosine and thymine. The specific order of these bases, like the order of letters in words, is meaningful. In effect, the 18 coded “message” of heredity is writ- ten in a four-letter alphabet. DNA molecules can duplicate themselves. This property plays a key role, not only in the passage of heredi- tary characteristics from one genera- tion to the next, but also in the devel- opment of multicellular organisms. The DNA in a fertilized human ovum reproduces thousands of billions of times to form the myriad cells of the mature individual. And each cell con- tains a complete copy of the genetic “specifications” for the human species, as well as those for the individual’s unique collection of hereditary char- acteristics. This duplicative process was re- produced in the test tube by Dr. Arthur Kornberg of the Stanford Med- ical Center, who won the 1959 Nobel Prize in medicine and physiology for the accomplishment. He prepared so- lutions containing the four DNA bases and other ingredients, added a dash of “natural” DNA as a primer—and found that the primer would assemble the bases into exact copies of itself. So far as is known, DNA functions only in the nucleus, like an original blueprint kept in the foreman’s office. From its headquarters, the DNA transfers its genetic information to ribonucleic acid (RNA), which con- stitutes the “working drawings” used in the cytoplasmic factory. RNA, like DNA, also consists of chains built up of four bases. Three of the bases—adenine, cytosine and guanine — are the same as those in DNA, but instead of thymine, RNA has uracil. (Dr. Severo Ochoa of the NYU College of Medicine shared the 1959 Nobel Prize with Kornberg for     NATURAL RNA     BIOCHEMICAL CODE depends on sequence of four kinds of units (gray) in RNA mole- cule. Various sequences select different amino acids (colof) for protein synthesis. MEDICAL WORLD NEWS developing methods of synthesizing RNA.) At NYU, the University of Chi- cago, and the University of St. Louis, researchers are now studying trans- mission of coded information from DNA to RNA. One NYU team, Drs. John Furth and Jerard Hurwitz, told the American Association for the Ad- vancement of Science that they have now transferred, in vitro, the letter- for-letter nuclear code to a material which is known to pass through the nuclear membrane into the cytoplasm. Exactly as Predicted They prepared a solution contain- ing quantities of the four RNA bases —unassembled components of the long molecules—and a special enzyme obtained from E. coli. Then they add- ed various species of DNA. The DNA, presumably acting as a sort of tem- plate, assembled the bases to form RNA molecules. And in each case, the proportion of the bases in the syn- thesized RNA reflected the propor- tions in the DNA, precisely as the theoretical model predicted. For ex- ample, for every hundred adenine units in the DNA, there were a hun- dred uracil units in the RNA. Not only the proportion, but also the order of the bases was transferred. In one experiment, the two investi- gators used a synthetic DNA in which every other base was adenine, In the RNA, every other base was uracil. This is only the first step. Once the coded plans reach the cytoplasm they are translated into action—syn-   DR. NIRENBERG spells out an explanation of his work in biological cryptanalysis. thesis of enzymes and other proteins. The properties of these proteins are determined by the sequence of their hundreds or thousands of component units. Each of these units is one of the 20-odd amino acids, and biochemists have speculated that each amino acid is specified by a different genetic “code word”—a group of three or four bases in the RNA molecule. A sequence of several thousand such “words” could specify the complete structure of even the most complex protein. But the pivotal question has been: Which word signifies which amino acid? The answer to this is the major contribution just now emerging from the laboratory of Drs. Nirenberg and Matthaei. For the first time, they have brought about controlled RNA man- ufacture of amino-acid chains in the test tube. And by utilizing known   SYNTHETIC (“POLY-U”’) RNA         DECODING EXPERIMENT uses RNA with only one kind of unit, uracil (dark gray). This simple sequence forms chains of only a single amino acid, phenylalanine (dark color). January 5, 1962 sequences of RNA bases, they have succeeded in equating certain sequen- ces with certain amino acids. Drs, Nirenberg and Matthaei are using E. coli solutions containing the 20 amino acids, energy-rich adenosine triphosphate (ATP), ribosomes (tiny intracellular particles where proteins are synthesized) and cytoplasmic flu- ids (cell sap). To this mixture. they add ‘“‘poly- U,” a synthetic RNA containing only one kind of base: uracil. The RNA triggers the formation of amino-acid chains (polypeptides), But most sig- nificantly, out of all available amino acids, the poly-U selects just one — phenylalanine—to incorporate in the polypeptide. Evidently, the code-word U-U-U  (uracil-uracil-uracil) trans- lates as “phenylalanine.” Within a few months, the NIAMD researchers expect to report on the code sequences associated with at least ° 15 different amino acids. Nirenberg and Matthaei’s tech- niques, which have yielded such ex- citing results, are now being taken up by half a dozen other groups. At least two Nobel laureates—Ochoa at NYU and Fritz Lipmann at Rockefeller In- ‘stitute—are heading research teams that are tackling protein synthesis. Ochoa’s group, including Drs. Carlos Basilio, Joseph F. Speyer and Peter Lengyel, has already achieved a -pre- liminary picture of RNA sequences associated with 14 amino acids. For each sequence they have determined the names and proportions of the bases, but not their order. Dr, Nirenberg predicts that “within another six months or so most of the genetic code will be cracked,” = 19", "Medical World Publishing Company", null, "Medical World News", "Medical World Publishing Company", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9jmz~sctd_j4jx", "00000000-0000-0000-1151-3A77B4EADD51", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Findings May Speed Solution to Genetic Code", "101584910X453", null, "1962", "15 January 1962", "This article details how RNA stimulates amino acid incorporation into protein, concluding that the sequence of bases in nucleic acid appears to be the key to the genetic code.", "Articles", null, "Public Reactions to the Genetic Code, 1961-1968", "3", "pages", "Text", "English", "Reprinted with permission from Chemical & Engineering News, 15 January 1962, 40, 3, 44-46. Copyright 1962 American Chemical Society.", "Copyright may apply", null, null, "RESEARCH Findings May Speed Solution to Genetic Code RNA stimulates amino acid incorporation into protein; sequence of bases in nucleic acid appears to be key to code More and more information is gather- ing which soon may permit chemists to solve the mysteries of the genetic code. Two teams of scientists in the U.S. have shown experimentally that ribonucleic acid (RNA) stimulates amino acid incorporation into cell pro- tein. They have also prepared a syn- thetic RNA that is specific for phenyl- alanine. A team of British scientists has evidence that the code is related to the sequence of bases along the nucleic acid of genetic material (C&EN, Jan. 8, page 43). Two chemists at the National Insti- tutes of Health, Bethesda, Md., have found new evidence showing that the hereditary instructions for the synthe- sis of proteins in living cells are car- ried by a “messenger” RNA. NIH’s Dr. Marshall W. Nirenberg and Dr. J. Heinrich Matthaei have devel- oped a stable, cell-free amino acid- incorporating system dependent upon messenger or soluble RNA (sRNA). They have also been able to substitute a simplified synthetic messenger for the natural product. RNA has a long chainlike polymeric structure consisting of ribose units linked by phosphate bonds. Attached to each ribose unit is one of four key nucleotides: adenylic acid (A), guanylic acid (G), cytidylic acid (C), and uridylic acid (U) (C&EN, May 8, 1961, page 81). Chemists think that the sequence of these nucleotides on the RNA chain governs how RNA directs amino acid incorporation into protein. Chemists soon found that the amino acids form covalent links to sRNA and that the reaction is catalyzed by the same preparations that contain the activating enzymes.         NO SN ON, oO OO KBCABCKBCABCABCABCABCABC, LL. Liiriliti LIL ItItitlIIIttll tl DO NON ON ON ON os KGCKRBEABEAB EAD EAD EABEAG. oee@#e Liitittirtit litt | PiLlit scan first mutant LON NON ON Ser gan ABCKABBCABCAGGCH BE) CABCA,. 4... Littrit ttt TIT It Titi titi tty tt eT a Reading restored ZO NOT OTL om OD ABCKABECAE BE ABCA BCABEAE GC. 6.50 LiiriltitittitI ITLL III itt ide Addition of- third mutant TRIPLETS. Chart prepared by Dr. F. H. C. Crick and co-workers shows type of code involved in the nucleic acid of genetic material. They found evidence for a coding ratio of 3 by examining six triple mutants of the FC family. In all the six, the combination of three mutants restored the function of the gene-product. With all three mutants in combination the order of triplet reading is restored. A, B, and C each represent a different base of the nucleic acid. The sequence is such that it is read in sets of three starting on the left 44 C&EN JAN. 15, 1962 Controversy, however, exists con- cerning the role of sRNA in protein synthesis. Little information, too, is on hand as to the nature of the code on the sRNA chain (the number and sequence of nucleotides) that signals which amino acids will be incorporated into cell protein. Dr. Nirenberg and Dr. Matthaei have succeeded in preparing from the colon bacillus (Escherichia coli) cell- free extracts which actively incorpo- rate amino acids into protein. Be- cause of its stability, the preparation is a stimulus to further zesearch. In the past, fresh cell-free E. coli extracts had to be made for each experiment. The NIH chemists’ preparation can incorporate amino acids into protein, as evidenced by the following charac- teristics (typical of amino acid incor- poration into protein): ®Both ribosomes and a certain fraction of the supernatant liquid after centrifugation (105,000 x g) are needed for amino acid incorporation. ® Adenosine triphosphate (ATP) and an ATP-generating system are necessary for protein synthesis. Incorporation of a particular amino acid is stimulated by a mixture of other amino acids. ® Certain substances known to in- hibit amino acid incorporation into protein (puromycin, chloramphenicol, and RNAase) strongly inhibit the ac- tivity of the extract. The NIH team’s studies show that soluble RNA from E, coli stimulates amino acid incorporation into protein. In addition, they find that messenger RNA preparations from E. coli strongly stimulate incorporation of C14-tagged valine into protein. When low mes- senger RNA concentrations are used, they obtain a linear relationship be- tween messenger RNA concentration and C!4-valine incorporation into pro- tein. The linear relationship between messenger RNA concentration and ac- tivity provides a valuable assay for messenger RNA. STIMULATION. When Dr. Nirenberg and Dr. Mat- thaei add a mixture of 20 amino acids to their preparation containing the ribosomal RNA, the amino acids stim- ulate the incorporation of valine into protein. Addition of small amounts of DNAase inhibited amino acid incor- poration, suggesting that the genetic material was directing protein synthe- sis in this cell-free system, In studying the effect of E. coli ribosomal RNA upon the incorpora- tion of seven different amino acids (valine, threonine, methionine, argi- nine, phenylalanine, lysine, and leu- cine), the NIH team finds that the added messenger RNA increases the incorporation of every amino acid tested. RNA from other sources stim- ulates C14-valine incorporation into protein, too. Tobacco mosaic virus RNA, for example, strongly stimulates amino acid incorporation. Phenylalanine Incorporation. The NIH team prepared a synthetic RNA composed of only one of the four nu- cleotides to see if it might specifically incorporate one amino acid. They made polyuridylic acid (poly U) and added 10 micrograms of it per ml. of reaction mixture. Poly U, they found, strongly stimulates C14-phenylalanine incorporation. They prepared other polynucleotides such as polyadenylic acid (poly A) and polycytidylic acid NIH graph shows that polyuridylic acid strongly stimulates phenylalanine incorporation into protein (poly C) but found that no other polynucleotide tested can replace poly U in activating phenylalanine. A solution of poly U and poly A (which forms triple-stranded helices) had no activity at all. Thus, single-stranded- ness is a necessary requirement for activity, the NIH chemists say. Omitting a mixture of 19 other amino acids does not inhibit phenyl- alanine incorporation, showing that polyuridylic acid stimulates the incor- poration of phenylalanine alone, Dr. Nirenberg and Dr. Matthaei say. Poly U, they find, has little effect in stimulating the incorporation of 17 other radioactive amino acids. Because the addition of sRNA can’t replace template RNA in their system, the NIH team says that tem- plate RNA (in the cell ribosomes) is a requirement for cell-free amino acid incorporation. Dr. Nirenberg and Dr. Matthaei’s work shows that poly U contains the information for making polyphenylal- anine. One or more uridylic acid residues therefore appear to be the code for phenylalanine. Poly U, it seems, functions as a synthetic tem- plate or messenger RNA. And _ this stable, cell-free E. coli system may well synthesize any protein correspond- ing to meaningful information con- tained in added RNA, they add.   CELL-FREE. Dr. Marshall Nirenberg removes sample of cell- free extract which incorporates amino acids into protein Another team of chemists is work- ing with E. coli systems. Peter Lengyel, Dr. Joseph F. Speyer, and Dr. Severo Ochoa of the New York University school of medicine also find that various synthetic ribonucleic acids are active as messengers in an E. coli system and determine in- corporation of different amino acids into an acid-insoluble product. The NYU team studied the incor- poration of different C14-labeled amino acids into an acid-insoluble product by the E. coli supernatant plus ribo- somes system, with and without the addition of poly U. Out of 19 amino acids tested individually, only the in- corporation of phenylalanine is strongly stimulated by poly U. This agrees with the NIH team’s findings. They also find that poly U exerts a small stimulation on the incorporation of leucine and isoleucine. Adding E. coli transfer RNA causes a further pro- nounced increase of phenylalanine in- corporation. This indicates to them that the polymers affect the transfer of activated amino acid residues from transfer RNA to ribosomes and act as messenger or template RNA in this system. In one experiment they found that with poly U and phenylalanine, without addition of transfer RNA, rat liver ribosomes can be substituted for their E. coli counterparts. JAN. 15, 1962 C&EN 45 Other Polynucleotides. The NYU group also studied the effects of other homopolymers on amino acid incorpo- ration. Poly A, they find, does not stimulate incorporation of any of 19 amino acids tested. Addition of poly A to a system containing poly U com- pletely inhibits poly U’s effect. This, they say, is undoubtedly due to forma- tion of the double-stranded, helical poly A+ U complex. Polythiouridylic acid (which appears to be multi- stranded) has no effect, and poly- fluorouridylic acid, which like poly U is single-stranded, had only a small effect on phenylalanine incorporation. Polycytidylic acid (poly C) has a small but consistent effect on the incorporation of proline but has no influence on that of any other amino acid. Poly UC (U:C = 5:1), they find, promotes the iacorporation of phenylal- anine and serine, and poly UA (U:A = 5:1) stimulates the incorporation of phenylalanine and tyrosine. Year-End Goal. The genetic code for proteins may be deciphered before the end of 1962. This is a prediction of four scientists working at the Cav- endish Laboratory of the Medical Re- search Council Unit for Molecular Bi- ology, Cambridge, England. They base it.on the results of experimental work indicating the type of code that is involved in the sequence of bases along the nucleic acid of genetic ma- terial. The scientists are Dr. F. H. C. Crick, Leslie Barnett, Dr. S. Brenner, and Dr. R. J. Watts-Tobin. Here’s how they typify, in general terms, the genetic code: * A group of three of the four bases (or less probably, a multiple of the three) codes one amino acid. ® The code-is nonoverlapping. ®A fixed starting point is used to read the sequence of bases. This, the scientists say, determines how the long sequences of bases are to be cor- rectly read off as triplets. There are no special “commas” (every fourth base) to show how to select the right triplets. Displacing the starting point by one base displaces the reading of triplets, and becomes incorrect. ®The code is probably “degener- ate,” or one amino acid can be coded by. one of several triplets of bases. Nonoverlapping Code. Pointing to the existing evidence that the genetic code is not overlapping, the Cam- bridge scientists contend that their 46 C&EN JAN. 15, 1962 experimental results rule out all over- lapping simple codes. But if there is no overlapping, how can the correct triplets along the con- tinuous sequence of bases be selected? Of the possible answers, Dr. Crick and his group favor this possibility: The correct choice may be made by start- ing at a fixed point and working along the sequence of bases three (or four, or whatever) at a time. The genetic experiments were done on the bacteriophage T4, which attacks strains of E, coli. A region of this bacteriophage consists of two adjacent genes, gene A and gene B. The mutant (designated FC O) in a seg- ment of the B gene produced by the action of proflavin was used. Assuming that the B gene pro- duces a polypeptide chain (probably through an RNA intermediate), Dr. Crick and his co-workers imagined that the string of nucleotide bases be read, triplet by triplet, from a_ starting point left of the B gene. They then supposed that the mutant FC O be produced by inserting an addition of a base in the sequence. This addition of a base at the FC O site will mean, the Cambridge work- ers say, that the reading of all triplets to the right of the FC O will be shifted along one base, and will be incorrect. So the amino acid se- quence of the protein which the B gene is assumed to produce will be completely altered from that point. This. is supported by experimental evidence where a gene undergoing such a mutation lacks any function. Dr. Crick and his colleagues postu- late that a suppressor of FC O (say FC 1) is formed by deleting a base. So when the FC 1 mutation alone is present, all triplets right of FC 1 will be read incorrectly, and so the function of the gene will again be absent. But when the two mutations are present in the same piece of DNA, as in the double mutant FC(O + 1), then although the arrangement of triplets between FC O and FC 1 will be altered, the first reading will be restored to the rest of the gene. Dr. Crick and his group claim to have convincing evidence that the coding ratio is three or a multiple of three. They constructed triple mutants having two forms, involving either three additions or three deletions. Taking a triple set of mutants, all of the addition type, they found that either mutating singly or in pairs re- sulted in an absence of function in the gene. But when the three mutants were combined in the same gene, the function of the gene protein was restored or partly restored. This, the Cambridge workers claim, is what would be expected if the coding ratio were three or a multiple of three. Ability to find the coding ratio, Dr. Crick and his group say, thus de- pends on the fact that at least one amino acid must have been added to or deleted from the polypeptide chain without greatly disturbing the function of the gene product. Degenerate Code? Assuming a triplet code, then there are 64 (4 X 4 x 4) possible triplets. Dr. Crick’s group found results which suggest that it is unlikely that only 20 of these represent the 20 amino acids, and the other 44 make no sense. If this were so, they say, the region of the gene over which suppressors of the FC O family of mutants occur (about a quarter of the B gene) would be smaller than was observed. But it depends on the size of the pro- tein which Dr. Crick and his colleagues assumed the B gene to produce. But, they say, the length of the B gene suggests that the protein may have about 200 amino acids; so the code is probably “degenerate,” or gen- erally more than one triplet code for each amino acid. It’s well known, the British scientists say, that if this were so, it would also account for the major dilemma of the coding prob- lem—namely, that while the base com- position of the DNA can differ in dif- ferent microorganisms, the amino acid composition of their proteins changes by only a moderate amount. How- ever, exactly how many triplets code amino acids and how many have other functions isn’t known. “Dr. Nirenberg and Dr. Matthaei’s work at NIH,” say Dr. Crick and his co-workers, “implies that a sequence of uracils codes for phenylalanine, and our work suggests that it is probably a triplet of uracils.” Dr. Crick and his group recog- nize that it is possible by various de- vices to synthesize polyribonucleo- tides with defined or partly defined sequences. If these will produce specific polypeptides, then they ac- knowledge that the coding problem is wide open for experimental attack. “If the coding problem is indeed three, as our results suggest,” they say, “and if the code is the same throughout nature, then the genetic code may well be solved within a year.”", "American Chemical Society", null, "Chemical and Engineering News", "American Chemical Society", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ierz.eawe~b39n", "00000000-0000-0000-1D52-DA593E40CE32", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "No Life in Test Tube Expected by Russians", "101584910X454", null, "1964", "30 July 1964", "This wire service article reports on Russian scientists who maintain the \"creation of life and modification of heredity in a test tube will not be possible for many years, if ever.\"  Nirenberg's collaboration with Russian scientists is mentioned and his early work in genetics is summarized. The reporter concludes that because a given nucleotide triplet always specifies the same amino acid in all species, \"it appears that nature has evolved from one ancestor.\"", "Articles", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "3", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "SCIENCE SERVICE @ WASHINGTON, D. C. 20036 ® OBSERVE FUTURE RELEASES ® HANDLE LIKE WIRE COPY © USE \"BY SCIENCE SERVICE\" @ NO LIFE IN Tu ST TUBE 7/30/ 64W EXPEOTED BY RUSSTANS By FAYE MARLEY Science Service Medical Writer NEW YORK, -- Creation of life and modification of heredity in a test tube will not be possible for many years, if ever, a Russian scientist told Science Service here. Dre Lev Kisselev of Moscow, who at the 1961 International Congress of Biochemistry translated the code-cracking speech of Ameri- cats\\\\ pre Marghall W. Nirenberg | said that he had been working with mae A A Seer me CS er Ley. rm Tm a eee other Russian scientists in Leningrad and Moscow on problems of   correlating function and structure of RNA, or ribonucleic acid. ‘Many results lie ahead when this work 1s done,\" he, said, but he only Laughed at the idea of higher life in a test tubee Dre Edgar Lederer, director of the new French institute of natural substencess15 miles south of Paris, said he hoped human life would not be created in a test tube, The creation of tobacco mosaic virus from inert chemicals led to an. announcement two years ago that life had been created in a test tube. But creating Life in a tiny infective virus is a far cry from creating human life as we know it, Progress is being mede in deciphering the genetic code, how- ever, end the results Dr. FKisselev foresees when all the tedious work of correlation in function and structure is finished coulda well ine clude correction of hereditary discases —- even a cure for some forms of cancer, ‘ Dr. Nirenberg, with Dr. J. Heinrich Matthei at thelMational Lami anne meena Institutes of Health, Bethesda, Md., partially unraveled the mystery ee, eA A Pe Een °   oS Ae a 2 + ence of the genetic code, They are, however, the first to agree that years cf work remain to be done before all the necessary sequences can be determineds Dre Nirenberg's work has shown that in all species, a given nucleotide triplet always specifies the same amino acid. Thus it appears that all nature has evolved from one ancestor ~~ DNA, or deoxyribonucleic acid, with RNA, or ribonucleic acid, carrying out its eod-Like bidding. MORE @ pe EG ue he 4 @ SCIENCE SERVICE ® WASHINGTON, D. C. 20036 © OBSERVE FUTURE RELEASES ® HANDLE LIKE WIRE COPY ® USE “BY SCIENCE SERV! NO LIFE -- CONTINUED SHED 2 74 °30/ GL Both Dr, Nirenberg and Dr. Severo Ochoe, president of the wnational Union of Biochemistry, spoke (Friday) at a, symposium rd ny ot wo 5 of the Sixth International Congress cf Biochemistry here, Dro Je De Watson of Harvard University, Cambridge, Mass., who shared the 1962 Nobel. Prize in Medicine and Physiology with Dr. Francis H. C. Crick of Cambridge University, England, was another speaker at this meeting. Dr. Ochoa shared the 1959 Novel Prize in Medicine end Physiology for his studies in the biosynthesis of RNA, Dr. Nirenberg!s early experiments were made possible by the use of mixed polymers of RNA that could be synthesized by the bacterial RNA polymerase, or enzyme, previously isotated by Dre Ochoae After intensive work, esnecially in the laboratories of Drs. Ochoa and Nirenberg, it 1s now possible to assign triplet RNA base codes to.all the amino acids. The corresponding DNA triplet code should then be the complement of the RNA code. Tests of this code in bacterial, plant ead animal systems have indicated that the Escherichia coli,code is universal, a fact of considerable evolutionary significance. The actuel definition of which triplet snecifies which amino acid is pursued at present by Drs. Ochoa, Nirenberg end others, principally by constructing messenger RNA molecules of known sequence to see which amino acid they snecify. ; By making and testing a large variety of messenger RNAs, a genetic code is being mapped oute In another eoproach, certain nucleotides in the master molecule, be 1% DNA or virus RNA, are exchanged, and the effect of this exchange is reflected in an altered pattern of amino acid incorvoration. The Indian—born Dr. H. Gobind Khorana, researching at the University of Wisconsin, Madison, has synthesized small DNA. models after ten years of work. From 10 to 20 years lie. ahead, Dr. Khorana feels before the full sequence can be established. Improvement of human heredity may be at the tip of the scientist's fingers, but the fingers must reach far into the future. Another Russian scientist interviewed py Science Service was Dre Ge Fe Géuse of the Academy of Medical Sciences in Moscow, who reported his work with Laboratcry animals using some antibiotics Shas fight cancer. e SCIENCE SERVICE ® WASHINGTON, D. C. 20036 ® OBSERVE FUTURE RELEASES ® HANDLE LIKE WIRE COPY ®@ USE \"BY SCIENCE SERVICE\" e NO LIFR ~~ COMTPINUED SHEET 3. 7/30/64W Working with Dr. Ae V. Laiko «t the Institute of New Antibiotics, Dre Gause found that they inhibit the synthesis of nucieic acids, Mitomycin C, porfircmycin and degranol, a representa tive of the chlcorethylamines, were the entibiotics used, These anti- biotics inhibited the synthesis of DNA in the cells of normal and ?mnu- tant staphylo-wuccus organisms, Staph is a cause of boils and other pus-—forming infections. Progress in basic knowledge reported at this congress gives hope for mankind's fight against disease as well as for understanding the puzzle of lifes In the words of Dre Ochoa, the human intellect will eventually solve the puzzle of the nature of life. , \"But will it ever solve the riddle of the meaning of life, of the existence of the universe, or even of its prerequisite, matter, and of the essence of the intellect itself?\" the MNobelist questioned. Dro Ochoa said the exploration of space might provide clues for further inquiry into the origin of life. Re IG Te Ee FUTURE RELEASE: Saturday morning, Aug. 1 7/30/64 CUE TOOTHPASTE CETS DENTISTS! APPROVAL By SCIENCE SERVICE CHICAGO, —- A second fluoride-gontaining toothpaste has American Dental Associntion { ADA) approval, Cue, made by the Colgate~Paimolive Company, joins Crest, Procter and Gamble Company product, as en \"effective decey~preventive dentifrice.\" Both Cue and Crest contain stannous fluoride, but the ADA says from the standpoint of cost, effectiveness and,convenience, water fluoridation is the best way to prevent tooth decay, Another method is by topical application of fluoride on the teetn by a dentist. Sia be Se SE ele deeds Sele Se Sh So sett eg St", "Marley, Faye ; Science Service", null, "Science News-Letter", "Science Service", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8pfc.ap4a-yad7", "00000000-0000-0000-88A1-D53B921468A8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Maurice Niehuss, University of Michigan to Marshall W. Nirenberg", "101584910X455", null, "1961", "19 May 1961", "Niehuss, vice-president and dean of faculties at the University of Michigan, informs Nirenberg that he has been appointed Assistant Professor of Human Genetics at the Medical School from January to June 1962.", "Letters (correspondence)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Reproduced with permission of the University of Michigan Medical School.", "Copyright may apply", null, null, "May 19, 1961 To Marshall W. Nirenberg, Ph.D. You are hereby notified of your appointment as Assistant Professor of Human Genetics, Medical School Term of Appointment: January 1 to June 30, 1962 University Year 19 -- 19 First Semester 19 -- 19 Second Semester 19 -- 19 With compensation at the rate of $12,000 a year, twelve-month basis Please return the employee oath at once to the Academic Appointments Office so that your employment may be certified to the Payroll Department. Maurice Niehuss Vice President and Dean of Faculties Note If You Do Not Accept this appointment, please notify the dean or department head under whom you planned to work. Upon Acceptance of this appointment, please note your privileges and obligations under the University procedures applying to the following: Any cards or forms enclosed must be filled out and returned immediately to the Academic Appointments Office, 4601 Administration Bldg.  Please note that the Employee Affidavit card must be notarized. The University provides for old age retirement allowances under two separate plans:  TIAA for those of the teaching staff (Professors, Instructors and Lecturers) and ERP for non-teaching academic appointees. All those of Assistant Professor rank or above should apply at once, as participation is a condition of employment.  In the case of full-time Instructors and Lecturers, participation is optional during the first three years of service, and mandatory thereafter. On the basis of this appointment you are eligible for the plan designated below: Teachers Insurance and Annuity Association Plan x Employee Retirement Plan Neither Plan Information concerning retirement plans, group life insurance and medical insurance may be secured from the Office of Staff Benefits, 3057 Administration Bldg.", "University of Michigan ; Niehuss, Maurice", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9viv~cf5s~v6uv", "00000000-0000-0000-6196-C6155B583923", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Notes on possible codons", "101584910X456", null, "1962", "[ca. 1962]", null, "Notes", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "5", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "© x sgt fF 8 CW he Mah Gn “gy ‘ xD { \\\\-   a | Gad, Bu ee m & Wage DNA ar “26 AA [A Bg Aud weu Ecc = & KAS Aee e MAMA AxBA ukAWA CECE GAY @ @ aD AA AG CC 66 c uA 6 uu RA CF cc C AY 6 Rw 6c 6 @ c\\\\u Uy UIC che @ ha z RIB ka \\\\ chee. Aa a, SB CAbU euch&  6AcyY ey A CHCE Aa CU UC STAG eter cc Ute ewhe™e ac * ! oe : eh Exe SOC ptr Yel CWU66 “6S PES —, ! Ze é Kuc UA6 AUC 4 4@ ——-UC ucts CMe UAL CRE AUC @64@ AN N cc 3 3 RA ce yO      Awe KOS 4 vw UW w C. GA a u ec f, : Mu & we & 6 a . 6 wa . FR are hah \" ape KR AAS 2 aw \" 6a Uk la mewic cA mA 3 ™ oom ve GU *2 1 oA Sf ee, ba OA ea ME Ba p KE AC ceAy ye pu amawic ASE — 2y ucé 2 ; ity ceih BE of W uth A a y | Q « Ua 3G due : cl. o @. - . uu C 6 G bn ~ 2 FAplagg. MRA Shee dig AMM Van Guu ARYA ee CL) , | ~~ AWA Wnpe _ . >» A Ke : UPR he . (2| x o _. « . i 7 a ) w des ish it éJ uf AG’ ay 4% Th = 1 AK wee a 4s {¢ poe ? : ; wee | AAG 2s Apa AUG ACE | wt © UA 4 fc ann varies ucc \\\\ ° by MH fe BR. ph” uec — WE nates us | O AUC = 3a AN 3 ki AUG . AW Cre Guc By C6 <6 AR . as frp : yYce = BAA GP UAY \\\\\\\\ Lubec » a)     ce cp 3h sae 3¢C Puy C64 —", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-29ws~6826.9ie2", "00000000-0000-0000-E9A1-249EDE6E4961", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Anne Messer to Marshall W. Nirenberg", "101584910X457", null, "1962", "22 October 1962", "Messer, a high school junior from New Jersey, asks Nirenberg about discrepancies she has seen in articles about the genetic code.  She requests more information about the theory of triplet combination and suggestions for work that can be done in the field of DNA research without specialized facilities.", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of Anne Messer.", "Copyright may apply", null, null, "Dear Dr. Nirenberg, I am a high-school junior, very much interested in science, particularly DNA. Through all of the magazine and newspaper articles I could find, I have seen what tremendous progress has been made in this field. However, I have found many discrepancies in the articles, especially on the matter of the doublet vs the triplet combinations of adenine, thymine, guanine, and cytosine. A recent article of The New York Times said that you had done extensive research on the theory of the triplet combinations, but did not go into any detail. I was wondering if you could send me more complete information on this. Also, could you please suggest some kinds of work which can be done in the field of DNA without the benefit of specialized research facilities. I would greatly appreciate any help you could give. Thank you very much. Yours truly, Anne Messer P.S. My aunt, Toni Nirenberg Messer, gave me your address, and I hope that you do not mind my writing to you for this information. Thank you again.", "Messer, Anne", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-zc4f_f2ai-3qeq", "00000000-0000-0000-3CBC-7E94BD40DB35", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Summary of Dr. Marshall W. Nirenberg's Work", "101584910X458", null, "1965", "[ca. 1965]", "This three page summary of Nirenberg's work, dating form the mid-1960s, begins with a discussion of his studies at NIH in 1959 and concludes with the statement that he has deciphered the genetic code.  Major research findings and their significance are included.", "Biographies (documents), Drafts (documents)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "The thousands of different proteins which are found in nature are composed of only 20 or so subunits, called amino acids, the exact characteristics of each protein molecule being determined by the number, kinds and arrangement of amino acids within the molecule. The production of proteins is directed by the genes, which are made up largely of deoxyribonucleic acid (DNA). Every living cell, and hence all living things, are what they are primarily because of the proteins they contain.  A specific protein is made at the direction of a code which is contained within the DNA molecule and is determined by the sequential arrangement of subunits within it. DNA does not act directly in protein synthesis but goes through an intermediary, ribonucleic acid (RNA), implanting the code in RNA. The messenger RNA, which is thus formed can be one of a great variety of patterns corresponding to one of an equally great variety of DNA molecules. The subunits of both DNA and RNA, called nucleotides, in turn contain still simpler substances belonging to the chemical classifications of purines and pyrimidines combined with sugar and phosphoric acid. The names of the nucleotides are derived from the four possible purines and pyrimidines, which in the case of RNA are adenine, guanine, uracil and cytosine. It is the composition and arrangement of the nucleotides which dictate the final composition of the protein molecules. In 1959 Marshall Nirenberg began studies at the National Institutes of Health on protein synthesis in cell-free system, and with amazing rapidity reported a series of observations which are now known throughout the world. He started by preparing from the bacterium, E-coli, a cell-free system which synthesized protein in the presence of amino acids and energy sources (ATP). Then he showed for the first time that messenger RNA is required for cell-free protein synthesis. Synthetic RNA and natural RNA prepared from a virus, greatly stimulated cell-free protein synthesis. Dr. Nirenberg discovered a synthetic RNA containing only a single pyrimidine, uracil, serving as a template for the cell-free synthesis of the protein containing only one amino acid, phenylalanine. This single experiment may be said to have \"cracked the genetic code.\" Further experiments with other synthetic RNAs containing various proportions of purines and pyrimidines revealed codes for essentially all the known amino acids normally present in protein. Although the base composition of RND codons and many properties of the genetic code were clarified with the use of synthetic polynucleotides, the sequence of the bases within each codon remained unknown. More recently, a general method of great simplicity was found by Nirenberg and co-workers for determining the base sequence of codons. With this it demonstrated directly that since tri- but not dinucleotides serve as templates in this system, a triplet code exists. The sequences of virtually all codons have now been determined by Dr. Nirenberg's laboratory. It has been shown that the patterns of synonym codons differ markedly in both template activity and specificity. The biological consequences of these and other findings remain to be assessed. It would seem logical, however, that some synonym codons must play special roles in protein synthesis, such as specifying the beginning or end of the genetic message; others may be necessary for the synthesis of certain proteins or may selectively influence the rate of protein synthesis. Studies are now in progress in Dr. Nirenberg's laboratory to define possible consequences of selective modification of components required for codon recognition. Particular attention is being focused upon mechanisms which may selectively control the rate of protein synthesis during viral infection and embryonic differentiation. Dr. Nirenberg has deciphered the genetic code. His work has given us understanding of much of the mysterious way information is coded into the nucleic acids and used to direct the incorporation of specific amino acids into proteins. It represents a major contribution toward understanding on a molecular basis how the chemicals of the cell nucleus carry the hereditary message from one generation to the next.", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-88c8_w4er_c2kx", "00000000-0000-0000-986D-E7736595F4AA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Lewis J. Brown to Marshall W. Nirenberg", "101584910X459", "101584910X460", "1967", "9 February 1967", "Brown, Nirenberg's former roommate, congratulates Nirenberg on the receipt of the 1966 Research Corporation Award and offers belated congratulations for the President's Award of Merit.  Personal details and an offer for Nirenberg to visit the Barrow Neurological Institute in Phoenix are included.", "Letters (correspondence)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "2", "pages", "Text", "English", "Reproduced with permission of Lewis J. Brown.", "Copyright may apply", null, null, "2-9-67 Dear Marshall -- Just a brief note of congratulations from an old roommate on receipt of the 1966 Research Corporation Award. (Including the cash award that accompanies this honor) -- there was a brief squib in the AMA journal but my brother (Monie [?]) sent me a full page from one of his journals about same. Also, a belated congrats on the Presiden'ts [sic] Award which I had not heard about and may be more of an honor than your [sic] more recent acquisition. You have done wonderfully well since we last visited with you in Chicago and no doubt will continue in the same manner in the future. I have no doubt you will be making that trip to Stockholm soon to meet the King of Sweden. My folks were in D.C. last fall and talked with you via phone so I'm told. They are getting along nicely although both have had their share of illnesses the past few years. I'm actively engaged in practice with the other men listed above. We have two children (1 female -- 1 male) and are expecting a third this summer. I was supposed to enter the Army (and Vietnam) in February but they have delayed the call now until the summer -- perhaps (hopefully) this dirty conflict will be over by then. [END PAGE ONE] [BEGIN PAGE TWO] I'm a staff member of the Barrow Neurological Institute in Phoenix -- devoted to clinical care, clinical and basic research in nervous system illnesses. We have several biochemists on our staff who would be interested in hearing your work. Might give you an opportunity to come to this part pf the country for a vacation away from the snow and cold of the East -- plus providing a tax deduction. Would like to see you again soon and meet your spouse. In any event, give it a little thought. Again, the true [?] purpose of this communication is to convey our warmest good wishes and congratulations on your recent award(s) and for continued good success in the future. Lew", "Brown, Lewis J.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3idw~69he~p63u", "00000000-0000-0000-7BD5-CC712756E558", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Lewis J. Brown to Marshall W. Nirenberg [transcript]", "101584910X460", "101584910X459", "1967", "[9 February 1967]", "Brown, Nirenberg's former roommate, congratulates Nirenberg on the receipt of the 1966 Research Corporation Award and offers belated congratulations for the President's Award of Merit.  Personal details and an offer for Nirenberg to visit the Barrow Neurological Institute in Phoenix are included.", "Letters (correspondence), Transcripts", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Reproduced with permission of Lewis J. Brown.", "Copyright may apply", null, null, "Dear Marshall: Just a brief note of congratulations from an old roommate on receipt of the of the 1966 Research Corporation Award.  (including the cash award that accompanies this award)-  There was a brief squint in the AMA Journal but my brother (Marnie) sent me a full page from one of his journals about same.  Also, a belated congrats on the President's Award which I had not heard about and may be more of an honor than your most recent acquisition. You have done wonderfully well since we last visited with you in Chicago and no doubt will continue in the same manner in the future.  I have no doubt that you will be making that trip to Stockholm soon to meet the King of Sweden. My folks were in D.C. last fall and talked with you via phone so I'm told.  They are getting along nicely although both have had their share of illnesses the past few years. I'm actively engaged in practice with two other men listed above. We have two children (1 male - 1 female) and are expecting a third this summer. I was supposed to enter the Army (and Vietnam) in February but they have delayed the call now until the summer - perhaps (hopefully) this dirty conflict will be over by then. I'm a staff member of the Barrow Neurological Institute in Phoenix -- devoted to clinical care, clinical and basic research in nervous system illnesses. We have several biochemists on our staff who would be interested in hearing your work.  Might give you an opportunity to come to this part of the country for a vacation away from the snow and cold of the East -- plus providing a tax deduction.  Would like to see you again soon and meet your spouse. In any event, give it a little thought. Again, the basic purpose of this communication is to convey our warmest good wishes and congratulations on your recent award(s) and for continued good success in the future. Lew", "Brown, Lewis J.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qaaj~nfki.qssu", "00000000-0000-0000-C6C9-298622EC293B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from G. Montalenti to Marshall W. Nirenberg", "101584910X461", null, "1967", "23 October 1967", "Montalenti informs Nirenberg that he would like, as a foreign member of the Royal Academy of Sciences of Sweden, in collaboration with A. Monroy and A. Liguori, to put forward Nirenberg's name for the Nobel Prize in Chemistry.  Montalenti requests Nirenberg's approval and related documents so that he may elaborate his proposal to the Academy.", "Letters (correspondence)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Rome, 23rd October 1967 Dear Dr.Niremberg, Although I have not had the pleasure of meeting you, may I be allowed to apply directly to you for the following reason. I am a foreign member of the Royal Academy of Sciences of Sweden, and in such a capacity I am entitled to submit to the Academy proposals for the award of the Nobel Prize for Physics and for Chemistry. In discussing the matter with some friends, in particular A. Monroy and A. Liguori, we decided that I should put forward your name for Chemistry. Which I will do with great pleasure. May I ask you if you have no objection against my proposal, to send me the necessary documents, i.e. a curriculum vitae of yours and a brief illustration of your work, with bibliographical references. I will then elaborate my proposal and send it to the Academy. Dead line for nomination to the Academy, Jan. 31st 1968. With kindest personal regards, Yours sincerely, Prof. G. Montalenti", "Montalenti, G.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j83r_at5v_n43u", "00000000-0000-0000-25E5-FA1EBA364686", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Genetic Code", "101584910X462", null, "1967", "15 September 1967", "This manuscript, edited on February 9, 1968, includes a summary of work on the genetic code, how it was deciphered, and the apparent logic of its design.", "Articles, Drafts (documents)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "19", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "THE GENETIC CODE by M. W. Nirenberg National Heart Institute National Institutes of Health Bethesda, Maryland Iv — ta ee at DRAFT OF THE MANUSCRIPT 9/15/67 INTRODUCTION I. IN . _ ve years studies on the genetic code, protein synthesis, and regulation ‘et protein synthesis have expanded to such proportions that     \\\\ . \\\\. cee. we Co investigators in other fretds-and-graauace™ Students often find it difficult ? * 4 f ro : tt. Cees lp rin eZ ua aS yyage og 4, wap Sam. aan hy 5 ey on ae Por, , to assimilate uitell facts ta stent them. fos og! r Kae a a ps \"Awe objectives have been tjovfrest rk 7 : o., ¢ 1 e      In writing this chapter tn ,. oe oe 2 a Lg “re a PF subeichomereciised ES decipher Nhe codes Secor the nature of the code Ane the ee ANE, Lila Zen evitencefer-it; “aid third, iin Logic yet them codes “designs An Yies apparent , attempt is made to formulate generRy principals and the/logical design of ‘, the code. iscussion of the code, how it was This chapter is meant to be a ‘ 2 In writing the chapter an attempt has translated and the experimental data. \\\\ fe; 2% als from the data and to state the been made to formulate general pn cif Coverage of topics apparent logic which underlies the design of the cod \\\\ ways, the chapter has been selective rather than comprehensive, amr in:so more of is/an essay than a review. Qs, The data which d#-now available on the code, the structure and function of nucleic acids and protein, and the process of protein synthesis -2- tw Wo ec Cue we cf is so extensive that Hewas~deemed essential from the outset to be selective \\\\ rather than conprehensivelhcdiseussing thie datas-— ~ . - ¢ Loe et a 7 : a wer ’ An attempt has been wade in this chapter to survey the Tengen code, Gh ‘¢ ‘Cf    oe to concentrate on fundamental A code-XxxampikExee andthe ‘logic-which-isemployed. . Coverage is selective rather than comprehensive. wf we B. Base Compositions of Codons Synthetic RNA preparations containing all possible base com- a binations have been used as templates for we. synthesis in vitro. In practice, the major cactor’ wetchoake the sensitivity of the assay is the presengdé of endogenous mRNA in E. coli extracts. As shown in Fig. > the level of endogenous mRNA is greatly reduced by incubacta’ of E. coli extracts in the presence of DNase and all components required for protein synthesis until amino acid incorporation ceases, Bxtrects then — tatized. Perey Protein synthesis then is almost completely dependent upon the addition of mRNA. Optimal conditions for in vitro protein synthesis stimulated by synthetic mRNA were determined ( ), and methods were devised for rapidly washing radioactive protein precipitates on cellulose nitrate ent filters ( -). Most radioactive, in products are washed with 10% tri- chloroacetic acidgy Fhose rich in proline are washed with 20% trichloroacetic acid ( dA, whereas lysine~rich peeseins are washed with a solution containing $s , sc im tungytate and trichloroacetic acid ( ). oF The specificity of ¢ randomly-ordered me meneeef i amino - Tide clare acid incorporation into§ ‘has been studied extensively with - Jaen consponlinng perder K      ! OAC) ere a) ee Folynucleotides whengone kind of base usvebiyts-a reuplates for he   amino acid» ; Little template activity wan detected with poly G; pre- sumably G-G interactions inhibit the template activity of RNA (discussed in a later section). A polynucleotide with two kinds of bases contains eight triplets; six criplets with two kinds of bases, and two triplets with onedfinis “pF al f Bager For example Poly Ui UC contains: ae wees uUC vt uuu\" \\\\ UCU ccc anne cuuU ccU cuc UCC gach Lem ppg oy Three preparations, poly Ue, poly CG, and poly AG, amenaciiwe tempiates i Caley '   ey for four amino acidsiy ree ‘other polynucleotide preparations ( poly UA, Keach ned poly UG, and poly c)servosreuptate for six amino acid syuakiay Tie os cola entp There~are four,polynucleotides pmen three kinds of basesganyg i \\\\ \\\\ poly UAG, poly UCG, poly UCA, and poly CAG. A polynucleotide with three =5- [BE os aa | kinds of bases¢ for-examph =2, poly UAG, resembles a mixture of seven eee NAE AUN Fim oe - oa Soe, wh poner,   potymucecesdf as follows: poly U, poly ¥ poly G, poly UA, poly UG, Rowden albetece: poly AG, poly UAG. X VAG contains tripletss wien one kind of base, eighteen ¢eiplets with two kinds of bases, and six ertplets with three kinds of bases,~e-tetel—ci—twenty—seven—tripteta, 2 Each ype: 7 pase polynucleotide stimulates the incorporation lat BF . _akhe.t ae wer teen §. Lee oe ‘into Proceso of. ,ben opemcne, amino acid Sh Four #2nocebeo 5 codons{ were ef — . efi wrt | f } . Cte ee tle o. Cyn ory. Nemes pe of x wf found which n could not be accounted for Oe ESS: Pe , eS Ane Bona! a Ata Se a) 7 ANC AE Be Pe ND Fo PY vag} WASmndents mphate—fox hh and, ,aspartic acid,   \\\\ a   Me y whe + sentence af pe            andepol ‘CAG / ’ 3 ©» . - ¢ was a template for serine wit ae artic acid, ___ adgitional three? base P P ‘ Bcd. ee - codons were assigned to ONE acid Se root onLeg: “lydenansisate~ . i me the .minimum kinds of bases which q P meyrbe present in: ‘codons for each amino acid, Jomcaases ones phe-resiilts. i.           snot Shes-che- “code i's” degenerate~because~-amino..acids.. -such~ ‘as--Leucine, rou A aca cet “arginine -and -serine-respond..to..several.. -polynucleatides which differ _in Wargame base-content:: do daes, With additional data it is possible to Yexivd the samt f peer riens vases Caney codongf as well as the kinds of bases whietr— are present. Base compositions of RNA codons are derived as follows: y “Ss je base composition of a polynucleotide ceanehe determined   i A fre expected frequency of each doublet or triplet is calculated easily once the base ratio of a randomly-ordered polynucleotide is known, By _ & synthesizing a series of polynucle¥eides, each containing identical bases, A ‘ . but with different proportions of-beses, and,’ etermining the relative poly poplide maleuol proportions of amino acids directed intoypwetetn by each polynucleotide,   base compositionsof the codon, as well as the number Larsalid be of nucleotides per codon can—be estimated. ch. here ot Coren. SOAAo eos leis dtfficuit to-compare-directly=the/ template efficiency of ntf wont, ryeiew- the efficiency of each diffe rast s- eg polynacleotide preparation    preparation may be influenced strikingly by factors other than base composition, such as, the conformation of the RNA in solution, the presence pon Aof terminal phosphate, its motecular—weighty the number of base residues _per molecule and so forth. | These factors will be discussed in later ' 7 GREP Per hr net ons, 4 sections. However vn amount of each amino acid incorporated into protein due to the addition of a,polynucleotide preparation canbe determined Unrsardasre led the Yelarive ProperTicNs oF warns Aerts epeebeet/ rR tendip athe rn teh Lonty Anand of different polynucleotide preparations can be compared. fH Table ia show#an example of data obtained with a poly AC preparation; similar data were obtained with four other poly AC Nea my, Pl pet i ye é af i : . preparations, each different.in base ratio ( ). The four possible doublet permutations do not contain enough specific information to code for the six amino acids directed into protein by poly AGA whereas, the infor- mation content of the eight possible triplets is adequate, If every -plet were read, some amino acids would respond to two or more codons. In such cases the sum of the triplet frequencies would then be compared with the corresponding amino acid incorporation data. For example, if (ACA) and (ACC) both corresponded to the same amino acid, the sum of their frequencies would be 24.9 percent, which could not be distinguished from the frequency of the doublet, (CA), which is also 24.9 percent. the relation between theoretical frequency and the experimentally determined frequency of amino acid incorporation into protein is shown Cheers, in Fig. . The.~data demonstrate that} istidine, asparagine and A Lai ree ion ; sr Ag ¢. . on f x LS iget  1. pee AP oO . a é 4 OES oe oh f “MM > Oa Fe ee Eh a ae glutamine’ composition of a histidine codon is (CAC)A an asparagine codon, 23 (AAC) { and a glutamine codon, (CAA). triplets, one of base composition (ACA)¥the other (ACC), or to the doublet (AC). Threonine responds either to two As shown in Fig. __s proline responds to two triplets, CCC and (CCA), or to the CC doublet, and tip lysine responds to the triplet AAA, this ' ' as follows: Proline Histidine Threonine Glutamine Asparagine Lysine AAA in poly AC was assigned to an amino acid ssay every triplet base compuEtesen In tzis way the nucleotide compositions of approximately 30 ccdons were F n shown in Table . Tentative base compositions were estimated for many codons containing three different bases. Most amino acids were found to se (Taplola) coded by muitiple words » Since synonym codons often differ by only one bese, a ie . . . Bs estimated by Ochoa ( ), Nirenberg ( ) and their coworkers. A summaryjis 7 % =”   a ~ ; S “ the bases which were common to each synonym codon were assumed to occupy une same position within eacn triplet. Similar results were obtained in both laboratories although extracts > in were prepared from E. coli B in the Ochoa laboratory and from E. coli ~asd W3100 ~24- (a K12 strain) in the NIH laboratory. C. Nucleotide Sequenceft of Codons ui t= uusfect-of- Trinucieotides—upon-\\\\AutRNAGBinding-te-Ribosomes, Each of the 64 trinucleotides have been synthesized, and assayed t oe i template’ fee binding of E. coli AA-tRNA. Since the initial studies showed that AA-tRNA for some amino acids binds to ribosomes in response to trinucleotides at 0,.02-0.03 M Mgt, but not at a PO one, 0.01 M Mgtt, (Nirenberg and Leder, 1964; Leder and Nirenberg, PNAS) ¢ / Snes 7 toe erate gamer ere 7 a relatively high Mgt? concentrationf 0.03 W WRG vas selected fox _/ ~ ere the initial survey of trinucleotide-ribosome~AA-tRNA(\\\\- All responses found at 0.03 M Mgtt then were reassessed at 0.01 and 0.02 M Mgt, Summaries of responses of unfractionated E. coli AA-tRNA are voy : id lB ape ‘ i \\\\ we EN kt ‘Hype thee Tart? Ree f ewe uh ee cS shown in Tables and . , —roe f N . -25 a+ Most trinucleotides have been assayed for template specificity pet ad with 20 AA-CRNA preparations from E. coli, each acylated with one radioactive and 19 unlabeled amino acids ( )}. In surveying Poke etl trinucleotide specificity, unfractionated AA-tRNA Geuatty ts hoy hea used,i itially, becauseaspecies of tRNA compete with one another during the formation of AA-tRNA-codon complexes and the specificity of codon recognition can be altered by changing the concentrations of two or more species of tRNA,   26 - ‘ (ee all triplets wexe-—feund—te correspond to amino oe) Synonym codons weresfoUmi=te-be logically related to one another, and in most cases, sytonym-eedonms differ only in the _— occupying the third position of the triplet. Only four unique patterns of degeneracy were found, each pattern determined by the kinds of bases which occupy the third positions of synonym triplets. Patterns of alternate third bases are: 1) G - b 2 wv-=c Pyted™ | 3) ATG Tee geen, Se OO 4) DFecla Ot EAdAD A £iith pattern, UFC Ss Ae G, te-feund which is not necessarily unique, because this pattern would result if two simpler patterns were present, such as [(U = C) + (A= G)] oc [WU = C = A) + (G)]. Codons specifying the initiation of protein synthesis may contain alternate bases at the first rather than the third position of the codons, For example, N-formyl-Met-tRNA responds to AUG, GUG, and possibly also UUG (discussed under Punctuation). 27% Ke. . aL degewerney 1S that many mfatiows One Cons © ¢yence oF logical LCG 1 Y leading to single base replacements in DNA at sites corresponding to third bases of mRNA codons may not result in amino acid re- ll it placement in protein, Hence, many mutations thus are silent, The code appears to be arranged so that the efZccts of base rex placements in DNA, or erroneous translation of a base in mRNA, of cen is minimized, Possible amino acid replacements in protein which would occur as a result of single base changes can be read in Tabie >y moving horizontally or vertical ly from the amino ac-.d in cuestion, but not diagonally,        b ge her. cha effects cfusom ‘CPPOTS “Nay be nimi S t 8 Tht amino ¥ Eten yecrrespend { teSimdLog, RNA codons’ Ni For example, ssp-eodons, GAU and GAC, are similar to Glu-codons, GAA and GAG; Ser=codons are related to threw codons, and so forth,   ~nlese veSultsi <   Polynucleotides and the cell-free prot ein Synthesizing Systems -(L52~of.the pyelaie ad oer 2 r ¥ pon AR-tRNA binding to. i besemes A the eftects of sixteen po lynucleotide preparations, each with ¢ one hundred different 5'-terminal doublet followed by approximately S@® C residues,   such as CO 1008 + ‘ omined-~ C(Matthaei), The results are shown in Table - Each RNA preparation mor trad day 4 ra aise h stimulated binding of Pro-\" and Ser-tRNA. Ala-tRNA,responded to most of the polynucleotides, &i™. The high- cesponhg of Pro-tRNA to every oe Oly polynucleotide demonstrates a nigh wt nase response to CCC residues, The very. high responsef of Ser-tRNA to polynucleotides a= frexeee- un- expected, Each RNA preparation contains three triplets, depending upon , poly the phase of codon recognition. For examp e, /UUCCC(C) 499 contains the trip.cts UUC, UCC, and CCC according to the reading phase and stimulates binding of Pro- “Ser-t *phe-t and Ala-tRNA to ribosomes. In some cases, bases one to three are recognized preferentially; in other cases, bases two co four are preferred, Therefore, phasing preferences depend con the triplet rather than the exact location of the triplet in the 5'-terminal   regard-te-the-preferred-modee-ef-phas ing-codon..cecOotittony-cinteroretation of -cadon-base—sequonce-oftenis-complicated-by” the-phasing--problem: LA Neverthetess, ffesponses of AA-tRNA to approximatély half of the sixteen codons tested are sufficiently high a that base sequence assignments can . ate o be derived readily, However, Anterpretation of the remaining data is complicated by the phasing problem, Responses of AA-tRNA to polynucleotides . ‘ fos A OF An, Ahraeg org, Ah whee agree well with the responses of AA-tRNA to crinucleotidesaif the assumption is t i i made that almost every postniapiRe in each polynucleotide is recognized to a greater or lesser extent. However, three differences in ressonse of AA-tRNA to polynucleotides and trinucleotides should be noted; Gly-tRNA does not respond to GGC(C)4 99> but does respond to the trinucleotide, GGC; Leu- Cc -tRNA does not respond to PEC) 100 but does respond to randomly-ordezred - Ce G PVP Ae poly. “ altscugh responses to the trinucleotide,/ cuc jf ofeebece difficult Cord fp Ekicclby , tha to cetect with unfractionated Leu-tRNA ( );, high responseg of Ser-tRNA to polynuclectides are not observed with rari piberetmn’. a: apie, KArarsie, and Lio cotleaqucs C) A foiyaucleotides with eatin, doublet, ‘riplet, or tetramer sequences thom were synthesized by Khorana—am-hts-eoiteapiey-t~ ) and were used,to stinu- Jota — — eemmonges late amino acid incorpsoration in E. coli extracts, wA-summery—ef the results, \\\\ _is_snewn-infepte—___, RNA preparations which do not contain an inictiato: i codon, such as AUG,/ or GUG, are translated in almost every possible phase during protein synthesis in E. coli extracts, RNA with a repeating doublet 30 7 eee and eee a ’ sequence contains two triplets in alternating sequence, and therefore is eeeaag® wha peopled 0 | a template for Croteen containing two amino acids in alternating sequence. Most RNA preparations with a repeating triplet sequence are read in three phases,each phase corresponding to a different triplet. For example, poly UUC stimulates the incoxvporation of radioactive phenylalanine, serine and leucine into proteing) Fherefore, poly UUC resembles a mixture of poly UUC, poly UCU, and poly CUUZ if the reading phase were (-UUC*UUC-)_., the protein procuct would be polyphenylalanines; if the/phasd #€ reading were (-UCU*UCU-),, “he product would be polyserine; and if the reading phase were (-CUU-CUU~)_, he expected product would be polyleucine.      £ four triplets and therefore serves as a template for ith repeating tetrapeptide sequences, Polymers which stimulate incorporation of less then Oran the expected number of amino acids contain terminator twhpke™, such as UAL, - ) weG-UAG, or the-beeetemetrigbe UGA (discussed under Punctuation). LO ; Results obtained with polynucleotides containing repeating, sequences directly demonstrate nucleotide sequences of texminator codons, Polynucleo- “ides without initiator codons are translated in almost every possible phase and the preferred mode of phasing apparently depends upon the triplet vathe: al than the exact distance of the tripl@t from the 5'-terminus of the poly- evcteotida. In most cases, base sequences of cuuuus Ca Le dexvivec ouiy if the reading phase of mRNA is known and is correlated with the phase of amino acid incorporation into protein. The number of codons per pmino acid is shown in Table . oix., aml en Gthone Ove mmm eed “> . mala uae Wen,            d sering, ix to, | Cogn OT p appeetregy and om four to one codons jeer each of the remaining amino acids. ! we “A . It should be nected the: only one codon corresponds to tryptophan and one to weston Eherne (a Nefommsf mt coe hie fg . ef nw hen Pow ox x D", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-62it-phps-vshv", "00000000-0000-0000-7AE3-883F619E6485", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Genetics and the Future of Man", "101584910X463", null, "1968", "1968", "Nirenberg considers the possibilities for genetics for the future a \"question of evolution.\"  He discusses the three main areas of genetic manipulation and their implications: molecular supplementation to remedy disease, euthenics to modify gene expression, and eugenics--the attempt to improve the genetic quality of the genes through selectivity.  A broader discussion of the mechanisms of genetics is followed by mention of the moral and ethical implications of research.", "Speeches, Drafts (documents)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "8", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Yrery HE“ lady OC prker, Jo CL, Ae As Dr. Jacobs said I am a biochemist but I have the feeling and I really should state now that a topic such as this - to really do justice to it--one needs a guru of sorts--some wise man, and I am afraid that I can perhaps pose some questions but there are no pat answers to these questions--at least I have none. My only objective today will be to, at least, raise these questions eo that they can be considered and perhaps you may have some answers. -Now, in considering the possibilities for genetics for the future this is really a question of evolution--biological evolution--and there are three broad areas that fall into this tpic.. The first is that a molecular supple- mentation, which is a very familiar area; that is, an individual, for example, has a deficiency disease and by simple supplementation, insulin, for example, for a diabetic, or vitamins, or what-have-you, it's possible to at least remedy this disease. Now, obviously a great deal more remains to be determined about the nature of the deficiencies, or the nature of the various diseases before it will be possible to supplement in any way. Now the second broad area really is the topic of euthenics. This is a term coined by Joshua Letterberg in Stanford, some years ago, Euthenics means to simplify--to modify gene expression, not the genes themselves but the expression of the genes. It is a sort of engineering, human engineering and to alter either the embryonic development of the individual or the develop- ment of the adult. Included in this topic would be organ transplantations as well. The essential thing is that the genes are not modified. It is simply thé individual. The third topic, or obviously for euphantics one must again learn much more about the nature of embryonic differentiation. This is an area that“€xtraordinarily topical and interesting to anyone who has worked in molecular biology today because most of the people in the field have the ae -2- very, very strong feeling that many things can be attempted--that it is possible to do many studies now and that a great deal. of information should be forthcoming related to embryonic development and the mechanisms involved. The third broad area is that of eugenics and this is simply an attempt to improve the genetic quality of the genes by selective breeding and this, of course, has had wide application in agriculture and in certain societies as well--human societies--a voluntary type of eugenics has been applied. Also in this topic all the questions of population, population explosion, and the third aspect of eugenics, I think is a genetic manipulation; that is, to program cells with artificial genes, and it is really this last aspect that I would like to address myself to. It really would be impossible to do justice to the many exciting possibilities that can be envisioned for the future, so I would really Like to focus on the possibility of using synthetic genes to program cells; ;where we stand today, what the problems are, and what the potential is, at least for the future, but before I can do that it is essential really to first point ‘out the basic strategy that the cell employs in order to store the information, how it is stored and how the information is read. Now, may I have the first slide FIRST SLIDE PLEASE: Now on this slide is shown very diagramatically a very simple protein. Each circle represents an amino acid. There are 20 varieties of amino acids and they are linked together in different sequences and essentially the protein is a linear sequence then composed of hundreds building blocks and there are 20 varieties of building blocks. Now the information that is passed on from generation to generation tells the cell how to build this protein. This particular protein is an enxyme and it's really a molecular machine--a beautifully designed machine. The funazxkx function of this particular protein is to aid in digestion. It simply cuts a certain type of foodstuff into very small pieces which can easily be -3- ‘digested and then reused. The cell will reuse the building blocks to build new molecules. Now the secret of each amino acid is the all important thing because although one can make some changes in sequence without drastically. effecting the function of the molecule, other changes-~even removing a single amino acid, or substituting a single kind of amino acid for another variety of amino acid may completely inactivate the function of the protein, NEXT SLIDE PLEASE--Now an essential cell will contain perhaps 3000 or 5,000 kinds of proteins and there may be many molecules of each kind of protein and each one will perform | a different function so the information that specify about how to build this machine is encoded in DNA, and the top line shows DNA in a highly diagramatic fashion, Actually this particular slide came from that great scientific journal Fortune Magazine. It illustrates the point I think diy well, It is a backbone and there are four kinds of letters, &xtxHx@ C A and C G are the initials of each letter. Now it is a linear sequence-- a very long linear sequence. In an average mammalian cell, for ES pt Ne single strand of DNA, a single cell may contain about three bé-kitor-vetty—, ~ v on the strand. That is enough information to specify something like for 3 million kinds of proteins. New-the sequence of the otter EER apnea thing that specifies the ‘sequence of amino acid in protein. ‘First the DNA message is transcribed. It is rewritten in a different form--in the form of RNA, messenger RNA which is indicated diagramatically in the bottom and the important fact here is that there is a complementarity, that a C, the first letter in DNA correspond to G in-RNA; A in DNA corresponds to U, etc., as shown here, and it is the RNA that really is the message that is translated, Nor how is the RNA message read? We will show this on the NEXT SLIDE NEXT SLIDE PLEASE: The cell is filled with gray particles as illustrated here. The message--the long RNA message attaches to one of the particles as shown and the amino acid which is indicated in the upper, right-hand portion of the diagram are linked enzymatically to specific adaptors. The letters in the RNA message are not read directly by the amino acids but there is an’. RNA adaptor--that coil, hair-pin like structure with the red amino acid fastened to it at the top and three letters in RNA corresponding to one amino acid in protein as shown. You will see in the upper left hand corner of the slide is a growing peptide chain and amino acids are added on one by one starting from the left and proceeding toward the right, reading three bases at a time, three letters in RNA at a time. The next slide shows this in a little more detail. The messenger RNA is starting there and the ribosome, the particles attach to it at one e@ and start reading down. One space will hold many translating units or many particles and as the particles proceed down the message the protein synthesis comes along and is finally released. Now the translating particle is in effect a robot. It can read any message if it is written in the correct form—~in the correct molecular Language and this is really how the code, which is really of the translation between the letters in a nucleic acid sequence and the sequence of amino acids in protein, It would prove to Be relatively simple to décipher this language by simply adding to the robot particles of synthetic messages composed of one or two kinds of bases and then determining what kinds of proteins, what kinds of amino acids that were incorporated into proteins. One could even determine more by simply using three letters alone and just looking to see which adaptors carrying the particular amino acid associated with the ribosomes. Now the next slide shows--this is the last slide»- and it simply summarizes the language. My intention is not to go into detail here, other than to make one or two points. That is, that the language is a very, very Simple language--and a logical language. There are many synonyms and the ~ -5- words in nucleic acid are listed on the upper left hand; for example, one UUG is equal to phenylalanine, so is UUC. Now both are synonyms and synonyms differ only in the letter occupying the third position of the triplet and there are only certain kinds of letters of permissible combinations of letters so it is a very simple, logical kind of language and it has been translated, and there are also some words for start and other words for stop because it is a continuous message. LIGHTS PLEASE: Now, it is not only a logical kind of language. All the available information now indicates that it is a universal language. That is, that all, or virtually all living forms on this planet at least uses essentially the same genetic language. There may be slight dialects or slight differences, but a considerable amount of evidence has been accumulated fl, very recent evidence, which does indicate that the same language is used by all living things. There are a few general principals, <ngieeries—tirat~ ore ‘cam-state, First, changes in the way the logic that the cell apparently uses the simple principal the cell uses. Use ,the principal of standardization. There are few kinds of parts and they are standardized. But great variety is achieved and great diversity also. By combining the part in different sequences and making many different kinds of sequences. Secondly, well, I mantioned the universality; but the important thing that I want to stress is that the particles will read any massage that is given to them. Now this raises the possibility that synthetic messages could be inserted into the cells and the particles will follow the instructions. This is essentially what happened in viral infection, Virus by and large is simply a strand of information of genetic information with a code around it to protect it. The cocmation goes--a simplified vérsion obviously but in essence is correct-- the information goes inside itself and is copied or translated, by the cell's machinery. Now, genetic surgery is a very sophisticated reality. In the -6- jate 20's it was discovered - and a truly spectacular discovery, that one colud prepare DNA from one strain of microorganism and simply add it in volume to another related strain of microorganism and under ideal conditions 10-15% of the individual's bacteria would take up the DNA and their enzymes inside the cell's other machines that will insert this DNA into the chromosome, genetic material of the host so that this organism is changed and it is changed genetically, the progeny then would have the inherited change. So this raises the question then that synthetic information could be used to program cells in much the same way as natural information has already been — used to alter bacteria, Now I should say right now that this has not been done really with mammalian cells. That there are great technical diffi- culties in doing this. Although there have been some recent experiments that -ggest that there are ways really of doing this. For example, it is possible and this already has been done, to prepare DNA from a virus added to mammalian cells and tissue culture. The DNA will get inside the cell and a virus is formed. Recently there have been technjques in which one can take chromasomes from one organism and two cells even from widely different species, fuse the cells together, the nuclei fuse and one has a mixture of chromasomes from two different organisms or 2 different species. The techniques are available for doing this kind of thing so I think it is really a matter of technical difficulty to find ways in which one could take a nucleic acid message and insert it into cells. Now the major difficulty really is in the synthesis of the information.. The chemistry is quite difficult and complicated. Thus far techniques are available for synthesizing very simple messages, By synthesizing them, I don't mean simply copying preexisting messages. because this can be done and has been done for 10 or 12 years and there are enzymes--molecular machines available that one can obtain from the cell that will copy a pre-existing strand of DNA with great stability. It is not a matter of copying but really a matter a J- of composing synthetic messages. The chemistry, as I said, is still rather primitive, but it has been possible to synthesize long strands of DNA or RNA containing simple repeating known sequences and technology is bound to improve vastly in the near future. Now, it wouldn't surprise me, for example, if the synthetic messages that are available today even quite simple used to transform or to program bacteria and I think that this is potentially a reasonable experi- ment even to do today and surely it will be tried and as I said technology will almost surely “improve greatly along these lines. So the main purpose of the talk this afternoon is to point out these possibilities and the current status of work along these lines, the probable future development of the experiments and ask questions because I think that when it becomes possible to do these things then man may be in a position to in some way control, alter or shape his bwn biologic evolution; i.e., to add information and to compose genes. Now this power I think could be used for great good. I think that eventually man stands to benefit greatly by techniques such as these. But I think it obvious that a great deal - an emense amount of.basic information must be learned first before such information could be applied. I should emphasize again that it is not possible to do this today with synthetic information but I think that it will be possible. My rather conservative estimate is in bacteria it is virtually possible to at least try it today and it may be successful in 5-10 years. In mammalian cells a conservative estimate would be 25 years. I think if you were to ask a dozen knowledgeable molecular biologists their estimate of time you would get varying answers but my estimate would be in 10-25 years. I could be off very easily. Now it's necessary I think to formulate the possibilities, state these questions because of the a ethical considerations, moral considerations as well as the technical problems that are involved in this because if and when man becomes able to shape his own evolution by programming -8- cells with synthetic information the question will arise \"which way to go? What are the desirable goals. I think everybody will agree on certain goals but there are many others of which a great diversity of opinion obviously will arise, I think it very important to state this problem now, well in advance of the need to state the problem because it requires time to think the thing through to get reasonable answers to it and to begin to perhaps shape the institutional framework in which this kind of approach would be used, This essentially is the reasons that I've come here and tried to describe some of these developments and I can only say in closing that I think that this potential has great possibilities for good. I think that eventually man will benefit greatly by it but there are a great many facts and a gread deal of information that must be obtained first before it is applied. Thank you very much,", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hy3q_3tcb.wthi", "00000000-0000-0000-F73E-6821D4C8188B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Manuscript for Pomona lecture", "101584910X464", null, "1967", "[1967?]", "In the pages selected here, Nirenberg addresses future prospects of genetic research.  Nirenberg makes some new predictions for future research and once again quotes virologist Salvador Luria on the impact of science on human affairs and the responsibility of scientists--the same quote is used in his 1967 Science article.", "Lectures, Drafts (documents), Excerpts", null, "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "3", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "- 23 - nie em ago. Ndwthe point [| would like!fo make_] i °     put the research perhaps in proper prospect in the long oe of things to come. serene Pah Tn it seems clear that wo most Lt . {x fon vA he aan tebe 3 ¢ Bentuat1y a té& com", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-smum_zvez_bvqt", "00000000-0000-0000-46A0-BF5816817EAE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Robert F. Murray Jr. to Marshall W. Nirenberg", "101584910X465", "101584910X181", "1969", "28 October 1969", "Murray encloses a copy of the text of the talk that Nirenberg gave at the Howard University seminar \"Genetic, Metabolic, and Developmental Aspects of Mental Retardation.\" Nirenberg had asked specifically that the copy not be published due primarily to its hypothetical nature.", "Letters (correspondence)", null, "Transition to Neurobiology, 1965-1969", "1", "pages", "Text", "English", "Reproduced with permission of Robert F. Murray Jr.", "Copyright may apply", null, null, "October 28, 1969 Dear Dr. Nirenberg: Enclosed is a copy of the text of the talk you gave at our seminar \"Genetic, Metabolic and Developmental Aspects of Mental Retardation\" .  I understand that it is not to be published, but we would like an accurate copy for our files on this conference.  Please review it and make any corrections or modifications you see fit.  I will see that you receive a final copy of the talk for your own records. Thank you for your participation and cooperation in this matter. Best regards, Robert F. Murray, Jr., M.D. Chief, Medical Genetics Unit", "Murray, Robert F., 1931- ; Howard University. College of Medicine. Department of Pediatrics and Child Health", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3fqf_f7f5_9iua", "00000000-0000-0000-480C-971F2AEB8D2C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Genetic Versus Neural Information Processing Systems", "101584910X466", null, "1969", "[1969?]", "In this talk, which was part of Howard University's seminar on \"Genetic, Metabolic, and Developmental Aspects of Mental Retardation,\" Nirenberg includes his speculations on information processing by neurons, the history of the genetic code, accuracy of protein synthesis, neural memory, and the process of learning.", "Speeches", null, "Transition to Neurobiology, 1965-1969", "9", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Dr. Marshall Nirenberg Genetic Versus Neural Information Processing Systems I'd like to talk very, very informally and do something that a biochemist should never do, does always in private and never publically if talking and speculate about something which one doesn't know very much about. I do this all the time with friends, especially in discussions but not in public so I want to emphasize that,rather than misleading anybody. I spend most of my time thinking about genetic information processing, mechanisms, and so forth and only recently have I really begun thinking seriously about information pro- cessing by neurons and so I just wish to say that it may sound a little rash to speculate, but, at any rate, if one views a simple cell, suchas E. coli, I guess E. coli, one sees the flow of materials and interconversion of molecules of various sorts, a flow of energy and also a flow of information. Now the infor- mation processing machinery in rapidly growing bacteria will account for 50 or 60% of the total dry weight of the cell. The information is encoded, as you know, in a special class of molecules. It's memory has a specific address and DNA is a very long macro-molecule consisting of only 4 kinds of characters in repeating sequence. A linear string of characters. In E. coli the information may be approximately 3 million letters in length in bacterial chromosomes. A human cell with DNA would consist of approximately 1,000 or 1,500 times as much information. The sequence of the letters in DNA correspond to the sequence of amino-acids in protein and as you know there are 20 amino-acids in protein, 4 bases in DNA and the information in DNA is decoded as a linear form, a one dimensional form and it's decoded by the reading apparatus starting at one point -2- in reading a word sequentially from left to right. The average protein contains some 400 amino acids in length so bacteria may contain about 3,000 kinds of proteins,probably maximally,and for each chromosome there is approximately a million and a half molecules with protein in bacteria so the number of mole- cules in each kind of protein may vary quite considerably from one molecule up to 100,000 or more molecules. The properties of the template, of the DNA template or an RNA template are really very, very simple. The nucleic-acid sequence, base sequence specifies a kind of object; one molecule of a particular kind. It differentiates, it selects an example of that kind of molecule from many other types of molecules. It specifies the relative position of the molecule, that is relative to the previous molecule it selected and it also specifies the time of the event, relative to the previous event that occurred. So the template really serves both as a template for other molecules and as a biological clock because the words are read in sequential fashion. The principle really isa Touring machine principle. That is in the 30's you say British mathematician, Charles Touring devised a very simple kind of machine that would have a reading head that was attached to a tape. The program would travel along the tape and would follow only one instruction at a time and it could read only one space at a time. Basically, this is the same principle employed in all of genetic coding processing and it has some really unique characteristics, for example, one can program one kind of reading machine, Touring machine, to perform any kind of task that any other Touring machine can be programmed for. One of the major principles is that there are relatively few kinds of units,and that great diversity is achieved, and also great complexity is obtained simply by varying the sequence of the units. The units are rather standard ones. I should emphasize that all the information available today indicates that the language is largely a universal language, that all species employ essentially almost the same genetic language. I think that this is important when one considers the origin and the evolution of the language and also,considering the problem of neural memory,’ information processing by the nervous system. The origin of the code is an interesting question. One may ask why is the code set up the way it is? Is there any rational reason for it, or is it the result of a unique set of circum- Stances that happened only once in the dim and distant past? Then after this unique set of circumstances the code that we're aware of may have evolved from this but the original event may still have control of the format or the form of the code as we see it today. The oldest fossils that are known are micro- organisms. They've been estimated to be 3 billion years of age, found by Barcorn and his colleagues. Fossil micro-organisms 2 billion and 1 billion years old have also been reported but the first abundant fossils are found only about 600 million years ago. About 500 million years ago all the invertebrate phyla had evolved and the first vertebrates were formed and mammals rather soon after the code evolved, that the code was probably fixed, the language was fixed because once a sufficient amount of information had been put into the system to form a bacterium, I think that probably it would be difficult to make terribly major changes in the code without destroying the information that -4- had already been put into the system unless it was possible, simultaneously to alter the translation apparatus so that the information that's stored can be retrieved. I think this is a fairly reasonable argument and one may well apply this same type of thinking; I wonder if it can be applied at least, to the problem of information processing by the nervous system. There are several differences, I think obvious differences: First, the genetic code must have evolved before any neural code could have evolved. The genetic code probably evolved as the first primitive cells evolved, perhaps between 600 million and 3 billion years ago. Whereas, the neural code or neural information processing mechanisms must have evolved at a later date when cells were more adept. They were probably quite sophisticated chemically during the transition, perhaps between single cells and multi-cellular forms of life. And this has another consequence, that is, that almost surely, I think, the basic mechanisms for processing neural information undoubtedly were selected from a large population of precursors or precursor mechanisms whereas one doesn't really know. If this is true in a case of genetic code it could have been one extremely rare set of circumstances. Probably the basic mechanisms for processing neural information had evolved and perhaps were probably fixed by 500 million years ago. All of the information that is available that I can see, at least that I'm aware of, which is not very much, but in regard certainly to the neural information processing mechanisms, but from all the available evidence that I see indicates rather strongly that universal mechanisms are at work, that the neural transmitter substances, neural hormones one finds in very simple forms and pretty much the same substances are found in flatworms as one finds in mam- mals. And similar kinds of mechanisms; synaptic vesicles and nerve endings and -5-+ so forth. So I would think that the basic mechanisms, there are relatively few basic mechanisms, operative, that they must be systematic mechanisms, and quite simple and logical mechanisms, and that probably they're almost universal similar to the genetic code and that after enough information had been put into this system, into the nervous system, in terms of basic mechanisms, that it's difficult; one can add on new mechanisms, yes, but to change old mechanisms would be difficult because you destroy all stored information if you did do this. I suppose one of the most important aspects of living organisms is the reliability of the mechanisms, of the machinery, and the accuracy also of the machinery. It's really remarkable if one considers that the average protein is 400 amino-acids long, in any series of sequential calculations obviously the accuracy decreases quite markedly as the number of serial steps increases. The: accuracy of protein synthesis, from everything that we know is usually quite high and I think that the basic reason for this is, well,there are two basic strategies, I think, that the cell employs to enhance the accuracy of protein synthesis: First, redundancy is a logical kind of redun- dancy, alternate words, in most cases, have identical first and second letters but only alternate, vary, the third letters so it's systematic redundancy. Secondly, one must consider the number of serial steps and the number of parallel steps here. Protein synthesis is largely a parallel operation. There are serial steps, but interestingly the serial steps enhance the accuracy of protein synthesis. There's one very, very interesting example of this that was reported by Berg and Norris. It was an error-correcting mechanism. As you -6- are probably well aware, the amino-acids are activated and the cell contains that enzymes/are specific to each amino-acid, different enzymes, one for each amino acid. The enzyme is a device that selects a particular, appropriate species of amino acid and the appropriate species of transfer RNA, the adapter molecule and also ATP. It catalyzes first the formation of an intermediate, an activated amino acid. Then the same enzyme will select a species of transfer RNA and it will transfer the amino acid from the activated state that's in to the transfer RNA. Berg and Norris found that a mistake could be made in the first step, the enzyme would make a mistake and it would recognize the wrong amino acid. In the second step it would correct this error by hydrolyzing, by breaking down this erroneous intermediate. It would not catalyze the transfer of the wrong amino acid to the correct tRNA. This is a beautiful error-correcting mechanism and a perfect example of a mechanism whereby two serial steps enhance the accuracy rather than decrease the accuracy of protein synthesis. Although the other steps are sequential steps, in almost all cases the accuracy of selecting one amino acid or adapter molecule with an amino acid linked to it is independent of the previous selections so an error in one selection usually does not affect the accuracy of further selections. I think that this is the crux of the strategy, right here. There are certainly places where the accuracy of selecting one amino acid does affect the accuracy of selecting subsequent amino acids. This is in initiation, in starting. If the first three bases are incorrectly selected, incorrectly phased, then all subsequent reading will obviously be out of phase and will be erroneous. Secondly, if an error is made and a word that corresponds to stop, terminate or a protein synthesis is terminated because a word is recognized incorrectly, obviously subsequent selections, there will be no reading, so this will affect it. But, in all other cases that I'm aware of an error of selecting one amino acid will not affect the accuracy of subsequent amino acids . The efficiency of this system is quite remarkable when one considers that the average rate of reading of the reading head, the ribosome along the tape, is relatively slow. The rate of reading is somewhat faster than the average person reads, perhaps in the neighborhood of 1,000 words a minute are read. The efficiency is enhanced very greatly because the cell contains many ribosomes, many reading heads, about 15,000 sites for protein synthesis per bacterial chromosome and one message can be read simultaneously at many different sites, by many dif- ferent ribosomes. Ina relatively short time a great many words are translated. E. coli have a generation time, under optimum conditions, of 20 to 25 mirlutes. During this time, 25 minutes, about 500 million amino acids are incorporated to protein. So it's a relatively efficient process. We come to the question, really, of learning and of memory. As I said, in the genetic memory, memory has a specific address, corresponding to each memory is a specific molecule, I don't know what the answer is, obviously, when applied to the nervous system. In recent years there has been a great deal of speculation and extrapolation that it may be each neural memory corresponds to a specific molecule of RNA or a kind of molecule of RNA. I don't know, as I say, the answer, but my bias is probably against this possibility although I think it's too early, really to evaluate. I doubt if there's sufficient information available to judge. All cells require nucleic -8- acid, have protein synthesis. It's possible, for example, that a specific permease of some sort can be induced, that one kind or just a limited number of kinds of molecules, proteins must be synthesized to facilitate memory, to lay down the memory traits. It's clear that a chemical reaction of some sort, I would think it highly likely, must take place. The nature of the chemical reaction remains to be defined. In genetics, for many years there was a great deal of confusion about the various theories of learning. One can think of learning on the genetic level, memory, of immunologic memory, and memory in the nervous system. The two basic theories were the instructive theory and the selective theory. By instruc- tive theory one means that something must be present. I think that the easiest thing to do is just to give an example of it. This was clarified by a very famous experiment by Luria and Delbruck during the '40's. This experiment was to take a petri dish, to put some E. coli on this and to grow up a uniform lawn of the E. coli on the petri dish. It was known that if in a flask, for example, in a liquid medium that if one adds an antibiotic streptomycin to a flask, that it will initially kill most of the bacteria, the next morning one sees resistant population of bac- teria. So the question really was, is the streptomycin necessaty for the apparent mutation that took place? Ina very clever and simple experiment they showed that the mutation would not require, that rather the mutation was independent of the streptomycin. Streptomycin was not required for this. They grew up a lawn of streptomycin sensitive to E. coli. Then on two plates here which contain strepto- mycin - no streptomycin in this plate, then by pressing a velvet cloth on this and then going directly here and directly here one would transfer colonies from -9- here to here to here, and here. And the orientation of colonies would be similar. One found on a plate like this that one little colony, a clone, the descendents of a single cell would grow, let's say here, here, and here. Then on this plate, with the same orientation, one found exactly the same orientation of the three bacteria that had spontaneously become resistant to streptomycin in the absence of streptomycin. It was very clear that the resistant bacteria were already there and were simply selected for by the streptomycin. This is a very simple experi- ment but it's almost precisely the same kind of situation that can be found in the antibody production mechanism. There seems to be cells that are present. Their descendents are selected for and the antibody production is due to the selection of the population of cells. One wonders, really, whether learning is due to selection pracess also as it is in genetic information processing and immunologic information processing. The philosopher, Locke, thought of the mind as a completely blank slate that experience writes on but Socrates thought that you can't learn anything that's not already in the mind. This is -very similar to a selective type of hypothesis, that one simply selects out from some- thing that's already there. The reason he thought this was because by questions only one can teach. I think that these are problems that one should think about. Thank you very much,", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7p47_6kj2.vi4g", "00000000-0000-0000-B334-19FD7D9775EE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Interview with Philip Nelson", "101584910X468", null, "2001", "20 June 2001", "This interview covers Philip Nelson's impressions of Marshall Nirenberg's work, their collaboration in neurobiology and electrophysiology, the state of the neurobiological field in the 1960s (including the influence of molecular biologists), the nature of tissue culture work, and major contributions in the field.", "Transcripts, Interviews", "Neurobiology,Research Design,Cooperative Behavior,Genetics,Electrophysiology", "Neuroblastoma Research, 1967-1976", "6", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "Interview with Phillip G. Nelson Wednesday, June 20, 2001 Topic: Collaboration with Marshall Nirenberg Interview by Jim Tabery Jim Tabery (JT): What did you think of Dr. Nirenberg’s work on the genetic code? Phillip Nelson (PN): His findings were of a somewhat peculiar sort in providing a lightning flash of insight and an instantaneous solution to a profound problem. There have been many big discoveries, but it seemed that his was the most comprehensive and clear-cut solution to the most fundamental problem of 20\" and now 21 century science. That’s my reading of it. I’m not a molecular biologist, but it has that for me: there had been an enormous gathering of evidence but with just this one set of experiments he showed clearly and decisively the nature of the code. JT: When I look over the work that Dr. Nirenberg has done both on the genetic code and on neuroblastoma with you, it seems that he was very interested in finding simple model systems that could get those decisive answers. PN: Right. He liked things that didn't require statistical analysis. I’m sure you’ve heard this from other people as well. It’s not that he didn’t use statistics (standard deviations and so forth), but the phenomena he liked to deal with were either on or off, black or white, qualitative things. This was to ensure that you weren't dealing with contributory, minor modifiers but that you were really as close as possible to identifying causative phenomena. I think that was his motivation in going to the neuroblastoma. One of his students, Nick Seeds, wrote a paper considering whether or not the neuroblastoma system was the E.coli for genetic neurobiology. The idea was that you could dissect in genetic terms the processes that were going on with neuron generation. That didn’t turn out to be the case. The neuroblastoma system was a complex one that was very polyploidal. It just wasn’t a clean, all-or-nothing kind of system because there was heterogeneity among the cells. Subsequently, the genetic approach has proven to be indeed the key to a lot of progress in neuroscience, but it’s been more a matter of producing mutants in drosophila or mouse or other organisms. So, somewhat paradoxically, the whole organism has turned out to be a cleaner genetic system, with the effort there going to finding simple and single dimensional aspects of the phenotype which are produced by a very clear, clean, single gene knockout or alteration of gene expression. The trick has been to get clear-cut correlates of what it is that that one gene is doing, and that’s been very successful. As the general neurosciences have progressed we have a number of nice systems: factors for growth, mechanics of axonal elongation, markers for various kinds of cell death, and markers for neurodifferentiation. Analysis of these sorts of phenomena has generally progressed very well, and this information has been used by the genetic manipulators of the organism to produce a genetically based developmental neurobiology. And Marshall has been very active in that area as well. The neuroblastoma system was one of the first he studied, but he also had roundworms in the lab at the same time that he was setting up the neuroblastoma. JT: He said that he started with nematodes and neuroblastoma, but it was eventually too much to do both, so he had to pick one. Brenner was working with the nematodes, so he went with neuroblastoma. When did you first meet each other? PN: I was thinking about that after you called. It was 33 years ago. We were out at a summer symposium put on by Frank Schmitt in Boulder, CO (NRP). At the time, our son was just twenty months old, and he’s now 34, so it must have been 33 years ago. I remember quite well that we were standing around after one of the talks, and Marshall and Eric Kandel were there in a little group, and Marshall said, “I’m really getting interested in some electrophysiology. I want to get into neurobiology.” And he asked Eric if he knew any electrophysiologists, and Eric tured to me and said, “Well, Phil's just down the hall from you in building 10!” And that started the interaction. We got together when we got back to Bethesda. JT: And then you soon moved over into another building, didn’t you? PN: We were in Building 10 together for a while, and then I went over to Building 36. Marshall subsequently came over to 36 as well. We continued that collaboration for a number of years, with a number of co-workers. JT: What was the collaboration like? How did you work together? PN: Marshall was doing the cloning and purification of different cell lines, and then got into production of hybrid cells with John Minna. Takahiko Amano was doing the cell cloning, so there was a variety of cell lines that were intended to be used as separate phenotypes. They wanted to be able to do a genetic analysis to determine the basis for electric excitability, cholinergic expression, etc. The hope was that they could be used as specific synapse formers. So he had this large amount of material generated by tissue culture techniques, and was doing a number of neurochemical studies on them. I then started working with electrophysiology with the major goal being to get synaptically competent lines, so that the process of synaptogenesis could be observed and analyzed. That was really the subject of our interaction. Some of the people in his lab worked with me, and some of the people in my group worked on the electrophysiology (John Peacock, and a number of others from my lab worked on those experiments). An interesting sidelight for me arose when we were trying to produce the genetic diversity with various hybrid cell lines which included some fibroblastic cell lines also. As part of the study of those lines, we found one very characteristic response in the non-neuronal cell lines, which then segregated and was seen partly in the hybrids and partly not in the hybrids. That research has led to investigation of the non-neuronal phenotype, which turned out to be quite interesting. JT: Did you pursue this in your research? PN: A ways, and then it was picked up by some others. I think the phenomenon is clearly seen in the oscillatory calcium transients in a variety of cells, but we didn’t push that direction of the work to its full biological capability. We jumped the track then to primary cell lines when Gerald Fischbach was in the lab, and he and I started working on primary muscle. We would make a culture of muscle cells from fetal chicks or mice, and then he started working on the spinal cord to go with the muscle cells to provide a synaptic system, and that worked. And then I went ahead with the neuroblastoma and muscle and was able to produce some synapses there. A lot has been done with that preparation of a neuroblastoma cell line in conjunction with either another neuronal cell line or muscle cells which can form cholinergic or other kinds of connections. But then I started to concentrate on the primary. neuronal material, and that was where Marshall and I separated because I started to work on that more exclusively, and he continued with the cell lines and other preparations. JT: So what were the years of the collaboration? PN:. Roughly, the late 1960’s (1967/1968) up to the late 1970's. JT: What was neurobiology like in the 1960’s? PN: Well, there hadn’t been nearly as much genetic dissection of the nervous system; we were still studying various aspects of plasticity. Long term potentiation ( LTP) came in during that period. Also, a lot of work in my area of neuroscience was occurring on modeling individual neurons to get their electrical characteristics, network analysis was coming in too, and there was a lot of developmental work. Richard Sidman and other people were starting that work by cataloguing genetic variants of mouse lines. Kandel was studying various forms of plasticity in the mollusks. Ron McKay, now here in the Neurology Institute, and others were starting to use neuronal material to produce monoclonal antibodies. A lot of that work was done in Marshall’s lab as well. The idea was that these antibody systems would — prove to be powerful probes for specific molecules, and they would permit characterization of neurons, and would also potentially be used as probes for disrupting function: if you block a function with a given antibody, then that would be a clue to what the molecular basis for that function was. Again, that’s proven to be a major plus in enabling identification of subsets of certain neurons. Thomas Jessell and many other people have used that very productively. But I think it’s fair to say that it has not proven to be as powerful a probe for neurological function as has the genetic dissection methodology. JT: Dr. Nirenberg was one of several from molecular biology who made the migration to neurobiology. Do you think that they were at all influential in taking neurobiology to the genetic dissection phase? PN: Oh yes. That’s absolutely true. Benzer and his drosophila work have obviously been hugely influential. Maybe the even more important thing was that fundamental approach—the view that you can analyze things, and the notion that there is going to be some discreteness. I think many of us more traditional neuroscientists, had a feeling of the complexity and the difficulty of analyzing neural function, and that’s sort of coming back to some extent. But perhaps we didn’t appreciate the power of these tools and just how far you could get with them. Now, whether they’re going to be enough is another question. I think that they probably aren’t. So there will be some additional kind of research that is more integrative and allows for the ability to accept small, incremental changes as coming together to produce the total phenomena. But there is no doubt that these discrete techniques and approaches have enormous power. JT: So the reductionism was an important step, but it can only get the science so far, and now we’re coming upon a whole new set of problems? PN: Maybe. I’m not sure that’s established, but that’s my bias. Now, I was receptive to reductionistic approaches. I started out studying spinal cord physiology on intact cats, and went to the tissue culture because I wanted a simple system too. I saw just how difficult it was to get at what was going on inside the intact system. With the new techniques people like Thomas Jessell and Marc Tessier-Lavigne have made tremendous progress in the intact systems as well. But at the time I switched over, the central nervous system was pretty opaque and didn't allow really mechanistic analysis, so the notion of tissue culture was enormously appealing to me because you could get at and see the preparations manipulated directly. JT: Were tissue cultures being done in the late 1960’s? PN: Some beautiful work was being done by Margaret Murray and Stanley Crain and colleagues, for instance. I think my lab, with Gerald Fischbach, and Marshall’s lab were instrumental in establishing that these systems could show a wide and interesting range of behavior, including synaptogenesis. They could show the full physiology and morphology of the neural tissue. So that was part of the excitement of seeing how far these could go. Gerald Fischbach and I had done some developmental studies on the muscle, and Gerry started using some preparations that had been developed by Moscona and used in morphological studies. He didn’t start from scratch, but not much had been done, and there had been nothing physiological involving synapses. Gerry was interested in whether these preparations things could make synapses or not. He took the muscle and dissociated spinal cord, and he would grind up the spinal cord, strain it through lens tissue, and wound up with just a bunch of single cells, Then he’d put the cells in a culture dish. One night he came running out of this little dark room where he did his physiology yelling, “They’re there!” He’d seen these synaptic events. There was a lot of significance implicit in this because it showed that this drastically reduced system could exhibit what anyone would say was an interesting and important phenomenon, namely synaptogenesis. I had the same reaction with the continuous cell lines. Here was a tumor that had been growing and being transplanted in various mice for 30 years. It had been discovered in the 1940’s. So you get these cells out and put them down in culture and then treat them with cAMP or other differentiating agent, and you’re looking for synapses. We’d been looking very hard unsuccessfully for a long time, but when we put the cholinergic line together with some muscle we found for the first time physiologically functional synapses, and that was very exciting. Marshall was very excited by the initial findings that these cells were electrically excitable. At that rather primitive stage, the notion was that the neuronal signature was an action potential, so it had been far from obvious that these cell lines that had been growing and multiplying could differentiate this way. I think he saw the possibility of dissecting these processes. JT: Is tissue culture work fairly common now in neurobiology? PN: Yes, it is widely, widely used. Tissue culture doesn’t exactly have a controversial aspect, but it does have its opponents. It’s now settled down to a pragmatic discussion about the degree to which the in vitro system will capture the phenomena you want to look at. I’ve been studying tissue cultures for 30 years now, but it is true that some processes don’t get expressed as much in culture as they do in vivo, so you have to realize the limitations. It’s an indispensable tool, but it has to be used with some degree of restraint and modesty. Getting hubristic about it isn’t a good idea because you can be lead seriously astray by it. It’s not a shortcoming; it’s the fact that the full expression of a phenomenon probably relies on many clues in many situations. The molecular biologist may say that there is one causal factor that can be sorted out, which may or may not be true. But, probably for many things, many causes produce the same effect or participate in producing a well-balanced, stable, non-oscillatory, well-regulated effect. And some of those causes may be absent in vitro, and then you see some distorted form of the phenomenon, so you just have to keep that in mind. JT: How did you get into neurobiology? PN: I had just finished medical school at the University of Chicago, and my wife was a year behind me. I had been interested in physiology all through medical school, so I stayed during her final year and worked with Julian Tobias, who was also a molecular neurobiologist. He had wide-ranging interests in many aspects of nature and science, particularly the nervous system, and the nervous system was just one of those very interesting things. I presented my thesis work at a FASEB meeting, which was kind of the only biology meeting in those days. Then Karl Frank, who was here at NIH, offered me a job. About that time, the Vietnam War was raging. Frank’s laboratory was excellent; he was doing pioneering work in intracellular recordings, so I came to his lab, and it was very rewarding. JT: Was this in the Child Health and Human Development (NICHD)? PN: No, that was in the Neurology Institute at the time. It was the National Institute of Neurological Diseases and Blindness. And it was in the Laboratory of Neurophysiology, which was jointly sponsored by Mental Health and Neurology. I think Seymour Kety was the overall head. Wade Marshall was the head of the Laboratory of Neurophysiology. Eric Kandel was there, Alden Spencer, Stanley Rappaport, Ichiji Tasaki, and Walter Freygang, and a lot of other very good people. So it was a very intellectually rich and personally rewarding place to work. JT: What years were you there? PN: I came in 1958 and switched over to Child Health in 1969. I had, meanwhile, moved into building 36. I maintained a lot of the contacts in the Neurology institute, and Marshall came over, and so we had a lot of traffic up and down. Mark Fishman came to work in his lab and then spent a year or so working with me on some of the projects that I’m still continuing. JT: And then what brought you over here to building 49? PN: Well, this was opening up. There had been a drive to get a building for the Child Health Institute. They were going to build it over in the north west corner of the NIH over by the firehouse, but then politics got in the way of that, as I understood it. Then the animal care facilities for primates desperately needed a new building because the facilities in building 10 were inadequate, and I think a good effect of the animal rights people came from their putting pressure toward acquiring good facilities for primates. This building was designated for that purpose and for the Child Health Institute. So there was some good space available, and with various and sundry space manipulations we got here. JT: To wrap it up, when you look at the research collaboration between you and Dr. Nirenberg, what do you think was your most important contribution to neurobiology? PN: Well, I think the clonal analysis approach has been very widely used. And the PC-12 cell line has been used by many neurobiologists for studies of a broad range of problems. Lloyd Greene developed that in Marshall's lab. JT: What is the PC-12? PN: PC-12 was a cell line isolated from a tumor. It turned out to have some very interesting properties, a primary one of which is that if you have them growing and treat them with nerve growth factor (NGF) they differentiate tremendously. They develop very long processes, and they’ll make synapses and do all sorts of things. So it’s been an extremely good model system for studying NGF action. It’s been used by Gordon Guroff, who was here in the Child Health Institute, and many others too. I think that Marshall’s insight towards this kind of approach, while it didn’t lead to the total revolution that he thought it might, has been extremely useful. And that notion of genetic dissection of elementary properties is very strongly captured by the clonal analysis, and that’s been part of the culture of being able to ascribe particular neurobiological functions to individual genes and their products. That’s been a major thing. People in his lab were also involved in some of the positional cue molecules that specify interactions in retina. And I think it was no small part due to the association with Marshall that we got involved in the in vitro approach, and that primary neuronal cell cultures (Gerald Fischbach and others in my lab) have been extremely useful. So, Marshall was one of the driving forces in that whole reduced preparation and genetic analysis approach JT: But now you are starting to see the pendulum swing back the other way towards the original approach to the whole organism? PN: Well, I wouldn’t say that it’s swinging back exactly. The genetic approach is going to continue. But there are other sorts of scientists who want to look at neurobiology from the stand point not of simplicity but of how you deal with complexity. And that’s not just neuroscience; it’s science in general. I think that there’s increasing interest in that approach as well. But I don’t think it will supplant the more discrete approach. JT: In just the last few years the NSF has put together a whole task force on biocomplexity, so there obviously is a major interest in what’s going on. PN: Well, it’s not either or. In fact, one of the people in the lab here (Doug Fields) has become interested in using the gene chip approach: to look at up to several thousand genes. He’s continuing with some studies along this line but using a reduced system where he can take a uniform cell type, stimulate it with varying patterns of electrical impulses, and then look for genes selected by their responses. So he’s still looking at the molecular level, but he’s also attracted by the notion of looking at complex responses. I don’t think that’s atypical. But it’s not easy to do. JT: So the question now is how do these separate approaches become integrated? PN: Right. I think that’s it: How do you use this discrete information you get from the gene manipulation approach on the problem of a more complex, balanced, multi-determinate, total organism? I wonder how Marshall would feel about it. Did you talk to him about it? JT: I didn’t bring it up. Do you still keep in touch with him? PN: A little bit, but not much. I see him occasionally, but we’re not interacting on a real scientific basis anymore. JT: Well, thank you Dr. Nelson. This has been extremely helpful. PN: My pleasure.", "Nelson, Phillip G.", null, null, null, null, null, null, null, null, null, null, null, "Tabery, Jim", null, null, null, null, null, null, null, null, null, null ]
, [ "row-xt3k.tbfy_37r3", "00000000-0000-0000-FBAD-4240B73F9CCE", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "On the Ribonucleic Acid Synthesized in a Cell-Free System of Escherichia Coli", "101584910X469", null, "1962", "15 March 1962", "This article describes experiments in which RNA is synthesized in an in vitro system derived from E. coli. The synthesized RNA is found to behave essentially like the \"undegraded\" messenger-RNA detectable in growing E. coli cells. The ability to synthesize this process was crucial in deciphering the genetic code.", "Articles", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "6", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "ON THE RIBONUCLEIC ACID SYNTHESIZED IN A CELL-FREE SYSTEM OF ESCHERICHIA COLI* By E. Oraxa, H. Mirsul, ann 8. Osawa INSTITUTE FOR MOLECULAR BIOLOGY, NAGOYA UNIVERSITY, NAGOYA Communicated by A. E. Mirsky, January 8, 1962 In a previous communication,! we described a method for the preparation of ‘‘undegraded”’ messenger-RNAs from bacterial cells (#. colt). Messenger-RNAs have been described before’ and the selective synthesis of this type of RNA under special culture conditions has been reported by Hayashi and Spiegelman.? These RNA fractions are rapidly labeled in isotope experiments and have base composi- tions resembling that of the bacterial DNA.? In our experiments the ‘‘undegraded”’ messenger-RNA fraction was prepared by the phenol method after first breaking the cells in the presence of Duponol in order to assure the immediate inhibition of ribonuclease activity. Messenger-RNA could be distinguished from ribosomal RNA by chromatography on columns of methylated serum albumin or by centrifu- 426 BIOCHEMISTRY: OTAKA, MITSUI, AND OSAWA Proc. N. A. 8. gation in a sucrose density gradient. The messenger-RNA was shown to be hetero- geneous, with sedimentation constants of 23-28 8, 19 8, and 12 § respectively, in contrast to the 23 S and 16S of H. coli ribosomal RNA.* This method of prepara- tion yields RNAs with sedimentation coefficients considerably larger than the 8 S value previously reported for messenger-RNA*® The experiments to be described in this paper are concerned with similar RNA fractions synthesized in an in vitro system derived from E. colt. Care has been taken to assure that the RNA is synthesized under conditions where degradation of the product is adequately prevented. A net synthesis of RNA in vitro has been achieved, and the behavior of the newly synthesized RNA on methylated serum albumin column chromatography or in sedimentation analysis is in essential agreement with that of the messenger-RNA fraction synthesized in vive. The in vitro synthetic system is a continuation of work begun in other laboratories*—!” in which it was shown that RNA synthesis from the nucleoside triphosphates of adenine, guanine, cytosine, and uracil, requires DNA asa “primer” and yields RNA fractions of base composition complementary to that of the DNA used. The cor- respondence between DNA and RNA base ratios in both messenger-RNAs prepared from living cells,' ? and in RNA synthesized 2m vitro’ *. 1! suggests their identity. This viewpoint is supported by observations that both types of RNA can form hybrids with DNA under appropriate conditions.*: 2» The work to be described below offers further evidence for the identity of the RNA synthesized in vitro with the messenger-RNA of the cell. Under conditions in which degradation of the product is prevented, the zn vitro system is found to produce RNA molecules considerably larger than the earlier estimates of Geiduschek et al.1? and Tissiéres and Hopkins'? who reported the RNA synthesized in their cell-free systems to be comprised of 5.5 8 and 10 8 components. Materials and Methods.—Exponentially growing H#. coli B (H) cells in a modified medium of Cowie et al.!4 were collected, washed twice with cold 0.025 M tris buffer (pH 7.5) containing 0.01 M Mg acetate, 0.001 M Mn sulfate, and 0.05 M KCl, ground with quartz sand, extracted with 3 volumes of the above buffer, and centrifuged 2 hr at 105,000 X g. The resultant supernatant which contains, in 1 ml, about 2 mg protein, 120 ng DNA, and 200 we RNA (mainly RNAs) was used as the enzyme preparation. The synthesis of the RNA was estimated by the incorporation of radioactivity from 8-C'4-ATP into RNA in the presence of the additions described in the jegend to Figure 1. Incubation was carried out at 37°C. After cooling, cold 10% trichloroacetic acid (TCA) was added to the reaction mixture, and the acid insoluble precipitate was washed with 5% TCA twice. The radioactivity left in the residue was measured. For the direct measure- ment of RNA, the TCA precipitate was treated with 10% perchloric acid at 70°C for 20 min. The RNA content was estimated on the acid supernatant by the orcinol reaction. Nucleic acids which contain the newly synthesized C1+*RNA were isolated in the cold by the duponol-phenol method‘ from the reaction mixture which had been incubated for 60 min under the same conditions described in the legend of Figure 1. At the beginning of the isolation several volumes of Duponol treated, nonlabeled 105,000 X g supernatant were added as carrier. Under the conditions the yield of the radioactive RNA was about 70%. The nucleic acids prepared were fractionated by a methylated serum albumin column chroma- tography”. 18 with or without added ribosomal RNA as “reference.’’ No difference was found between them. The RNA contained in fractions No. 72-120 of Figure 2a was precipitated with two volumes of ethanol and dialyzed 18 hr. Thesample was examined by sucrose density gradient centrifugation according to the method of Britten and Roberts.° The centrifugation was carried out at 2°C either at 25,000 rpm for 10 hr with No. SW 25 rotor or at 39,000 rpm for 4 hr with No. SW 39 rotor. RNA synthesis in the 105,000 X g supernatant from E. coli: Tt was observed Vou. 48, 1962 BIOCHEMISTRY: OTAKA, MITSUI, AND OSAWA 427 Fie. 1.—Time course of RNA cpm 4ygRNA synthesis in the 105,000 x* g super- natant of Escherichia coli. Reaction | vessel contained in 0.5 ml: Mg acetate 600 io 4160 (5 wM), Mn sulfate (1 »M), KCI (25 | uM), tris, pH 7.5 (12.5 uM), 8-Cu | 140 ATP (0.5 uM = 84.4 muc), GTP, 500 CTP, and UTP (each 0.25 uM), J pyruvate kinase (20 ug), phosphoenol- 4 pyruvate (2.5 4M), salmine sulfate 400 | oy (15 ug), and 0.2 ml of the 105,000 x g pf 100 supernatant of EH. coli. ——®@ , Complete mixture without further 300 80 addition of GTP, CTP and UTP. ——O-——: Complete mixture to which 60 GTP, CTP, and UTP (each 0.5 »M) 20° were supplied at every 10 min as indi- os, 40 cated by arrow. —:—-— Ao. .— : Same, QM. net increase of RNA measured by !° orcinol reaction. ---X--~-: Com- plete mixture minus salmine, without a addition of GTP, CTP, and 99 20 40 0 26°   i20   * 20       that the RNA synthesis stops after 10-15 min, followed by degradation of the product if salmine is not present. One of the reasons for this may be due to the action of some kind of ribonuclease. It was found that salmine sulfate, at the op- timum concentration of 30 ug/ml, markedly stimulated the initial reaction rate and also effectively protected the product from degradation. Even in the presence of salmine, the RNA synthesis was terminated after about 10 min. It was then supposed that hydrolysis of the ribonucleoside triphosphates might be occurring in our crude system; shortage of the substrates would then result in the termination of the reaction. When three nucleoside triphosphates (guanine, cytosine, and uracil) were supplied at 10-min intervals, the RNA synthesis continued for a further 10-15 min. Simultaneous addition of C'4-ATP was not necessary. As shown in ee --0--0-~ OD.260 C.pm.   Lek I6s 71,000         O.8F oer ONA 4 500 oat SRNA oat if end o kee 2.0.0-00-00-.008 . °o 30 40 $0 60 70 60 90 100 Wo 120 Tube No Fia, 2a.—Fractionation of the RNA synthesized in the 105,000 > g supernatant of HE. coli with methylated serum albumin column chromatography. 4 ml/tube/10 min were collected at room temperature. 428 BIOCHEMISTRY: OTAKA, MITSUI, AND OSAWA Proc. N. A, S. —s =+0--0--                           OD.260 cpm, 4h : Approx. L2F GC Py/Pu “'s 460,000 I 46% Lo? 485 rot «=o L500 % «1.02 «12 Tl sow 101 19 ost TM 50% 100 26 Ja4qo00 ost o.4t 42000 o2b J 0 Aho ps 1 L 4 wk. 1 1 4. 20 30 40 50 60 70 ao 90 100 W98   TubeNo. Fic, 2b.—Fractionation of the RNA labelled with P2 for 30 sec in growing £. colt cells with the same chromatographic method asin Fig.2a. (From M. Takai, N. Kondo and §. Osawa.') Figure 1, addition of these three triphosphates at every 10 min supported a con- tinuous synthesis of the RNA at least for 60 min. Further synthesis of the RNA may be possible if the incubation is continued under the same conditions. Omission of any one of the triphosphates from the reaction mixture reduced the incorporation over 85 per cent. Addition of 5 ug/ml deoxyribonuclease completely inhibited the reaction. These results show that the RNA synthesis in this system, as in others,*—!% proceeds using four ribonucleoside triphosphates as precursors and is dependent on the presence of DNA. Usually 160-200 ug of RNA were synthesized after 60 min in 0.5 ml reaction mixture. The ratio of the newly synthesized RNA to DNA is between 1.3 and 1.6. The results suggests that a considerable part of the RNA formed is liberated from the DNA template. Characterization of the RNA synthesized in vitro with methylated serum albumin column and sucrose density gradient centrifugtation: A typical chromatographic pattern of the RNA on a methylated serum albumin column is reproduced in Figure 2a. For the purpose of comparison, the pattern of the messenger-RNA as demon- strated by P®? pulse labelling of growing JZ. col: cells is also shown (Fig. 2b). The radioactive RNA synthesized in vitro can be fractionated on the column into at least. 3 fractions. The first one is eluted just before the 16s ribosomal RNA. The second is located a little earlier than that of the 23s ribosomal RNA. The third one predominates in amount and is eluted after the 23s RNA. From the figure it is seen that no appreciable amount of sRNA or of ribosomal RNA is formed in this system. The chromatographic positions of these three radioactive RNAs cor- responds well to Peak II, Peak ITI, and Peak IV of the pulse labeled messenger- RNA respectively as may be seen in Figures 2a and 2b. Evidence for the presence of Peak I RNA is not clear. Examination of the RNA in sucrose density gradient centrifugation demonstrated the presence of three RNA peaks with approximate sedimentation coefficients of Vou. 48, 1962 BIOCHEMISTRY: OTAKA, MITSUI, AND OSAWA 429 10s-12s, 17s-19s, and 26s-30s, respectively. The s-values are in essential agree- ment with those found for the messenger-RNA. Considering the data on the mes- senger-RNA in which each radioactive peak isolated after methylated serum al- bumin column chromatography was separately examined in density gradient cen- trifugation, it appears that the first peak in the sedimentation analyses corresponds to Peak II in the chromatogram, the second one to Peak ITI, and the third one to Peak IV respectively. The third peak was found to be very unstable and is easily converted to the smaller units. It is of considerable interest to suppose that the third one is the functional messenger-RNA. In this connection we have recently found” that, in Z. cold normally infected with T,-phage, three main messenger-RNA peaks which are similar to those of noninfected H. colt can be detected on the methyl- ated serum albumin column chromatography; when protein synthesis is prevented by chloramphenicol virtually no Peak IV was detectable. Conclusion RNA can be continuously synthesized in the 105,000 X g super- natant of E. cold in the presence of four ribonucleoside triphosphates, DNA, an energy generation system, and salmine sulfate. The RNA synthesized is well protected from degradation by salmine sulfate. The addition of salmine to the reaction mix- ture therefore makes it possible to prepare the “undegraded” RNA by the phenol method. Examination of the RNA using methylated serum albumin column chromatography or sucrose density gradient centrifugation indicated that the synthesized RNA behaves essentially like the “‘yndegraded’’ messenger-RNA which is detectable by pulse labelling of growing EH. coli cells. These observations, to- gether with the identity of the base ratios (which are equivalent to those of DNA) of these RNAs support the view that the RNA synthesized in the cell free system is the messenger-RNA. Neither sRNA nor ribosomal RNA seems to be synthesized in the system described in this paper. The authors are much indebted to Dr. A. E. Mirsky and Dr. V. G. Allfrey for their interest and constant encouragement. They also acknowledge the helpful advices of Dr. M. Takai of our laboratory. * Supported by grants from the Rockefeller Foundation, the Ministry of Education of Japan, and the Asahi Press. 1 Takai, M., N. Kondo, and 8. Osawa, Biochim. Biophys. Acta, submitted for publication. 2 We adapt the term ‘“‘messenger-RNA” to this type of RNA (see ref. 5). 4 Hayashi, M., and 8. Spiegelman, these ProceEpinGs, 47, 1564 (1961). 4 Kurland, C. G., J. Mol. Biol, 2, 83 (1960). 5 Gros, F., H. Hiatt, W. Gilbert, C. G. Kurland, R. W. Risebough, and J. D. Watson, Nature, 190, 581 (1961). 6 Hurwitz, J., A. Bresler, and R. Diringer, Biochem. and Biophys. Res. Comm., 3, 15 (1960). 7 Furth, J. J., J. Hurwitz, and M. Goldmann, z67d., 4,363 (1961 ). ® Weiss, S. B., and T. Nakamoto, these ProcrgpiNas, 47, 694 (1961). 9 Ochoa, S., D. P. Burma, H. Kroger, and J. D. Weill, these ProcrEDINGs, 47, 670 (1961). 10 Burma, D. P., H. Kroger, 8S. Ochoa, R. C. Warner, and J. D. Weill, these PRocEEDINGS, 47, 749 (1961). 11 Stevens, A., J. Biol. Chem., 236, PC 43 (1961). 13 Geiduschek, E. P., T. Nakamoto, and 8. B. Weiss, these PRocrEpinGs, 47, 1405 (1961). 13 Tigsiares, A., and J. W. Hopkins, to be published. 44 Cowie, D. B., G. N. Cohen, E. T. Bolton, and H. Robichon-Szulmajster, Biochim. Biophys. Acta, 34, 39 (1959). 18 Huang, R. C., N. Maheshwari, and J. Bonner, Biochem. and Biophys. Res. Comm., 3, 689 (1960). 430 BIOCHEMISTRY: RISEBROUGH, TISSIERES, AND WATSON Proc. N. A. 8. ‘6 In a current experiment, we have found that a protein fraction isolated from the 105,000 x g supernatant can catalyze a similar RNA synthesis in the presence of four nucleoside triphos- phates and the Z. coli DNA prepared by the method of Marmur (J. Mol. Biol., 3, 208, 1961). In this system also, the addition of salmine enhances the initial rate of the synthesis and prevents the degradation of the product. The rate is about comparable to that of the crude system. However the reaction practically ceases after ten minutes even if the nucleoside triphosphates are further added at 10-min intervals. The ratio of the synthesized RNA to the DNA added is around 0.5. The result might be explained by assuming a factor necessary for the liberation of the synthesized RNA from the DNA. In the crude system, the RNA would be continuously liberated from DNA in the presence of the “factor;’’ in the “purified” system, the RNA would not be liberated because of the absence of the “factor.” 17 Mandell, J. D., and A. D. Hershey, Anal. Biochem., 1, 66 (1960). 18 Philipson, L., J. Gen. Physiol., 44, 899 (1961). 19 Britten, R. J., and R. B. Roberts, Science, 131, 32 (1960). 20 Ishihama, A., unpublished experiment (1961).", "Mitsui, H. ; Otaka, E. ; Osawa, S.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kfh3-3yie~ee45", "00000000-0000-0000-2349-5F85DAAFCA39", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Historical Review: Deciphering the Genetic Code -- A Personal Account", "101584910X474", null, "2004", "January 2004", null, "Articles, Reminiscences", "Genetic Code", "Beyond the Laboratory: Professional, Personal, and Political Life, 1967-2002", "9", "pages", "Text", "English", "Public Domain", "Public domain", null, null, "TRENDS in Biochemical Sciences Voi.29 No.1 January 2004 ELSEVIER Historical review: Deciphering the genetic code — a personal account Marshall Nirenberg Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, 9000 Rockville Pike, MSC - 1654, Building 10, Room 7N-315, Bethesda, MD 20892-1654, USA This is an autobiographical description of the events that led to the breaking of the genetic code and the sub- sequent race to decipher the code. The code was deci- phered in two stages over a five-year period between 1961 and 1966. During the first stage, the base compo- sitions of codons were deciphered by the directing cell- free protein synthesis with randomly ordered RNA preparations. During the second phase, the nucleotide sequences of RNA codons were deciphered by deter- mining the species of aminoacyl-tRNA that bound to ribosomes in response to trinucleotides of known sequence. Views on general topics such as how to pick a research problem and competition versus collabor- ation also are discussed. I would like to tell you how the genetic code was deciphered from a personal point of view. I came to the National Institutes of Health (NIH) in 1957 as a post-doctoral fellow with Dewitt Stetten, Jr, a wise, highly articulate scientist and administrator, immediately after obtaining a PhD in biochemistry from the University of Michigan in Ann Arbor. The next year, I started work with William Jakoby and, by enrichment culture, I isolated a Pseudomonad that grew on y-butyrolactone and purified three enzymes involved in the catabolism of y-hydroxybutyric acid [1]. There was a weekly seminar in Stetten’s laboratory in which Gordon Tomkins (Figure 1), who worked in a different laboratory, participated. Gordon was brilliant, with a wonderful associative memory and a magnificent sense of humor. His seminars were superb, especially his description of the step-by-step developments in the problem that he intended to discuss. Towards the end of my post-doctoral fellowship, Gordon replaced Herman Kalckar as head of the Section of Metabolic Enzymes and offered me a position as an independent investigator in his laboratory. The other independent investigators in the laboratory were Elizabeth Maxwell and Victor Ginsberg, who were carbohydrate biochemists, and Todd Miles, a nucleic-acid biochemist. It was a wonderful opportunity and I decided then that if I was going to work this hard I might as well have the fun of exploring an important problem. In my opinion, the most exciting work in molecular biology in 1959 were the genetic experiments of Monod and Jacob on the regulation of the gene that encodes Corresponding author: Marshall Nirenberg (mnirenberg@nih.gov). 8-galactosidase in Escherichia coli and that the mechan- ism of protein synthesis was one of the most exciting areas in biochemistry. Some of the best biochemists in the world were working on cell-free protein synthesis, and I had no experience with either gene regulation or protein syn- thesis, having previously worked on sugar transport, glycogen metabolism and enzyme purification. After thinking about this for a considerable time, I finally decided to switch fields. My immediate objective was to investigate the existence of mRNA by determining whether cell-free protein synthesis in FE. coli extracts was stimulated by an RNA fraction or by DNA. In the longer term, my objective was to achieve the cell-free synthesis of penicillinase, a small inducible enzyme that           Figure 1. Gordon Tompkins. Gordon was brilliant, highly articulate and very funny. He was a charismatic individual who created a stimulating atmosphere and encouraged exploration. In 1958, towards the end of my post-doctoral fellowship at the NIH, he offered me a position as an independent investigator in his laboratory. http://tibstrends.com 0968-0004/$ - see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2003.11.009     lacks cysteine so that I could explore mechanisms of gene regulation. I thought that in the absence of cysteine the synthesis of penicillinase might proceed, whereas syn- thesis of most other proteins might be reduced. In England, Pollock [2] had shown that penicillinase is inducible in Bacillus cerus and had isolated mutants that differed in the regulation of the penicillinase gene. In 1959, tRNAwas recently discovered but mRNA was unknown. At that time, the only clues that RNA might function as a template for protein synthesis were a report by Hershey et al. [3], showing that a fraction of RNA is synthesized and degraded rapidly in E. coli infected with T2 bacteriophage, and a paper by Volkin and Astrachan [4], which showed that infection of E. coli by T2 bacteriophage resulted in the rapid turnover of a fraction of RNA that had the base composition of bacteriophage rather than the DNA of E. coli. If mRNA did exist, I thought that it might be contained in ribosomes because amino acids were known to be incorporated into protein on these organelles. I estimated it would take me two years to set up a cell-free system to determine whether RNA or DNA stimulated protein synthesis, which was a pretty accurate estimate. I knew this was a risky problem to work on because starting out as an independent investigator you are supposed to hit the deck running and prove that you are an effective, productive investigator. One evening I saw Bruce Ames working in his laboratory. Because I thought he was one of the best young scientists at the NIH I described my research plan and asked for his evaluation. He just looked at me and said ‘It is suicidal’. Although we both agreed that it was a dangerous project to work on, I thought suicidal was a little extreme. On the one hand I wanted to explore an important problem, on the other I was afraid of failure, but the wish to explore was much greater than the fear of failure. As soon as I moved to Gordon’s laboratory I started to make cell-free extracts that incorporated amino acids into protein, and to prepare DNA and RNA from ribosomes of penicillinase inducible and constitutive strains of B. cerus. I devised a sensitive assay for penicillinase and starting with conditions that had been devised by Lamborg and Zamecnik and his colleagues [5], I tried to obtain the de novo synthesis of penicillinase following addition of either RNA or DNA fractions from either B. cerus or E. coli. Systematically, I explored the optimum conditions for cell- free synthesis and showed that RNA prepared from ribosomes of B. cerus that expressed penicillinase con- stitutively stimulated penicillinase synthesis by 10-15%, but RNA from either uninduced ribosomes or DNA had no effect. However, the stimulation of penicillinase synthesis was small and it was clear that I needed a more sensitive assay. Usually around noon, Gordon Tomkins would come into my laboratory with a sandwich and we would go into the hall and talk about my work, his work, and various exciting results that had been published. I always stopped to talk to him, even though the extract that I was preparing was slowly dying in an ice bucket, because these were wonderful conversations. Gordon encouraged me and created an exciting atmosphere for young investigators. http:/tibs.trends.com TRENDS in Biochemical Sciences Vol.29 No.1 January 2004 47                     200: 7 7 ' v J ' z a T + ONAase 1800} A z 5 - Qo ez a Oo € 1000F > - = ~ ww : 3 +ONAGSO™ Q 500+ / +—! MINUTES o 40 SO B60       Figure 2. Incorporation of 4¢~—labeled valine into protein in Escherichia coli extracts. Endogenous incorporation of radioactive amino acids into protein in £. coli extracts was high. However, amino acid incorporation ceased after incu- bation for ~40 min with DNase I. | found that | could freeze £. coli extracts and thaw them without loss of activity, so | incubated E. coli extracts in the absence of radioactive amino acids for 40 min, divided the extracts into small aliquots and froze them for use ‘ater in different experiments. Endogenous incorporation of radioactive amino acids was greatly reduced in such extracts, and addition of -mRNA preparations from ribosomes clearly stimulated amino acid incorporation into protein {16,34,49]. Reproduced from Ref. [16]. After working on this for about for about a year and a half, Heinrich Matthei came to my laboratory as a post- doctoral fellow. Heinrich was a plant physiologist from Germany who was a post-doctoral fellow at Cornell who wanted to work on protein synthesis. He came under the impression that, because the NIH is such a big institution, many people would be working on protein synthesis. He stopped in Roy Vagelos’s laboratory and Roy sent him to me because I was the only person at the NIH who was studying cell-free protein synthesis. We needed a more sensitive assay, so I suggested that Heinrich use the cell- free amino-acid-incorporating system that I had optimized to measure the incorporation of radioactive amino acids into protein. Heinrich insisted on preparing 20 M4C-labeled amino acids by growing algae in the presence of 4C.bicarbonate, hydrolyzing the protein and purifying each of the !*C-labeled amino acids, because this is what he had done previously. Using this more sensitive assay it was immediately apparent that RNA from ribosomes, but not DNA, stimulated incorporation of radioactive amino acids into protein [6,7]. I jumped for joy because this was the first definitive demonstration in vitro that mRNA existed and was required for protein synthesis. We fractionated RNA from ribosomes and found, as expected, that only a small portion stimulated amino acid incorporation into protein [8]. We made three trivial technical advances that had a tremendous effect on our work. First, I established conditions that enabled us to freeze and thaw E. coli extracts with little or no loss in the ability to incorporate     64900 ¥ t t t ¥ ¥   + POLY U 56,000 F- 4 29,900 4 COUNTS /MINUTE/mg,. PROTEIN 16,900 E 6000 F 7       ° a a. a s. rs s ° iS 30 45 & 7 90 MINUTES       Figure 3. Poly(U) greatly stimulates the incorporation of radioactive phenylalanine into poly-phenylatanine [8]. amino acids into protein. Second, as shown in Figure 2, basal, endogenous incorporation of 14C-labeled valine into proteins in the absence of mRNA was high, hence, the increase in amino acid incorporation caused by mRNA was relatively small. We confirmed the reports of Kameyama and Novelli [9] and Tissieres et al. [10] that DNase I inhibited the incorporation of amino acids into protein in cell-free E. coli extracts. Therefore, we incubated E. coli extracts in the presence of DNase I but without a radioactive amino acid for 40min until endogenous amino acid incorporation had almost stopped [16,34,49]. Then we divided the extracts were into small portions and froze them for use later. Because the endogenous incor- poration of radioactive amino acids into protein was low in these extracts, it was stimulated markedly by the addition of mRNA. Third, the standard method of washing radio- active protein precipitates in trichloracetic acid to remove radioactive amino acids involved repeated centrifugation and resuspension of protein pellets, which was very laborious and time consuming. One evening I compared this standard method with washing protein precipitates by filtration through Millipore filters. The results were identical. By using frozen—thawed, preincubated E. coli extracts and washing radioactive precipitates on Millipore filters we could do as much as in one day as had previously taken us 8-10 days. I then obtained yeast rRNA and tobacco mosaic virus (TMV) RNA and we found that both were as active as mRNA. However, RNA from TMV was 30-50 times more active than ribosomal RNA at stimulating amino acid incorporation into protein. I called Heinz Frankel-Conrat in Berkeley, a world expert on TMV who hada mutant with an amino acid replacement in the viral coat protein, to tell http:/tibs.trends.com TRENDS in Biochemical Sciences Vol.29 No.1 January 2004 him our results. He invited me to come to his laboratory to synthesize radioactive protein directed by RNA from wild- type and mutant TMV, with the intention that he and a colleague would purify and characterize the products to determine whether the radioactive protein synthesized was TMV coat protein. I felt like Marco Polo exploring a new area. Before going to Frankel-Conrat’s laboratory I obtained some poly(U) and instructed Heinrich to make 20 different solutions, each with 19 cold amino acids and one radioactive amino acid, to detect poly(U)-dependent incorporation of a single radioactive amino acid into protein. After working in Frankel-Conrat’s laboratory for about a month, Heinrich called me very excitedly to tell me that poly(U) was extraordinarily active in stimulating the incorporation of only phenylalanine into protein [8] (Figure 3). I immediately returned to Bethesda. We also showed that single-stranded poly(U) functions as mRNA, but double-stranded or triple-stranded poly(U)—poly(A) helices do not [8] (Figure 4). This was the first RNA antisense experiment. In addition, we showed that poly(C) directs the incorporation only of proline into protein [8]. I thought the poly(U) result wouldn’t be believed unless we characterized the radioactive polyphenylalanine pro- duct of the reaction very carefully. As shown in Table 1, hydrolysis of the 1C-labeled polyphenylalanine by HCl recovered stoichiometric amounts of ‘C-labeled phenyl- alanine. I also thought we should show that the solubility of the '4C-polyphenylalanine was the same as that of authentic polyphenylalanine, but because I knew nothing about this I went to Chris Anfinson’s laboratory, which was directly under mine, to ask for names of investigators who         ao 120 ul ke <t & & 100 fod S Poly U z Mo PolyU + Poly e us yw 80 a = pod 4 1 60 >» =z w x a. zt 40} oy x 20F Paly Ut Poly A 0 i? igh i 9% 12.5 25 37.5 mp MOLES [pA] or [pC}       Figure 4. Addition of poly(A) completely inhibits the mRNA activity of poly(U) by the formation of double-stranded and triple-stranded helices. By contrast, addition of poly(C) has little effect on the MRNA activity of poly(U). This experiment, done in 1961, was the first the anti-sense RNA experiment [8].     TRENDS in Biochemical Sciences Vol.29 No.1 January 2004 49 Table 1. Characterization of “C-labeled polyphenylalanine, the synthesis of which is dependant upon poly(U): comparison of characteristics of product of reaction and poly-L-phenylalanine [8]   Treatment 6 N HCI for 8 h at 100° 12 N HCl for 48 h at 120-130° Extraction with 33% HBr in glacial acetic acid Extraction with the following solvents: H2O, benzene, Product of reaction Poly-L-phenylalanine   Partially hydrolyzed Completely hydrolyzed Soluble Insoluble Partially hydrolyzed Completely hydrolyzed Soluble Insoluble nitrobenzene, chloroform, N,N-dimethylformamide, ethanol, petroleum ether, concentrated phosphoric acid, glacial acetic acid, dioxane, phenol, ecetone, ethyl acetate, pyridine, acetophenone, formic acid       might have characterized polyphenylalanine. Michael Sela was the only person in the laboratory at the time; I knew that he worked with synthetic polypeptides so I asked if he knew anything about the solubility of polyphenylalanine. He said ‘I do not know much, but I can tell you two things: one, polyphenylalanine is insoluble in most solvents; and second, it does dissolve in 15% hydrobromic acid dissolved in concentrated acetic acid.’ I looked at him in delight as well as astonishment because I had never heard of such a solvent. Fifteen years later I learned that Michael Sela was the only person in the world who knew that polyphenylalanine dissolved in this esoteric solution because it is used to characterize C termini of proteins and he had mistakenly added it to polyphenylalanine, which, to his surprise, dissolved. I was scheduled to give a talk in 1961 at the Vth International Congress of Biochemistry in Moscow. Just before leaving for Russia, I married Perola Zaltzman, a biochemist from Rio de Janeiro who worked with Sidney   Soe Figure 5. A picture of Heinrich Matthaei (left) and myself in 1962.       http:/tibs trends.com   Udenfriend at the NIH, and we planned to meet for a leisurely, two week vacation after the meeting. I gave my talk in Moscow to an audience of ~ 35 people [11]. However, Francis Crick invited me talk again in a large symposium that he was chairing on nucleic acids, which I did to an extraordinarily enthusiastic audience. After returning to Bethesda, Fritz Lipmann generously gave me a partially purified transfer enzyme and we showed that phenyl- alanine-tRNA is an intermediate in the synthesis of polyphenylalanine directed by poly(U) [12]. A picture of Heinrich Matthei and me taken in 1962 is shown in Figure 5. Soon afterwards I gave a talk at the Massachusetts Institute of Technology. A few years earlier Severo Ochoa from New York University had been awarded the Nobel Prize for his discovery, with Marianne Grunberg-Manago, of polynucleotide phosphorylase, which catalyzes the synthesis of randomly ordered polynucleotides. While I was answering questions from the audience, Peter Lengyel came up to the podium and told the audience that he and others in Ochoa’s laboratory had used randomly ordered synthetic polynucleotides that contained several different nucleotide residues to direct the incorporation of other amino acids into protein. I flew back to Washington feeling very depressed because, although I had taken only two weeks to show that aminoacyl-tRNA is an intermediate in protein synthesis, I should have spent the time focusing on the more important problem of deciphering the genetic code. Clearly, I had to either compete with the Ochoa laboratory or stop working on the problem. The next morning (Saturday) I went to the library in Leon Heppel’s corridor to look up methods of synthesizing, purifying and characterizing randomly ordered poly- nucleotides whose synthesis was catalyzed by polynucleo- tide phosphorylase. Robert Martin (whose wife writes the Miss Manners newspaper column) was in the library and, when I told him what had happened, he suggested we synthesize randomly ordered polynucleotides that week- end. And that is exactly what we did. Bob, who is a superb, energetic investigator, stopped his own work and during the next few months synthesized and characterized many randomly ordered polynucleotides. Bob Martin played a major role in deciphering the genetic code. Between 1961 and 1964 Bill Jones, Bob Martin and I determined the base compositions of RNA codons by directing amino acid incorporation into protein using many randomly ordered polynucleotide preparations that contained different combinations and proportions of bases [18-19]. In Figure 6 are the minimum species of bases required for mRNA codons. Although Severo Ochoa was a       Polynucleotides Amino acids vu PHE Cc PRO A LYS G a uc LEU SER UA LEU TYR ILE ASN UG LEU VAL cys TRP CA HIS THR GLN ASN cG ARG ALA AG ARG GLU UAG ASP MET CAG ASP SER       Figure 6. The specificity of randomly ordered polynucleotide templates in stimulat- ing amino acid incorporation into protein in Escherichia coli extracts. Only the minimum kinds of bases necessary for template activity are shown, so many amino acids that respond to randomly ordered polynucleotides composed of two or more kinds of bases are omitted. The base compositions of RNA codons were derived from these experiments [49]. Reproduced from Ref. [49]. fierce competitor during this time [20-28], we had never met. So, in 1961 I called him when I was in New York to arrange a meeting. I thought it would be more civilized to cooperate, or perhaps split the problem in some way, rather than compete with one another. Ochoa was very gracious, he invited me to his laboratory and introduced me his post-doctoral fellows, and we had tea in the library. However, there was no way we could collaborate. Later, to my horror, I found that I enjoyed competing. I focused on solving the problems that we were investigating and on working more effectively, rather than on winning or losing. The competition stimulated me to become more focused and I accomplished far more than I would have in its absence. From the beginning I vowed never to cut corners or reduce the rigor with which experiments were done to win the competition. Therefore, the quality of our work remained high throughout the deciphering process. Years after the genetic code was deciphered, Mirko Beljansky told me that during a year’s sabbatical in Ochoa’s laboratory, Ochoa suggested that he see whether synthetic polynucleotides could direct cell-free protein synthesis in E. coli extracts. Beljansky spent a year trying to direct cell-free protein synthesis with poly(A), but was unsuccessful because the polylysine product of the reaction is a basic protein and is not precipitated by trichloracetic acid. Similarly, in Jim Watson’s laboratory, Tissieres also tried to direct cell-free protein synthesis with poly(A), but did not detect the polylysine product for the same reason. Therefore, the idea of using synthetic polynucleotides to direct cell-free protein synthesis origin- ated independently in at least three laboratories. We showed that the code is formed of triplets by the amounts of radioactive histidine, threonine, asparagine, glutamine, lysine and proline that were incorporated into protein by five poly(A-C) preparations that contained different ratios of A and C [18,19] (Figure 7). The data for lysine and proline are not shown. The observed codon frequencies were compared with the theoretical frequen- cies of triplets or doublets in each poly(A-C) preparation calculated from the base composition determined for each http://tibs.trends.com TRENDS in Biochemical Sciences Vol.29 No.1 January 2004 polynucleotide preparation. The data showed that the codon for histidine is a triplet that contains one A and two Cs and the codons for asparagine and glutamine are triplets that contain two As and one C. The data also showed that two triplets or a doublet correspond to threonine. A group picture of the people in my laboratory and their spouses taken in early 1964 is shown in Figure 8. There were ~ 20 people in my laboratory in all who deciphered the genetic code and about half of them (and their spouses) are in this picture. The occasion was a party for Brian Clark (middle of the first row in a suit), who was returning to Cambridge, England after several years in my labora- tory. The post-doctoral fellows who came to my laboratory were superb and our work on deciphering the genetic code was very much a group project. Although we and Ochoa’s group had deciphered the nucleotide compositions of RNA codons, the nucleotide sequences were unknown. We tried several ways to solve this problem, but were unsuccessful. However, Kaji and Kaji [29] had shown that poly(U) stimulates the binding of radioactive polyphenylalanine-tRNA to ribosomes and Regina Cukier in my laboratory was using randomly ordered polynucleotides to direct the binding of aminoacyl- tRNA to ribosomes. I wondered how small a message one could use that would remain functional in directing     a----» Theoreticol Frequency of RNA Codewords crmmne Observed Frequency of 30F Ac Amino Acid fncorporation d or AACHACE nom mnm nn ” ~     PERCENT CODEWOROS IN POLY AC       ‘ ~.\\\\ o 16 20 3S 40 50 60 WD 80 90 100 PERCENT C IN POLY AC ° 225 1 i i i 4. L i         Figure 7. The theoretical frequencies of RNA codons in randomly ordered poly(AC) preparations that contain different proportions of A and C, compared with the observed frequencies of incorporation of radioactively labeled amino acids into protein. The codon for histidine contains one A and two Cs, and the codons for asparagine and glutamine contain two As and one C. These results showed that the code is a triplet code [18,19]. Reproduced from Ref. [18].     TRENDS in Biochemical Sciences Vol.29 No.1 January 2004 51           Figure 8. Going away party for Brian Clark who was returning to Cambridge, England after several years in the laboratory. The photograph was taken early in 1964. This is the only group photograph | have of the people in my laboratory while we were deciphering the genetic code. From teft to right: Sid and Joan Pestka, Mrs. Marshall and Dick Marshail, Tom Caskey (partially hidden), Ty Jaouni, Mrs. Rottman, Norma Heaton (the marvelous technician with whom | worked for 39 years), Fritz Rottman, Brian Clark, Phil Leder, Shirley Shapiro (the secretary for the laboratory), Joel Trupin, Mrs. Trupin, myself, Bill Groves, Perola Nirenberg, Mrs. O'Neill and Charlie O'Neill. aminoacyl-tRNA binding to ribosomes. Leon Heppel gave me a doublet, a triplet and a hexanucleotide that consisted only of U residues. I thought that ternary complexes, such as a°H-phenylalanine-tRNA—UUU trinucleotide—ribosome complex, would be retained by Millipore filters, whereas 3H-phenylalanine-tRNA would be washed through the filter. The first experiment worked beautifully; the tri- nucleotide, UUU, stimulated binding of 7H-phenylalanine- tRNA to ribosomes, but the doublet, UU, was without effect [30]. A similar experiment with oligo(A) prepar- ations [49] is shown in Figure 9. It was clear that we could use this technique to determine the nucleotide sequences of RNA codons. However, most of the 64 trinucleotides       [4C]-Lys-tRNA ADDITION BOUND TO RIBOSOMES umoles ApA 0.01 ApApA 1.92 ApApApA 1.92 ApApApApA 1.92 ApApApApApA 2.71         Figure 9. Nucleotide sequences of RNA codons were determined by stimulating the binding to ribosomes of an appropriate species of radioactive aminoacyl-tRNA which recognized the trinucleotide. The aminoacyl-tRNA-trinucleotide—ribosome ternary complex was adsorbed on a Millipore filter and unbound aminoacyl-tRNA was removed by washing [30,49]. http://tibs.trends.com had not been synthesized previously or isolated. Our major problem was to devise methods to synthesize trinucleotides of known sequence. Phil Leder saw an advertisement offering 0.5 g of each of the 16 doublets for $1500 per doublet. Although expensive (a few years earlier my salary for 6 months was $1500), we brought the lot. How- ever, we received only 15 doublets because the US Customs Service confiscated one and used it all to test for drugs. Marianne Grunberg-Manago was visiting Maxine Singer at that time, and both were experts on the use of polynucleotide phosphorylase. Phil Leder and Richard Brimacombe from my laboratory joined them in optimizing conditions for the polynucleotide phosphorylase catalyzed synthesis of oligoribonucleotides from doublet primers [31]. Trinucleotides then were separated from oligonucleo- tides of different chain lengths by electrophoresis. In addition, in 1964, Leon Heppel (who was one of the best nucleic acid biochemists in the world at the time) suggested an esoteric method for the synthesis of tri- nucleotides and higher homologues, reported in a single sentence in one of his papers [32]. He showed that, in the presence of a high concentration of methanol, pancreatic RNase A catalyzes the synthesis of trinucleotides and higher homologues from oligoribonucleotide primers and pyrimidine 2’-3'-cyclic phosphates. We used both methods to synthesize triplets, as shown in Figure 10. Polynucleo- tide phosphorylase catalyzes the addition of nucleotide residues to the 3'-ends of doublets and other oligonucleo- tide primers, whereas pancreatic RNase A catalyzes the addition of nucleotides to the 5’ termini of oligonucleotide primers. Maxine Singer, Marianne Grunberg-Manago, and Leon Heppel, as well as Phil Leder, Mert Bernfield and Richard Brimacombe made major contributions to deciphering the genetic code. Bob Martin, Leon Heppel, and Maxine Singer are shown in Figure 11, and pictures of Phil Leder and Tom Caskey are shown in Figure 12.     82 BReview | TRENDS in Biochemical Sciences Vol.29 No.1 January 2004   POLYNUCLEOTIDE PHOSPHORYLASE Mg* + (1) ApG + UDP —————=—————— ApGpu + ApG(pU},+ P RNase A (2) ApG + URIDINE-2',3'- meeenye a UpAnG + (Up ApG CYCLIC PHOSPHATE PAPG + (Up).Ap       Figure 10. In 1964, most of the 64 possible trinucteotides had been neither syn- thesized nor isolated. The major problem was to devise methods for the synthesis of trinucleotides. The two enzymatic methods that we used are shown. (1) Maxine Singer (an expert on polynucleotide phosphorylase), Marianne Grunberg-Manago (who had discovered polynucleotide phosphorylase while in Severo Ochoa's lab- oratory), Philip Leder and Richard Brimacombe discovered conditions for the addition of one or a few nucleotide residues to the 3'-end of doublet primers, cata- lyzed by polynucleotide phosphorylase {31]. (2) Leon Heppel suggested an esoteric method for triplet synthesis that he, Whitfield and Markham had discovered in which pyrimidine-2', 3'-cyclic phosphates are added to the 5’-ends of doublet pri- mers catalyzed by pancreatic RNase A in the presence of a high concentration of methanol [32]. Merton Bernfield synthesized half of the trinucleotides in our lab- oratory using this method. It took my colleagues, Philip Leder, Merton Bernfield, Joel Trupin, Sid Pestka, Fritz Rottman, Richard Brimacombe, Charles O’Neal, French Anderson, Don Kellogg, Ty Jaouni and myself a year to synthesize the 64 trinucleotides and test each against 20 different radioactive aminoacyl-tRNA preparations to decipher the nucleotide sequences of RNA codons [30,33,35—40]. Philip Leder deciphered the first. nucleotide sequence of a valine RNA codon using trinucleotide-dependent binding of aminoacy]- tRNA toribosomes [33]. We soon found that the third bases of synonym RNA codons varied systematically [33,35—40] and, using purified aminoacyl-tRNA fractions [39-41], we found four patterns of degeneracy, as shown in Figure 13. Francis Crick coined the term ‘wobble’ to describe the third-base degeneracy of RNA codons [42]. Generously, Robert Holley gave us a highly purified fraction of yeast alanine-tRNA that he and his colleagues had sequenced in what was the first determination of the nucleotide sequence of a nucleic acid (43]. Philip Leder and I showed that this species of alanine- tRNA recognizes the codons GCU, GCC and GCA, and that the nucleotide residue in the tRNA anti-codon that recog- nizes U, C or A in the third position is hypoxanthine [40].           Figure 12. Phil Leder came to my laboratory as a post-doctoral fellow and played a major role in deciphering the genetic code. He was the first to decipher the nucleo- tide sequence of a codon [33]. Tom Caskey was another post-doctoral! fellow in my laboratory. Together with Dick Marshall, he compared the genetic code of Escheri- chia coli with that of Xenopus and hamsters and showed that the code is universal [41]. Later, Tom Caskey and his colleagues worked on the mechanism of termin- ation of protein synthesis. After we had published the nucleotide sequences of 54 of the 64 RNA codons [", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-n52j~jedu-nfmm", "00000000-0000-0000-C7F1-7A18E36DCFD1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Theodore M. Bayless to Victor A. McKusick", "101584910X484", null, "1971", "12 February 1971", null, "Letters (correspondence)", "Congenital Abnormalities,Gastrointestinal Tract", "The Bar Harbor Course and \"McKusick's Catalog,\" 1960-1980", "2", "pages", "Text", "English", "Reproduced with permission of Theodore M. Bayless.", "Copyright may apply", null, null, "February 12, 1971 Re: Fourth annual conference on clinical delineation of birth defects, June, 1971 Dear Dr. McKusick: I will be pleased to participate in your conference. You had asked for suggestions as to topics or participants for the area of digestive and absorptive functions of the GI tract such as alterations in the digestive ability of the small intestine mucosa. You are covering this with the disaccharidase deficiencies. Some interests in this area include fetal development and changes in lactase levels after birth as well as the prevalence in adults. There is also some recently accumulating information on peptidase deficiencies and defects in amino acid absorption. Along the lines of absorptive defects, the glucose-galactose malabsorption is an excellent example. In terms of defects in mucosal synthesis, a-Beta lipoproteinemia is a good example. If you wish I could certainly cover all of these topics. Another area would be familial chloridorrhea. These patients are unable to reabsorb chloride and have a severe diarrhea and electrolyte inbalance. In terms of the pancreas, there are some children with hereditary pancreatitis and some with absence of the pancreas. I think that this could easily be covered by your cystic fibrosis people. The area of the development of the stomach is being explored by Dr. Julius Deren of the University of Pennsylvania. There are no major clinical problems that come to mind, so this area is probably not vital. It could also be covered very briefly in a discussion of the small bowel development. Dr. Deren is also interested in the small bowel. There are a number of other people who could easily speak on the small bowel, but I think that most of this could be included in the talk on disaccharidases. As I'm writing this, I wonder if celiac disease should not be mentioned because this certainly seems to be an inherited disorder and the defect is probably present at birth, but does not become clinically apparent until the child is about 8 months of age when he begins to consume large amounts of wheat, rye and oats. The current going thoughts are that this might be due to a peptidase deficiency and inadequate digestion of some of the wheat and peptides. This has not been proven but it does make a nice story. Obviously Tom Hendrix or I could talk about this. Dr. Dowd Kowlessar-Jefferson in Philadelphia is extremely interested in the peptide but has been unable to come up with a specific peptidase deficiency at this time. Pardon my rambling. I hope this is of some help to you. Sincerely, Theodore M. Bayless, M.D.", "Bayless, Theodore M. ; Johns Hopkins University. School of Medicine", "Alan Mason Chesney Medical Archives. Victor Almon McKusick Collection", null, null, "McKusick, Victor Almon, 1921-2008", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4f2y_eycc~9nnf", "00000000-0000-0000-C0AE-71AD4F1ACBAA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Characteristics and Stabilization of DNAase-Sensitive Protein Synthesis in E. Coli Extracts", "101584910X49", "101584910X50", "1961", "October 1961", "This innovative article is one of Nirenberg's most important early contributions to the coding problem.  Published along with \"The Dependence of Cell-Free Protein Synthesis in E. coli Upon Naturally Occurring or Synthetic Polyribonucleotides,\" this article provides experimental evidence that an extract prepared from bacterial cells can make protein even when intact living cells are not present.  This revelation is a crucial step in the history of breaking the genetic code.", "Articles", "Escherichia coli,Deoxyribonucleases", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "9", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Reprinted from the Proceedings of the Nationa, ACADEMY OF SCIENCES Vol. 47, No. 10, pp. 1580-1588. October, 1961. CHARACTERISTICS AND STABILIZATION OF DNAASE-SENSITIVE PROTEIN SYNTHESIS IN E. COLI EXTRACTS By J. Hemvrich Marruarr* Anp MARSHALL W. NIRENBERG NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by Joseph E. Smadel, August 3, 1961 It has been assumed for many years that in protein synthesis the base sequence of DNA specifies the base sequence of RNA and that RNA in turn controls the amino acid sequence of protein. In accord with this notion, several groups recently have observed an inhibition of amino acid incorporation into protein by DNAase in cell-free extracts.'~* One object of the present investigation was to study this phenomenon further. A major difficulty in the study of cell-free protein synthesis in H. coli systems has been the necessity for preparing fresh enzyme extracts for each experiment. Techniques have not been available for stabilization and storage of enzyme ex- tracts comparable to the techniques available for mammalian systems.‘ In the present communication, an amino acid-incorporating system stable to storage for several months will be described. The characteristics of amino acid incorporation into protein by the stored extracts were investigated also. A part of these data has been presented in a preliminary report.? Methods and Materials.—E. coli W3100 cells, harvested in early log phase, were washed by centrifugation and disrupted by grinding with twice their wet weight of alumina A301 (Aluminum Corporation of America) for 5 min at 5°. All subsequent steps were performed at this tempera- ture. The enzymes were extracted with buffer containing 0.01 M Tris(hydroxymethy])amino- methane, pH 7.8; 0.01 M magnesium acetate; 0.06 I KCl; and 0.006 M mercaptoethanol (standard buffer) equivalent to two or three times the wet weight of cells. The extract was centrifuged three times at 30,000 x g for 20, 20, and 60 minutes, respectively. The pellets were discarded after each centrifugation. The final supernatant fluid (S-30) was centrifuged at 105,000 X g for 2 hr in the Spinco Model L ultracentrifuge to sediment the ribosomes. The supernatant solution (S-100) was aspirated, and the ribosomes were suspended in standard buffer by gentle homogenization in a Potter-Elvehjem homogenizer and were washed by centrifuging again at 105,000 & g for 2 hr. The supernatant fluid was decanted and discarded, and the ribosomes (W-Rib) were suspended in the original volume of standard buffer. Fractions 8-30, 8-100, and W-Rib were dialyzed against 60 volumes of standard buffer overnight at 5° and were stored in small aliquots at — 15° until needed. DNAase I, RNAase, and trypsin were crystalline preparations obtained from the Worthington Vow. 47, 1961 BIOCHEMISTRY: MATTHAEI AND NIRENBERG 1581 Biochemical Co. The sodium salt of polyadenylic acid (Batch BC-5815-2, Miles Chemical Co.) had a molecular weight of approximately 30,000. U-C1+L-valine (6.5 mC/mM) was obtained from Nuclear-Chicago Corp. Polyglucose carboxylic acid (molecular weight approximately 30,000) was a gift from Dr. Peter Mora. Puromycin was a gift obtained from Dr. Arthur Weiss- bach. Chloramphenicol was obtained from Parke Davis & Co., and highly polymerized salmon sperm DNA from California Foundation for Biochemical Research. Proteolytic enzymes as contaminants of the DNAase were assayed by the method of Anson‘ using denatured hemoglobin (Nutritional Biochemical Co.) as the substrate. RNAase contamina- tion of DNAase was assayed by incubation of DNAase with purified RNA, precipitation with cold trichloroacetic acid, and determination of the absorbancy at 260 my of the supernatant solu- tion obtained after centrifugation. The DNAase digest of DNA was prepared by incubating 5 mg/ml salmon sperm DNA with 10 pg/ml DNAase for 6 hr at 35° in 20 wmoles/m] phosphate buffer, pH 7.0, and 20 umoles/ml magnesium acetate. DNAase was destroyed by three deproteinizations according to the method of Sevag,7 and traces of solvents were removed by bubbling N, through the solution. Protein was determined by a micro modification of the method of Lowry. The synthetic amino acid solution used contained 1 wmole/m] of each of the following L-amino acids: glycine, alanine, serine, aspartic acid, asparagine, glutamic acid, glutamine, isoleucine, leucine, cysteine, cystine, histidine, tyrosine, tryptophan, proline, threonine, methionine, phenylalanine, arginine, and lysine. The complete reaction mixture is shown in the legend for Table 1. TABLE 1 CHARACTERISTICS OF C!4L-VaLiInE INCORPORATION INTO ProTEIN By CELL-FREE, L. colt ENnzyYME PREPARATIONS STORED FOR SEVERAL WEEKS AT — 15° Experiment Counts/min/mg no. Additions protein 1 Complete 679 — 105,000 X g supernatant solution 74 “ ~— Ribosomes 15 “ — ATP, PEP, PEP kinase 38 “ + 10 we RNAase 9 “ — Amino acid mixture 365 “ + 0.02 wmole Puromycin 38 7 + 0.30 »pmole Chloramphenicol 82 “ — 0.03 umole GTP, CTP, UTP 583 “ — 0.03 pmole CTP, UTP 652 “ Deproteinized at zero time 4 2 Complete 2078 a + 10 ug DNAase 1223 . Deproteinized at zero time 5 The reaction mixtures contained the following in ~xmole/ml: 100 Tris (hydroxymethyl)aminomethane, pH 7.8; 10 magnesium acetate; 50 KCl; 6.0 mereaptoethanol; 1.0 ATP; 5.0 phosphoenolpyruvate, K salt; 20ug¢ phos- phoenolpyruvate kinase, crystalline; 0.05 each of 20 L-amino acids minus valine; 0.03 each of GTP, CTP, and UTP; 0.015 C!41-valine (~70,000 counts); 1.0 and 0.7 mg 8-100 and W-Rib protein, respectively were present per reaction mixture in Experiment 1. 2.1 mg 8-30 protein were present in Experiment 2. Total volume was 1.0 ml. Samples were incubated at 35° for 60 min, were deproteinized with 10 per cent trichloroacetic acid, and the precipi- tates were washed and counted by the method of Siekevitz.13 Planchets were counted in a Nuclear-Chicago gas flow counter with a Micromil window and a counting efficiency of approximately 30 per cent. All assays were performed in duplicate. Resulis.—Stabilization of cell-free extracts: The effects of dialysis and freezing upon the ability of the $-30 fraction to incorporate C'+-L-valine into protein are presented in Figure 1. No enzymatic activity was lost after overnight dialysis. If mercaptoethanol was omitted from the dialyzing buffer, rapid loss of activity was observed. Reduced glutathione was not quite as effective as mercaptoethanol in preventing loss of activity. Dialyzed 8-30 fractions were divided into aliquots and were stored at —15°. The results of Figure 1 demonstrate that the enzyme, after storage for twenty- four hr at —15°, was as active as fresh 8-30. Frozen preparations lost less than 10 per cent activity per month. 1582 BIOCHEMISTRY: MATTHAEI AND NIRENBERG Proc. N. A.S.     2400 DIALYZED 4 a FRESH DIALYZED FROZEN T T T T T 2000 [ T T T T i 1 |    1600 0 Qo °° ° + FRESH 1200 DIALYZED + 1600 DIALYZED 800 FROZEN 4 '200 400 COUNTS/MINUTE /MG. PROTEIN a Qo °       COUNTS/MINUTE/MG. PROTEIN       400 7 MINUTES | Fig. 2.—The effects of dialysis and o | bow L 1 freezing upon C!*-L-valine incorporation ° 1s 30 45 60 7s into protein using washed. ribosomes (W- MINUTES Rib) and 105,000 x g supernatant solu- tions (S-100). @ Fresh W-Rib and 8- Fig. 1.—The effects of dialysis and freezing 100; A W-Rib and 8-100 dialyzed sepa- upon C!_L-valine incorporation into protein in rately for 12 hr; oO W-Rib and S-100 30,000 x g supernatant layer fractions (S-30). after dialysis and storage separately at @ Fresh 8-30; oO 8-30 after 12 hr of dialysis: —15° for 24 hr. The composition of the A 58-30 after dialysis and storage at —15° for 24 reaction mixtures is presented in Table 1. hr. The composition of the reaction mixtures is 0.9 and 1.0 mg W-Rib and 8-100 protein presented in Table 1. 2.1 mg 8-30 protein were respectively were present in each reaction added to each reaction mixture. mixture. When washed ribosomes (W-Rib) and 105,000 x g supernatant fluid (S-100) were stored separately or recombined at —15°, some activity was lost compared to the 8-30 fraction (Fig. 2). No loss in enzymatic activity of fractions S-100 or W-Rib was observed after overnight dialysis. Again, addition of mercapto- ethanol prevented rapid inactivation. Storage of the fractions separately at —15° resulted in a loss of approximately 25 per cent of the activity. The enzyme fractions lost less than 5 per cent of their activity per week, and fractions stored for several months were routinely used in these studies. Figures 1 and 2 also demonstrate that of the total incorporation obtained after incubation for one hour, approximately 50 per cent occurred within the first 15 min. The characteristics of C'*-L-valine incorporation into protein by S-100 and W-Rib fractions stored at —15° for several weeks are presented in Table 1. The rate of incorporation in the absence of either S-100 or W-Rib fractions was negligible. Experiments of this type strongly suggested that W-Rib fractions were not con- taminated with intact cells, and this check was routinely performed with each enzyme preparation. Effects of additions and deletions: Incorporation was dependent upon addition of ATP and an ATP-generating system, and incorporation was completely inhibited by the addition of RNAase. Omitting a mixture of 20 L-amino acids from the reaction mixture resulted in a 46 per cent decrease in incorporation of C\\\\-valine into protein, suggesting de novo synthesis of protein. The dependence of C'4-L- valine incorporation upon addition of the amino acid mixture was observed only when well-dialyzed preparations were used. Addition of 0.02 umoles puromycin or 0.30 umoles chloramphenicol/ml to the reaction mixture depressed amino acid incorporation. Puromycin was a better inhibitor of C-valine incorporation Vou. 47, 1961 BIOCHEMISTRY: MATTHAEI AND NIRENBERG 1583 than chloramphenicol. Omission of guanosine-5’-triphosphate (GTP), cytidine- 5’-triphosphate (CTP), and uridine-5’-triphosphate (UTP) resulted in a slight inhibition of C'-valine incorporation. However, addition of GTP alone largely replaced the mixture of three triphosphates. Experiment 2, also in Table 1, demonstrates that addition of 10 yg of crystalline DNAase markedly inhibited Ci-valine incorporation. Further experiments dealing with this effect will be discussed later. That the incorporation of C1*-valine into protein depends upon the presence of the S-100 fraction is further documented in Figure 3. Little C'-valine was in- corporated into protein when 0.7 mg W-Rib protein alone was used. The incorpo- ration was proportional to the amount of 8-100 fraction added up to 2.5 mg S-100 protein. In Figure 4 are presented data demonstrating the dependence of C#4-valine             PI 23800 - 4 2600 }- 4 2400 [- 4 2200 F 4 2000 - 4 w ~ 1800 4 2a < 5 1600 + 4 2 1400 + 4 < Ld 3 1200 > . 4 2 tooo 4 = a Boo 4 S Zz 600 4 Ss 400 + 4 200 4 oO « L L { 1 oO 10 20 3.0 4.0 1 MG.105,000XG SUPERNATANT 6 \\\\ i Lt L FLUID PROTEIN 9 ! 2 3 4 MG. RIBOSOMAL PROTEIN Fic. 3.—The dependence of C1L- . valine incorporation into protein upon _ Fig. 4.—The dependence of incorpora- the amount of 105,000 X g supernatant tion of C1*-L-valine inte protein upon E. solution (8-100). The composition of colt ribosome concentration. The compo- the reaction mixtures is presented in sition of the reaction mixtures is presented Table 1. 0.7 mg ribosome protein in Table 1. 1.0 mg 105,000 x g super- (W-Rib) were added to each sample. natant solution protein (S-100) was added Reaction mixtures were incubated for to each sample. Reaction mixtures were 60 min at 35°. incubated for 60 min at 35°. incorporation upon W-Rib fractions. No incorporation by 1.0 mg 8-100 protein alone was observed. C1+valine incorporation was proportional to the amount of W-Rib added within the range of 0-1.0 mg ribosomal protein. The effect of pH upon C'-L-valine incorporation into protein is presented in Figure 5. A sharp pH optimum was observed with maximal incorporation at pH 7.8. DN Aase effect: The effect of DNAase upon C!-valine incorporation over a 90-min incubation period is presented in Figure 6. In the absence of DNAase, 1584 BIOCHEMISTRY: MATTHAEI AND NIRENBERG Proc. N. A. 8. COUNTS/MINUTE/MG. PROTEIN 1600 1400 1200 1000 800 400 200         L 4 Mo 7.0 7.4 7.8 8.2 86 3.0 pH Fig. 5.—The effect of pH upon C!*-L-valine incorporation into protein. The composi- tion of the reaction mixtures is presented in Table 1. 100 wmoles Tris(hydroxymethy!)- aminomethane were present in each reaction mixture. Samples were incubated for 10 min at 35°. COUNTS/MINUTE /MG.PROTEIN 2000 1500 500 ro ~ ~~ T 1 To TO      ™. - DNAase     Let. dod Fe Ld 20 30 +40 50 60 70 80 30 MINUTES Fic. 6.—The effect of DNAase upon C!*-L-valine incorporation into protein. © Minus DNAase, A + 10 ug DNAase. 2.1 mg 30,000  g supernatant solution protein (8-30) were present in each reaction mixture. The composition of the reaction mixtures is pre- sented in Table 1. Vou. 47, 1961 BIOCHEMISTRY: MATTHAEI AND NIRENBERG 1585   POT TTt 2200 100 4 2000           z S = 90 + 1800 < S _e—— COUNTS / MINUTE /MG. PROTEIN & 80 + 1600 3 ye z ° ao 7O 41400 © S 3 Ss a 6 4 ~ g 60 1200 = = Zz a c 4 = uw 80 1000 a z z S 3 i 40 Tf —~PERCENT INHIBITION = 800 |, a 2a z g Z 30 4600 m K 2 z WW & 20 4 400 ut a 10 4 200   Ll 1 | tf | | | |  S6 3 1 23 45 6 7 8 9 10 #o DNAase Fig. 7.—The effect of increasing concentrations of DNAase upon C!-L-valine incorpora- tion inte protein. A Counts/min/mg protein, @ Percent inhibition of amino acid incor- poration. The composition of the reaction mixtures is presented in Table 1. 2.1 mg 30,000 X g supernatant solution protein (S-30) were present in each reaction mixture. Samples were incubated at 35° for 60 min. the incorporation was more rapid during the first 30 min of incubation than during the next 60 min. At the end of 90 min of incubation, however, incorporation had not stopped. Addition of 10 ug crystalline DNAase/ml of reaction mixture did not affect. the initial rate of incorporation, but incorporation ceased after 30 min. These results demonstrate that reaction mixtures must be incubated for more than 20 min to obtain reproducibly the DNAase effect. The sensitivity of the system to DNAase is presented in Figure 7. When 0.1 ug. DNAase were added, approximately 70 per cent of the maximum DNAase inhibition was obtained. An inhibition that was almost maximal was obtained with 1.0 yg DNAase/ml. Increasing the concentration of DNAase 10-fold beyond this did not appreciably increase the inhibitory effect of DNAase, thus negating the presence of a contaminant inhibitor in the crystalline DNAase preparation. Contamination of DNAase with traces of proteolytic enzymes and RNAase seemed likely, since commercial DNAase is prepared from pancreas. Therefore, different preparations of crystalline DNAase were tested for both proteolytic and RNAase activity. Their purity varied widely. Crystalline DNAase obtained from the Worthington Biochemical Company (Lot No. D692-95-7) was the purest prepara- tion tested and contained less than 0.3 per cent by weight of material which had a proteolytic enzyme activity and less than 0.001 per cent RNAase activity, cor- responding to less than 0.05 yg trypsin and 0.0001 ug RNAase per 10 yg DNAase. In Table 2, the effects of these concentrations of RNAase and trypsin, singly and combined, upon C-valine incorporation into protein are presented. The system 1586 BIOCHEMISTRY: MATTHAEI AND NIRENBERG Proc. N. A. 8. TABLE 2 Tue Errect or RNAasE AND TRYPSIN UPON C-L-VALINE INCORPORATION INTO PROTEIN Experiment Counts/min/mg no. Additions protein L Complete 374 “ + 0.0001 ug RN Aase 352 “ + 0.001 yg RNAase 206 “ +0.01 yg RNAase 69 “ + 0.1 pg RN Aase 18 “ + 1.0 ug RNAase 9 “ Deproteinized at zero time 8 2 Complete 374 “ + 10 ug DNAase 234 “ + 0.05 ug Trypsin 394 “ + 0.05 ng Trypsin + 0.0001 pg RNAase 402 “ Deproteinized at zero time 8 The composition of the reaction mixtures is presented in Table 1. 2.8 mg S-30 protein were added to each reac- tion mixture. Samples were incubated at 35° for 60 min. TABLE 3 Tue Errrecr or PoLyANIONS UPON THE DNAass-INHIBITED INCORPORATION OF C44-L-VALINE INTO PROTEIN AND THE Errect oF 4 DNAass Dicest or DNA upon INCORPORATION Experiment Counts/min/mg no Additions protein 1 Complete 2,040 “ + 10 we DNAase 825 “ + “ “ + 100 xg polyadenylic acid 685 “ “ “ + 100 ug polyglucose car- boxy] derivative 810 “ + 100 ug polyadenyliec acid 2,430 “ + 100 pg polyglucose carboxyl derivative 2,150 “ Deproteinized at zero time 7 2 Complete 1,842 “ + 100 wg DNAase digest of salmon sperm DNA _ 1,825 3 Complete + 10 wg DNAase 604 “ + 1.0 ml reaction mixture incubated with 10 ug DNAase for 60 minutes 661 The components of the reaction mixtures and the incubation conditions are presented in Table 1. 2.0 and 1.0 mg W-Rib and S-100 protein were present in Experiments 1 and 2. In Experiment 3, 2.1 mg 8-30 protein was present in complete systems. 2.1 mg 8-30 protein was also present in reaction mixture incubated with DNAase for 60 min. Samples were incubated at 35° for 20 min. was extremely sensitive to RNAase. As little as 0.001 wg RNAase/ml of final reaction mixture depressed amino acid incorporation. The addition of 0.05 gg of trypsin and 0.0001 hg RNAase (the amount of trypsin and RNAase in 10 pg of the DNAase preparation used) had no effect upon the incorporation. This DNAase preparation was used for all subsequent work with DNAase. It should be emphasized that crystalline DNAase obtained from commercial sources varies widely in its content of trypsin and RNAase and thus should be assayed before use. The data of Table 3 demonstrate that addition of polyanions such as polyadenylic acid and a polymer of glucose carboxylic acid did not reverse the inhibition ob- tained upon addition of DNAase. Higher concentrations of polyanions than those shown in Table 3 were inhibitory. Addition of a DNAase digest of highly poly- merized salmon sperm DNA had no effect upon amino acid incorporation. In Experiment 3, Table 3, an additional experiment of this sort is presented. A reaction mixture containing 2.1 mg S-30 protein was incubated with 10 ug DNAase for 60 min. During this period the DNA contained in the enzyme would have been largely digested. Also, after 60 min, amino acid incorporation into protein had completely ceased. This reaction mixture, containing the endogenous, di- Vou. 47, 1961 BIOCHEMISTRY: MATTHAEI AND NIREN BERG 1587 gested DNA products, was then added to a fresh reaction mixture to see whether products of DNAase digestion were inhibitory. The results show that the prod. ucts of DN Aase digestion were not inhibitory. Discusston.—A considerable amount of evidence has been obtained indicating that C'-valine incorporation into protein in this system was not due to the pres- ence of contaminating intact H. coli cells. The extracts were repeatedly centrifuged at 30,000 X g, and the pellet containing intact cells and debris was discarded. No intact cells or protoplast-like bodies were microscopically visible in the supernatant fluid. Neither ribosomes nor 105,000 < g supernatant solution alone incorporated appreciable quantities of C'-valine. Both fractions were needed. In addition, combined extracts were almost totally inactive if ATP and an ATP-generating system were omitted. The rate of amino acid incorporation procecded rapidly for approximately 30 min and then gradually decreased. The incorporation had many characteristics expected of de novo protein synthesis. It required ATP and an ATP-generating system, was stimulated by a mixture of other L-amino acids, and was strongly inhibited by low concentrations of puromycin, chloramphenicol, and RNAase. In addition, the incorporation could be partially inhibited by addition of DN Aase. The initial rate of incorporation was not inhibited by DNAase, in contrast to the extremely sensitive inhibition of the portion of the incorporation occurring after 20 min of incubation. As low as 0.1 ug DNAase per ml of reaction mixture greatly inhibited the incorporation occurring after 20 min of incubation. Various com- mercial preparations of crystalline DNAase were assayed for contamination with proteolytic enzyme activity and RNAase. Some were heavily contaminated. The maximum amount of trypsin and RNAase present as contaminants in the crystalline DNAase preparation used in this study had little effect upon C1+valine incorporation when added to the system. Furthermore, if a trace contaminant in the DNAase were responsible for inhibiting amino acid incorporation, a cor- respondingly greater inhibition of amino acid incorporation would be expected when high concentrations of DNAase were used. The data of Figure 7 demon- strate that almost maximal inhibition was obtained with 1 yg DNAase per ml of reaction mixture. Increasing the DNAase concentration 10-fold did not appreci- ably increase the inhibition. The data of Table 3 demonstrate that the products of DNAase digestion were not inhibitory in this system and that polyanions cannot non-specifically reverse a DNAase-inhibited meorporation system, in contrast to reports of such non-specific reversal in thymus nuclei.® Although the mechanism of synthesis of template or “messenger” RNA has remained an enigma, enzymes possibly involved in this process are being studied, 8-2 It 1s not possible to say whether intact DNA is necessary for amino acid incorpora- tion into protein in the later stages of incubation in this system. One possibility, however, which is consonant with all of the known facts is that the initial rate of amino acid incorporation is primarily due to the completion of partially finished peptides linked to RNA templates. If template RNA were used only once, amino acid incorporation would cease as soon as the peptide chains were finished. Inhibition by DNAase observed in this cell-free system may be due to the destruc- tion of DNA and its resultant mability to serve as templates for the synthesis of 1588 BIOCHEMISTRY: MATTHAEI AND NIRENBERG Proc. N. A. 8. template RNA. Other explanations, however, are fully plausible, and it is not possible at this state to rule out alternative interpretations. In the following paper, further experiments on amino acid incorporation using the system described here are presented. It will be shown that in addition to the usual requirements, the system is stimulated by template RNA. Summary.—Cell-free H. cola extracts have been obtained which actively in- corporate amino acids into protein. Methods were devised whereby these extracts could be dialyzed and stored for long periods of time at —15° without undue loss of activity. The characteristics of amino acid incorporation by such stored ex- tracts were strongly suggestive of de novo protein synthesis, for incorporation required both ribosomes and 105,000 X g supernatant fractions, ATP and an ATP- generating system, was stimulated by a mixture of other L-amino acids, and was markedly inhibited by puromycin, chloramphenicol, and RNAase. The initial rate of amino acid incorporation was not inhibited by DNAase; subsequent in- corporation was greatly inhibited. The possible relationship of the DNAase inhibition of amino acid incorporation into protein to the synthesis of messenger” RNA was briefly discussed. * Supported by a NATO Postdoctoral Research Fellowship. 1 Tissiéres, A., D. Schlessinger, and F. Gros, these ProcrEepines, 46, 1450 (1960). 2 Matthaei, J. H., and M. W. Nirenberg, Ped. Proc., 20, 391 (1961). 3 Kameyama, T., and G. D. Novelli, Biochem. Biophys. Res. Comm., 2, 393 (1960). 4 Kirsch, J. F., P. Siekevitz, and G. E. Palade, J. Biol. Chem., 235, 1419 (1960). 5 Mora, P. T., E. Merler, and P. Maury, J. Am. Chem. Soc., 81, 5449 (1959). 6 Anson, M. L., J. Gen. Physiol., 22, 79 (1938). 7 Sevag, M. G., D. B. Lackmann, and J. Smolens, J. Biol. Chem., 124, 425 (1938). 8 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, zbid., 193, 265 (1951). 9 Allfrey, V. G., and A. E. Mirsky, these Procrgpinas, 44, 981 (1958). 0 Hurwitz, J., A. Bresler, and R. Diringer, Biochem. Biophys. Res. Comm., 3, 15 (1960). 4} Stevens, A., ibid., 3, 92 (1960). 12 Weiss, S. B., and T. Nakamoto, J/. Biol. Chem., 236, PC18 (1961). 18 Siekevitz, P., ibid., 195, 549 (1952).", "Nirenberg, Marshall W. ; Matthaei, Heinrich", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qqhm~6jq5~s4f5", "00000000-0000-0000-F823-BBBE051E13DC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Qualitative Survey of RNA Codewords", "101584910X51", "101584910X117,101584582X280,101584582X281", "1962", "1962", "In this article, Jones and Nirenberg provide a progress report on code translation efforts.  They find that almost every amino acid tested can be coded by a polymer containing only two bases.  The article includes approximations of nineteen triplet codewords and suggests that any theory concerning the physical basis of the code must explain the available data revealing the high coding efficiency of polynucleotides, marked codeword specificity, and degenerate codewords.  The degeneracy model of Gamov and Watson-Crick pairing are among some of the ideas discussed.", "Articles", "Amino Acids,RNA,Codon,Polynucleotides", "Translating the Code of Life and the Nobel Prize, 1962-1968", "9", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Vor. 48, 1962 BIOCHEMISTRY: JONES AND NIRENBERG 2115 QUALITATIVE SURVEY OF RNA CODEWORDS* By Outver W. Jones, Jr., anp MarsHatt W. NIRENBERG NATIONAL HEART INSTITUTE, NATIONAL [NSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by Richard B. Roberts, October 29, 1962 A degenerate genetic code was suggested a number of years ago by Gamow! and by Crick.? In such a code, an amino acid may be directed into protein by two or more codewords. Previous work demonstrated that C'-amino acids were directed into protein by synthetic polynucleotides in cell-free E. coli extracts.’ and that leucine incorporation was stimulated by either poly CUG,t UC. or UA) Thus the code was shown to be degenerate with respect to leucine.*~* Initially, all of the codewords found contained U. However. assuming a triplet code, the proportion of U compared with other nucleotides in codewords seemed unusually high, for natural template RAXA, such as viral RNA. did not contain such a preponderance of U. To resolve this paradox, a more degenerate code was pro- posed with both non-U and U containing codewords.» An alternative hypothesis was advanced by Roberts, who suggested a doublet code, for in such a code the proportions of nucleotides would be within the range found in viral RNA.” 3 The existence of non-U codewords was suggested when poly AC preparations were found to direct small amounts of proline and threonine into protein.® 7° Recently, in a careful study, Bretscher and Grunberg-Manago clearly demonstrated coding by non-U words. Several poly AC preparations were reported to code well for proline, threonine, histidine, and glutamine. This work indicated that other 2116 BIOCHEMISTRY: JONES AND NIREN BERG Proc. N. A. S. non-U polynucleotides might have template activities. In this communication. further qualitative analvsis of coding by such polynucleotides will be reported. Materials and Methods—The preparation of E. coli extracts (preincubated, DN Aase-treated 830 fractions), and the techniques used in washing, plating, and counting protein precipitates have been reported.*. Polvribonucleotides were synthesized according to the method of Singer and Guss.!! The base-ratio of each polynucleotide preparation was determined by incubation in 0.4 1 KOH at 25° for 18 hours. Under these conditions, little deamination occurred.'? Such mild conditions were not sufficient te hrdrolvze certain polymers: however. in such cases incubation in 0.3 NV KOH at 37° for 18 hours resulied in complete hvdrolvsis.1* Mononucleotide products were separated either by paper electrophoresis (Whatman No. 3 MM paper, 0.03947 ammonium formate. pH 3.7: or by descending paper chromatography (Whatman No. 3 M™ paper and a solvent system containing 0.1 M sodium phosphate, pH 7.0. and 3 M ammonium sulfate}. Two per cent or greater contamination of polynucleotides by U would have been detected. No contamination by U was found. Reaction mixtures used to determine C’-amino acid incorporation into protein contained the following components: 0.1 M Tris ( bydroxymethvlaminoethane} pH 7.8: 0.01 Af magnesium acetate: 0.05 M KCl: 6 x 10-* M mercaptoethanol; 1 x 107° M ATP: 5 Xx 10-3 M potassium phosphoenolpyruvate; 20 ng/ml of crystalline phosphoenolpytuvate kinase (California Biochem. Corporation); 0.8 X 10-4 M C-amino acid (approximately 60,000 counts /minute ‘reaction mix- ture); 3.2 x 10-4 M each of 19 C'%1-amino acids minus the C-amine acid: 10 xg of polwnucles- tide /reaction mixture where specified: and E. coli preincubated S-30 extracts (3-2 mg protein reaction mixture). Total volume of each reaction mixture was 0.3 mi. Reaction mixtures were incubated at 37° for 30 minutes; thus total incorporation rather than rate was measured. Stimulation of amino acid incorporation by polynucieotides containing four bases: The data of Table 1 demonstrate that synthetic polynucleotides containing four bases stimulate the incorporation of a large number of amino acids into protein. In TABLE 1 StimcLaTION OF AMINO Acrip IncorPoRATION By Poty UGAC Polynucleotide UGAC TGAC UGAC TGAC UGAC UGAC U 55 UC 56 G8 U 23 UC 2 U 27 G 32 G 25 G 45 G 21 G 22 G 43 Minus Base-ratio A 5 A 5 A @ A 4 A 22 A 2) polynucleotide moles per cent c 68 Cc 33 Cc 43 Cc 52 Cc 2 Cc 69 control Designation Ap231 Ap232 Ap2338 Ap234 Ju258 3u2510 C)camino acid Incorporation above control (Auumoles)* Alanine 110 127 62 152 31 5 10 Arginine 69 270 68 212 99 57 1] Aspartic acid™® 10 40 9 10 25 12 12 Glutamic acid* 16 52 12 9 14 a 2: Glvcine 62 663 25 40 12 6 13 Histidine 20 8 1 24 13 0 4 Isoleucine 68 301 60 90 0 0 22 Leucine - 168 1.243 125 418 12 0 41 Lysine 10 21 0 3 25 . 4 Methionine 9 64 0 9 15 4 12 Phenylalanine 152 606 86 64 14 0 10 Proline 50 140 125 1.007 121 10 7 Serine 179 ‘7 181 445 30 6 47 Threonine 15 54 19 78 44 0 7 Tryptophan 23 & 16 8 1 Lo. 45 Tyrosine 14 80 1] 12 6 0 7 Valine 100 602 57 70 43 13 7 Total 1,075 5.086 867 2,651 512 107 292 * Aupmolee represents the difference between C!t amino acid incorporation into protein in the presence and absence of polynucleotides. . The components of the reaction mixtures are presented under Methods and Materials. The specific radio activies of C4amino acids varied from 2.0 to 9.4 mcuries,/mmole. 100 ~pmoles of C}+aminc acid is equivalent to 420 counte/minute obtained with a Nuclear-Chicago thin- window, gas fiow counter. Basal incorporations obtained when polynucleotides were omitted are presente in the last column (Minuz polynucieotide). Vow. 48, 1962 BIOCHEMISTRY: JONES AND NIRENBERG 2117 the last column is given the basal level of C'*-amino acid incorporation obtained in the absence of polynucleotide; other figures refer to the net increase above basal incorporation due to addition of polynucleotide. The base-ratios of the polynucleo- tides vary widely. The fifth polynucleotide (J u-258) contains approximately equal proportions of U, G, A, and C, whereas the other polynucleotides contain predomi- nant amounts of two or three nucleotides. All of the polynucleotides were active in directing amino acid incorporation except polynucleotide Ju-2510. Although 10 ug of polynucleotide were added to each reaction mixture, the total amount of C'™- amino acid directed into protein by each polynucleotide varied more than 50-fold. As we have shown previously, the template activity of polynucleotides is dependent upon factors other than nucleotide sequence. For example, large polymers of-chain length greater than 100 units are considerably more active than shorter ones.\" Single-stranded polynucleotides are active, whereas double or triple stranded poly- mers are not. In addition, randomly mixed copolymers which have a high degree of secondary structure are inactive in coding.” In particular, polymers containing much G have little activity. possibly because of G-G interactions. Thus, the relative inactivity of the last poly UGAC preparation (Ju-2510) should not be ascribed necessarily to the presence of a high proportion of nonsense nucleotide sequences. Such considerations make it difficult to compare with validity the relative abilities of different polynucleotides to code for the same amino acid; thus, ‘such comparisons should be made with caution. The fact that polynucleotides con- taining four bases coded so well for so many amino acids strongly suggests that most nucleotide sequences can be read. In addition, a high proportion of U clearly was not required for messenger RNA activity. Stimulation of amino acid incorporation by poly ACG: The coding activities of polymers which did not contain U are given in Table 2. Base-ratio analysis of each poly ACG preparation failed to detect contamination by U. Poly ACG preparations TABLE 2 STIMULATION of AMINO Acip [NcorPoRATION 8¥Y POLY ACG Polynucleotide ACG ACG ACG ACG A 46 A 2 A 4 A 16 Minus Base-ratio Cc 32 Cc 32 C 77 Cc 72 polynucleotide moles per cent G 22 G 9 G lg G 12 sontrok Designation F257 M 76 M 75 M 74 Camino acid Incorporation above control ( A~psmoies) * Alanine 23 45 56 85 8 Arginine 128 30 40 74 9 Aspartic acid® 167 0 0 24 13 Glutamie acid* 326 0 9 33 21 Glyeine 3 . 0 0 0 13 Histidine 71 6 9 95 3 {soleucine 0 0 0 0 20 Leucine 0 10 0 0 40 Lysine 820 3 0 33 6 Methionine 1 4 0 0 10 Phenylalanine 0 0 1 6 9 Proline 147 320 185 41 3 Serine i82 24 30 53 45 Threonine 250 Ll 13 il 8 Tryptophan 1 0 0 0 43 Tyrosine 0 + 0 0 18 Valine 1 5 5 7 6 Total 2.222 464 339 454 282 * SuuMoles represents the difference between C'-amino acid incorporation into protein in the presence and absence of polynucleotides. Assay procedures are described under Wateriala and Wethods. 2118 BIOCHEMISTRY: JONES AND NIREN BERG Proc. N. A. S. stimulated the incorporation of many amino acids tested, including alanine. arginine. glutamic acid, lysine, proline, and threonine. Such high incorporations of glutamic acid, lysine, and threonine were not. observed previously. * Anumber of amino acids did not appear to be coded by any ACG preparations, which suggested that U may be an absolute requirement in coding for some amino acids. Stimulation of amino acid incorporation by polynucleotides containing two bases: The polynucleotides in Table 3 are listed in order of decreasing C content. In accord with the findings of Bretscher and Grunberg-Manago.\" poly AC stimulated incorporation of proline. threonine, and histidine. In adgition. poly AC was found to direct aspartic acid, glutamic acid, and lysine into protein.* Poly AC (J-104: contained 97 per cent C, yet actively directed proline into protein. Thus. it appears that one codeword for proline may contain only C. Relatively large amounts of lysine were directed into protein by AC J-109 and J-108 which contained 67 and 80 per cent A respectively. Thus, the lysine codeword may contain only A. The data of Table 4 demonstrate the effects of poly CG and AG preparations in directing amino acids into protein. The first three CG polymers contained high proportions of C and directed alanine, arginine, and proline into protein. The last poly CG preparation (F-135) contained 91 per cent G, and was inactive as template RNA. Poly AG directed incorporation of glutamic acid and lysine into protein. Quantitative aspects of data: A comparative study of polynucleotides of varying base-ratios is helpful in evaluating amino acid incorporation data, for relative amino acid incorporations can be easily correlated with changes in base-ratio. Occasional inconsistencies and the significance of minor incorporations become apparent. Isotope dilution experiments were performed routinely to detect the possible presence of radioactive impurities in Camino acids. The presence of C4-impuri- ties seemed unlikely, for incorporation of a C)*-amino acid was reduced sharply if the reaction mixture contained both a C-amino acid (0.05 »mole) and the same TABLE 3 STIMULATION OF AMINO AcTp INCORPORATION BY Poty AC Polynucleotide AC AC AC Ac AC AC Minus Baze-ratio A 3 A 6 A W A 30 A Gi A 80 polynucleotide moles per cent Cc 97 Cc 94 Cc 88 c 70 C 33 Cc 26 control Designs tion J104 3403 3102 3101 J108 J108 C)4amino acid Incorporation above contro] ( Auumoles)* Alanine 0 j 0 0 0 0 i Arginine 0 4 1 1 0 0 12 Aspartic acid®™ - QO. Og BI 157 53 24 Glutamie acid™ 4 19 24 53 135 53 15 Glycine 5 16 0 2 0 0 6 Histidine 0 0 5 198 85 17 23 Isoleucine 6 0 6 0 0 0 42 Leucine 0 0 a 1 3 Lysine 5 10 14 47 909 441 5 Methionine 0 10 0 0 0 0 10 Phenylalanine 0 0 1 0 4 0 11 Proline 625 1,132 643 1,102 140 20 9 Serine 11 19 18 16 9 8 46 Threonine 30 65 75 170 176 105 9 Tryptophan 23 0 1 1 1 Q 44 Tyrosine 14 2 2 0 0 0 14 Valine 0 U 0 0 0 0 5 Total 723 1,278 793 1,641 1,616 707 294 * AuuMolet represents the difference between Ccsmino acid incorporation into protein ir the presence and absence of polynvciectides. Assay procedures are described under Materiales and Methods. Vor. 48, 1962 BIOCHEMISTRY: JONES AND NIREN BERG 2119 TABLE 4 STIMULATION of Amino AciD INCORPORATION BY Poty CG anp AG Poiynucieotide CG CG CG CG AG AG Minue Base-ratio Cc 30 Cc 387 Cc 82 Cc 9 A 73 A 48 polynucleotide moles per cent G 10 G i G 18 G 91 G 27 G 52 control Designation M141 M 7i F120 F135 J106 J107 C!<amino aad {Incorporation above control ( Agssmoles)* Alanine 30 20 63 0 0 0 14 Arginine 39 16 36 1 10 8 13 Aspartic acid™* 0 0 6 3 12 10 26 Glutamic acid® 0 0 0 0 44 5 ll Glycine 5 0 8 0 2 0 4 Histidine 0 0 0 0 0 0 26 {soleucine 0 0 0 0 1 0 39 Leucine 0 0 0 5 0 li 7 Lysine 2 0 -0 > Oe 1) 8 3 Methionine 0 0 0 0 0 0 12 Phenylalanine 3 5 4 8 0 0 14 Proline it4 202 356 2 1 1 8 Serine 18 0 6 0 0 0 42 Threonine 0 0 1 0 i 0 3 Tryptophan 0 1 \\\\7 1 0 0 40 Tyrosine 2 6 2 0 0 0 i+ Valine t 1 0 0 0 0 4 Total 246 251 549 20 181 43 282 * \\\\yuMoles represents the difference between C!+amino acid incorporation into protein in the presence and absence of polynucleotides. Details of the assay procedures are described under Materials and Methods. C'-amino acid (1.0 umole). The purity of each C'-amino acid also was deter- mined by paper electrophoresis followed byradioautography as described previously .* Limiting amounts of polynucleotides were added to reaction mixtures and Jotal amino acid incorporations were measured rather than rates of amino acid incorpora- tions. E. coli extracts contain nucleases which rapidly degrade synthetic poly- nucleotides and the nuclease content may vary from one preincubated S-30 prepa- ration to another. Since many different enzyme extracts were used in this study, the data are not useful for quantitative analyses. Comparisons between theoretical frequencies of triplets, etc.. in polynucleotides and relative amino acid incorpora- tions have not been presented because the data do not permit such calculations to be made with accuracy. The data demonstrate only qualitative aspects of the code; that is, nucleotide compositions of words and the degree of code degeneracy. Summary of [ncorporation Data.—Table 5 summarizes all of the coding data previously published~§ ‘6 * and those obtained in this study. Only polynucleo- TABLE 5 Semwary or Copine DaTa CtLamino acid stimulated by C’%-amino acid Stimulaced bywgoly-* poly-* Phenylalanine U(983t a Isoleucine TA 8) Proline Ci?) CAST) CU(80) CG80) Tryptophan UG(6) Lysine At?) AC(33)—-AG(8O) ATCO) Tyrosine UA) Threonine AC(13) Arginine CG(U15) Serine UCi233) UCac23)? Methionine UAG(1) Valine UG(15) Histidine AC(10) Leucine UGil4; = UC) UAC?) Alanine CGULL Glyeine UGS5) Aspartic acid® AC(8) Cysteme UG(3- 15; Glutamic acid® AC(7) AG(20) * Polymers used for these calculations coutain the minimal number of bases necessary to direct the Ci4_amino acids into protein. a . : + Numbers in parentheses refer to: upmoles of amino acid incorporated X 100/Sum of incorporation of all amino acids at the optimal base ratio assayed. 2120 BIOCHEMISTRY: JONES AND NIRENBERG Proc. N. 4.8 tides containing the minimum number of bases capable of stimulating an amino acid into protein are given in Table 5. The coding of proline by poly C and lysine by poly A was suggested by the poly AC experiments presented in Table 2. The fact that poly C and poly A code so weakly may be due to inhibitory effects o! secondary structure. In solution at acid pH. poly A is double-stranded.’* * and at neutral pH, both poly A and poly C have partially ordered structure.”) A surprising conclusion drawn from this summary is that almost every amino acid tested could be coded by a polymer containing only two bases. Methionine couid be coded only by poly UGA as previously reported® “ but since the amoun: oF methionine directed into protein was small, this codeword remains questionable. Assuming a triplet code, a summary of codewords estimated thus far is presented in Table 6. Previously. poly UCG was found to direct alanine and arginine into TABLE 6 Tentative SumMaRy oF CopE Worps * C)4amino acid M-codewords Alanine CCG Arginine CGC Aspartic acid® ACA Asparagine® UAC or CAAT Cysteine UUG or UGGt Ghutamic acid® ACA AGA AGUS Glycine UGG Histidine ACC Isoleucine UTA Leucine GUU CUU AUUT (LTT) Lysine AAA AAC AAG AAU Methionine UGA§ Phenylalanine CUE Proline ccc 3 ccU CCA CCG Serine ucG Uct Threonine CAC CAA Tryptophan UGG Tyrosine UAT Valine UGU * Nucleotide sequence in codewords is arbitrary. ${ We cannot now differentiate between these poasihilitics + Proposed by Spever ei al.'* & itis not entirely clear whether theac codewords requir \\\\ protein and codewords containing U, C, and G were proposed for these amine acids.* > 17.38 The observed frequencies of incorporations’ suggest coding of alanine and arginine by either UCG or CCG. but not by both codewords. In addition, the data of Table 4 show that poly CG codes for alanine and arginine. thus, codewords corresponding to these amino acids do not appear to contain U. Since it is not possible at this time to distinguish between triplet and doublet codes, etc., the assignments in Table 6 represent current approximations of code- words. It seems probable that additional codewords will be found. Discussion.—Codeword specificity in protein synthesis: The term degeneracii refers to the phenomenon whereby one amino acid is coded by two or more code words. This term is inadequate when applied to the mechanism of coding. for it does not indicate codeword specificity. A degenerate code may have high or low specificity depending upon the fidelity of protein synthesis. In most cases the fidelity of protein synthesis in rivo appears to be high, and amino acid replacemens> other than those due to mutation, have not been found. However. although the amino acid sequence analyses would reveal mistakes at one site occurring with # Vor, 48, 1962 BIOCHEMISTRY: JONES AND NIREN BERG 21291 frequency higher than | or 2 per cent, they would not reveal occasional mistakes occurring at different sites. Thus, occasional coding errors of 1 or 2 per cent, dis- tributed at random over entire protein molecules, might not be detected. In the in vitro system, coding during protein synthesis displays striking specific- ity. In Table 3, for example, poly AC preparations do not direct the incorporation into protein of alanine, arginine, glycine, isoleucine, leucine, methionine, phenylala- nine, tryptophan, tyrosine, or valine. The specificity of coding by poly CG and AG preparations in Table 4 is equally apparent. Such negative data clearly demon- strate the very high fidelity of codeword recognition during protein synthesis. The codewords corresponding to both leucine and valine contain U and G.‘-* Although the nucleotide content of these codewords is identical, each word was shown to code only for the appropriate amino acid.22 Thus. nucleotide sequence as well as chemical structure confers specificity upon codewords. However, one unclarified example of ambiguity has been found. Poly U directs about 3-3 per cent as much leucine into protein as phenylalanine.* Bretscher and Grunberg-Manago also have reported this phenomenon. ® In the absence of phenylalanine poly U coded for leucine about 50 per cent as well as it would code for phenylalanine.” Efficiency of synthetic RNA in coding: In spite of the previously mentioned difficulties in comparing template activities of RNA preparations with different chain lengths and degrees of secondary structure, it seems clear that synthetic polynucleotides containing 4, 3, or 2 bases code as well in this system as natural template RNA obtained from viruses.*: **—* The efficiency in coding displayed by synthetic polynucleotides suggests that most nucleotide sequences direct amino acids into protein, and that few nonsense nucleotide sequences are present. Although alternative explanations of coding efficiency, such as nonrandom polynucleotides or nonsequential reading of template RNA, may be considered, such efficiency cannot be ascribed simply to random error in directing amino acids into protein, for amino acids are coded with marked specificity. Considerations such as these may be used to approximate the coding ratio. In a doublet code only 16 base permutations are possible. Thus. the information content would be insufficient to code specifically for all amino acids. Triplet and quadruplet codes would contain 64 and 256 eodewords respectively. Since almost every amino acid tested was found to be coded by polynucleotides con- taining only 2 bases, specific and efficient coding by quadruplet words would not seem likely. The data sugge either coding of all amino acids by tripiets or coding of some by triplets and o*hers by doublets (mixed doublet-triplet code). Recently Weisblum, Benzer, and Holley” have established a molecular basis of degeneracy by demonstrating that multiple species of transfer RNA recognize different codewords with specificity. Multiple peaks of transfer RNA correspond- ing to at least four amino acids have been found independently by Holley et al.,™ Sueoka ef al..2? and Doctor et al.” If a triplet code is assumed, each cell would require almost 64 transfer RNA species. Alternatives which do not require so many transfer RNA species deserve consideration. For example, Donohue and others have described many models other than Watson-Crick pairing.”* The demonstrated interaction between poly A and poly I,” and the type of base-pairing suggested by Hoogsteen** also might be 2122 BIOCHEMISTRY: JONES 4ND NIREN BERG Proc. NX. ALS cited. Theories which require recognition of either the 2- or 6-substituents of bases*4 are not supported by the demonstration that hypoxanthine functions in codewords like G.%-® The 2-amino group of G does not appear to be required. A triplet code may be constructed wherein correct hydrogen bonding between two out of three nucleotide pairs may, in some cases. suffice for coding. Correct pairing of a base at one position in the triplet sometimes may be optional. 11 should be noted that a triplet code of this type in some respects Ww ould bear a super- ficial resemblance to a doublet code and would he in accord with all of the data. Any theory concerning the physical basis of the code agnust aitempt to explain the following experimentally obtained data: (1) high coding efficiency by syntheuc polynucleotides: (2) marked codeword specificity: ©} degenerate codewords: (4) the 2-amino group of G is not essential for proper coding: (5) RNA with a high degree of secondary structure has little ability to code: and (6) almost al) amine acids tested can be coded by polynucleotides containing only two bases. Summary. —Synthetic polynucleotides containing 4. 3, or 2 bases have been found to direct amino acids into protein with high efficiency and specificity. Many additional RNA codewords which do not contain uridylic acid have been found Almost all amino acids could be coded by polynucleotides containing only 2 hasc>. These results have been discussed in terms of the genera! nature of the code. It is a pleasure to thank Lynn Hurwitz, Linda Greenhouse. and Glenna Bailey for valuable technical assistance. * Presented at the Symposium on Informational M acromolecules, New Brunswick, New Jersey. Sept. 1962 (proceedings in press ). + The following abbreviations are used: A, adenylie acid; G, guanylic acid; C, evtidvlic acid. U, uridylic acid. 1Gamow, G., Nature, 173, 318 (1954). ; * Crick, F. H. C., in Structure and Function of Genetic Elements. Brookhaven Symposia 1? Biology, No. 12, 35 (1959). 2 Nirenberg, M. W., and J. H. Matthaei, these PRocEEDINGS, 47, 1588 (1961). 4 Martin, R. G., J. H. Matthaei, O. W. Jones, and M. W. Nirenberg. Biochem. Biophus. Ree Comm., 6, 410 (1962). 5 Matthaei, J. H., O. W. Jones, R. G. Martin and M. W. Nirenberg, these PROCEEDINGS. 48. 666 (1962). 6 Spever, J. F., P. Lengvel, C. Basilio, and S. Ochoa, these PROCEEDINGS, 48, 63 (1962) ? Roberts, R. B., these PROCEEDINGS, 48, 897 (1962). 8 Tbid., 1245 (1962). 9 Jones, O. W., and R. G. Martin, Fed. Proc., 21, 414 (1962). »” Bretscher, M. S., and M. Grunberg-Manago, Nature, 195, 283 (1962). 11 Singer, M. F., and J. K. Guss, J. Biol. Chem., 237, 182 (1962). 12 We thank Dr. M. Grunberg-Manago for this protocol. 18 Davidson, J. N., and R. M. S. Smellie, Biochem. J., §2, 504 (1952). 4 Unpublished observations. % Singer, M. F.. 0. W. Jones, and M. W. Nirenberg. unpublished observations. % Lengvel, P., J. F. Spever, and 8. Ochoa, these PROCEEDINGS, 47, 1936 \\\\ 196] }. U Lengvel, P., J. F. Speyer, C. Basilio, and S. Ochoa, these PRroceEpINGS, 48, 1 Speyer, J. F.. P. Lengvel, C. Basilio. and S. Ochoa, these PROCEEDINGS, 48, 19 Fresco, J. R., and P. Doty, J. Am. Chem. Soc., 79, 3928 (1957). % Rich, A., D. R. Davies, F. H. C. Crick, and J. D. Watson, J. Mol. Bool.. 3, 73 | 1963 + 21 Fresco, J. R., Trans. N.Y. Acad. Sci., Series IT, 21, 653 (1959; 22 Nirenberg. M. W.. J. H- Matthaei, O. W. Jones, 8. H. Barondes, and R. G. Proc., in press. gg? 12 44) (1962) Martin Fed Vou. 48, 1962 BIOCHEMISTRY: KAPLAN AND CAHN 2123 *3 Nirenberg, M. W, J. H. Matthaei, and O. W. Jones, unpublished observations. 4 Ofengand, J., and R. Haselkorn, Biochem. Biophys. Res. Comm., 6, 469 (1962). * Tsugita, A., H. Fraenkel-Conrat, M. W. Nirenberg, and J. H. Matthaei, these Proceepines, 48, 346 (1962). %* Nathans, D., G. Notani, J. H. Schwartz, and N D. Zinder, these PRoceEprNes, 48, 1424 1962). ™ Weisblum, B., 8. Benzer, and R. W. Holley, these Procezprnes, 48, 1449 (1962). * Holley, W. H.. B. P. Doctor, 8. H. Merrill, and F. M. Saad, Biochim. Biophys. Acta, 35, 272 (1959). *9 Sueoka, N., and T. Yamane, these Proczseptnes, 48, 1454 (1962). * Doctor, B. P., J. Apgar, and R. W. Holley, /. Biol. Chem., 236, 1117 (1961). 31 Donohue, J., these PRocrEeptNas, 42, 60 (1956). 32 Rich, A., Vature, 181, 521 (1958). 3 Hoogsteen, K.. Adeta Cryst., 12, 822 (1959). 34 Woese. C. R.. Vature. 194, L1i+4 (1962). * Basilio, C.. A. Wahba. P. Lengyel, J. F. Speyer, and S. Ochoa, these Procespines, 48, 613 1962). %® Poly AC ig reported to code for glutamine.\"' We have been unable to obtain C'-glutamine and C'-asparagine. Therefore, we cannot differentiate between codewords corresponding to each free acid and its respective amide.", "Nirenberg, Marshall W. ; Jones, Oliver W., Jr", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ihkz~5dim-inmx", "00000000-0000-0000-3700-EA7FB02480E3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "RNA Codewords and Protein Synthesis, II. Nucleotide Sequence of a Valine RNA Codeword", "101584910X52", null, "1964", "1964", "After the poly-U experiments, the next major challenge was finding out what the remaining codewords were.  In this article, the authors use a rapid method for measuring sRNA binding to ribosomes and try to resolve some problems with ambiguous and synonymous codewords.  The experimental evidence suggests the binding of specific types of sRNA to ribosomes is directed by only certain corresponding trinucleotides, making sequencing efforts more feasible in the coding process.", "Articles", "RNA,RNA, Messenger ; Ribosomes,Amino Acids", "Translating the Code of Life and the Nobel Prize, 1962-1968", "8", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "RNA CODEWORDS AND PROTEIN SYNTHESIS, I. NUCLEOTIDE SEQUENCE OF A VALINE RNA CODEWORD By Pui Leper «xp MARSHALL NIRENBERG NATIONAL HEART INSTITUTE, NATION AL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by R. B. Roberts. Junc 36, 1964 Recent studies demonstrate that synthetic gelynucleotides. such as poly U, in- duce with specificity the binding of C 4amino-acvl-sRNA to ribosomes.?~* Char- acteristics of binding in the absence of poly U also have been reported! C1*- Phe-sRN A binding induced by poly U is thought to represent an early step in pro- tein synthesis before peptide bond formation; however, the precise nature of the interaction has not been clarified. To establish the sequence of nucleotides in RNA codewords, we devised a rapid, sensitive method for measuring C-amino-acyl-sRNA binding to mbosomes, and have investigated both characteristics of binding and the minimum oligonucleotide chain length required to direct such binding.§ pUpUpU, pApApA, and pCpCpC directed the binding of Phe-, Lys-, and Pro-sRNA, respectively, whereas dinucleotides had no effect. Trinucleotides with 5’-terminal phosphate were more active than those with no terminal phosphate, whereas trinucleotides with 2'.(3’)-terminal phosphate were inactive. In this report, trinucleotides of known sequence were used to direct sRNA bind- ing. GpUpU, but not its sequence isomers, UpGpU and UpUpG, was shown to induce Val-sRNA binding to ribosomes. These data indicate that the nucleotide sequence of an RNA codeword for valine is GpUpU. Materials and Methods.—Analyses of poly- and oligonucleotides: (a) Paper electrophoresis was performed on Whatman 54 or 3 MM paper in 0.05 M NH,COOB buffer, pH 2.7, at 80 v/cm for 0.5 hr with authentic reference markers. If a marker was not available, the expected mobility was calculated? (b) Descending paper chromatography was performed at room temperature with Whatman 3 MM paper and the following solvents: (4) eonc. NH,OH-N-propano)l-H.Q, 10/55/35, v/v; (B) 40 gm (NH4)SO, dissolved in 100 ml 0.1 M sodium phosphate, pH 7.0. Bands were visualized under UV light. (c) Ultraviolet absorption measurements were made in 4 Zeiss spectrophotometer, and spectra were obtained in a Cary recording spectrophotometer The absorbancy of eluates from paper was read against blank paper eluates. (d) The base ratio of the poly UG preparation (D-132) was 0.74/0.26 (U/G). The base ratio of UpUpG, UpGpt, and GpUpU preparations was determined by. incubating 2.0 A*® units with 3.5 x 107? unite of T, RNase’® in 0.02 ml of 0.5 M ammonium acetate, pH 4.5, for 2.5 hr at 45°. The digestion products were separated by paper electrophoresis, eluted, and the absorbancy of each at appro- priate Ame. Was determined. Preparation and separation of oligonucleotides: UpU war characterized as described. UpUpG and UpG were obtained by incubating 3 x 103 A units of poly UG with 1.3 x 10! units of T:- RNase (Sankyo Co., Ltd., Tokyo) in 4.7 ml of 0.1 M NH.HCO, at 37° for 6 br.\" The reaction mixture was lyophilized, dissolved in 0.1 M (NH,), COs, and terminal phosphates were removed by incubation with E. coli alkaline phosphatase, free of diesterase, as described by Heppe! et al.?! Oligonucleotide fractions were separated by paper chromatography with solvent 4 for 1& hr. Uitraviolet-absorbing bands with mobilities of{UpG and UplipG were eluted with H.0, lvophilyzed. and purified separately by electrophoresis. GpUpU and GplU were obtained by digesting poly UG with purified pork liver nuclease.® re- moving terminal phosphates, and separating fractions by paper chromatography and electro- phoresis a8 described above. This procedure vielded 4 fraction containing Up and GpU and also one containing GpUpU, UpGpU, and UpUpG. Paper chromatography with solvent B for 420 Vou. 52, 1964 BIOCHEMISTRY: LEDER AND NIRENBERG 421 36 br resolved each dinucleotide. The trinucleotide fraction was separated into two bands by chromatography with solvent B. The band nearest the origin contained GpUpU of high purity. Tt was eluted with H.O, absorbed on acid-washed Norite, and eluted with 45% ethanol 0.5 M NH.OH. GpUpU was purified again by chromatography with solvent 8, eluted, and desalted as described. A derivative of pancreatic RNase A was used by Dr. Merton Bernfield to catalyze the transfer of uridine-2’,3’-cyclic phosphate to GpU.'2_ UpGpU was purified from the reaction mixture by paper chromatography and electrophoresis as described for UpUpG. Characterization of triplets: GpUpU, UpGpU, and UpUpG preparations were purified as de- scribed. Only one spot was observed after each was subjected to paper chromatography with solvents 1 or B and to paper electrophoresis. Base-ratio and sequence analyses established the purity, chain length, and base sequence of each triplet preparation. Base-ratio analyses were as follows: GpUpU, 1.2 U, 1.0 Up, 1.0 Gp; UpGpU, 1.0 U, 1.0 Up, .09 Gp; UpUpG, 2.1 Up, 1.0 G. Base sequence was determined as follows: 2.0 A® unita of each trinucleotide was digested separately with T,-RNase (as described earlier) and in other reactions, with 2.0 ug of chromato- graphically purified pancreatic RNase (Sigma) in 0.02 ml of 0.1 M (NHi)2COs, at 37° for 6 hr. Reaction products were separated by paper electrophoresis. Dinucleotide products were eluted, lyophilized, and digested with the RNase to which they were susceptible, and the products again were separated by electrophoresis. Nucleosides and nucleotides were identified by their electro- phoretic mobility as well as by their spectra. After digestion of GpUpU, UpGpU, and UpUpG with pancreatic RNase, only the following products were obtained: GpUp + U; Up + GpU; and Up + G, respectively. After digestion of GpUp and GpU with T,-RNase, Gp + Up and Gp + U were obtained, respectively. T,- RNase digestion of GpUpU, UpGpU, and UpUpG yielded Gp + UpU, UpGp + U, and UpUpG, respectively. Digestion of UpU, UpGp, and UpUpG with pancreatic RNase yielded Up + U, Up + Gp, and Up + G, respectively. No other UV-absorbing material was found. Since a 2% contaminant could have been detected, the purity of each trinucleotide was estimated to be >98°%. GpUpU and UpGpU have not been purified previously. Ribosomes, sRN A, and E. coli extracts: E. coli W 3100 aRNA was prepared from cells grown to late log phase in 0.5°% nutrient broth, 1% glucose.™ £. coli BsRNA waa obtained from General Biochemicals, Inc. Each C'-amino-acyl-sRNA was prepared in the presence of 19 C}*-amino acids as described elsewhere. In additional experiments C\\\\-Leu-sRNA also was prepared in the absence of other amino acids. C1-L-Val, C“-L-Phe, and C'-L-Leu, uniformly labeled with radioactivities of 293, 229, and 435 cpm/sumole (Packard Corp. scintillation counter), respectively, were obtained from New England Nuclear Corp. Ribosomes for binding studies and E. coli ex- tracts (DNase-treated, preincubated, S-30 fraction) for amino acid incorporation into protein studies were prepared as described elsewhere.’. ' Assays: Each 50-,l reaction mixture contained 0.1 VM tris-acetate, pH 7.2; 0.02 M mag- nesium acetate; 0.05 WM KCI; and 1.0 A™ units of ribosomes unless otherwise specified. Incuba- tions were at 24° for 20 min unless otherwise indicated. C'-amino-acyl-sRNA binding to ribo- somes was determined. as reported elsewhere,’ by washing the ribosomes on Millipore filters. Ribosomes with bound sRNA remained on the filter. C™-amino acid incorporation into protein was determined aa reported previously.\" Results.—E ffect of GpUpU and Poly UG upon the binding of C'+-valine sRNA to ribosomes: The data of Figure 1 show that the binding of Val-sRN A to ribosomes is stimulated by the addition of GpUpU or poly UG and is dependent upon the con- centration of either template. More Val-sRNA is bound to ribosomes in the pres- ence of poly UG than in the presence of an equivalent amount of GpUpU. We have observed similar differences between the activity of pUpUpU and poly U.* In Figure 2, the rates of binding at 0° and 24° are compared, and the specificity of GpUpU and poly UG for amino-acyl-sRNA is shown. At 0°, poly UG markedly stimulated Val- and Phe-sRNA binding to ribosomes, whereas GpUpU stimulated only Val-sRNA binding. At 24° the rates of binding directed by poly UG were higher than at 0°. Slightly higher binding in control reactions was observed 422 BIOCHEMISTRY: LEDER AND NIREN BERG Proc. N. A. S.   12 Tr (especially of Leu-sRNA). Under the conditions employed, poly UG had little activity in directing Leu-sRNA binding —= to ribosomes, compared with its ability to direct amino acid incorporation into protein. + Codgword specificity and the effect of (Afg*%) concentration: The data of Figure 3 demonstrate that poly UG stim- +  ulates binding of Val- and Phe-sRN A to ribosomes optimally at 0.02-0.03 A/ (Mg++), whereas GpUpU directs bind- “= ing of Val-sRNA optimally at higher ee concentrations. At higher O4g++) eon- o4 uy WoLes B ise RESID us % % centrations, a small and apparently non- Fig. 1.—The relation between GpUpt 5?’ ecific increase in Val-. Leu-, and Phe- (©} and poly UG (()) concentration and C*%- sRNA binding was observed in control SSRN eunding to Tibogomes, Bach “s; reaction mixtures (no addition). Tn- ribosomes and 18.9 wamoles of ian attached and polynucleotides induce specific GpUpt were added oe specified. 3 anc’ sRNA binding. GpUpU directed only Val-sRNA binding, whereas UpGpU and UpUpG had ne effect upon either Val-, Leu-, or Phe-sRNA. The addition of tri- or polynucleotides did not appreciably increase Leu-sRNA binding over that of contro] reactions within the range of (Mg++) concentrations tested. The specificity of di-, tri-, and polynucleotides in directing amino-acyl-sRNA binding is shown in Table 1. Poly UG directed Val- and Phe-sRNA binding. whereas poly U directed the binding only of PhesRNA. UpU, GpU, and UpG did not stimulate Val-, Phe-, or Leu-sRNA binding. The effect of GpoUpU was tested upon the binding of 17 other sRNA preparations. each with a different C'*-amino accepted (C!*-asparagine and C'4-glutamine-sRNA not tested). GpUpU was found to direct the binding only of Val-sRNA. Amino acid incorporation into protein: Since poly UG had little effect upon Leu-sRNA binding to ribosomes, we investigated the ability of poly UG to direct ow a T                 pw MOLES C*¥*-VALINE-sRNA BOUND » & & 8 & tv I T t &     TABLE 1 Coprworp SprEciFiciry Addition, C!*Amino-Acyl-sRNA Bound to Ribosomes, zumoles Expt. mumoler base residues C'cVal-aRNA Ct-Phe-sRNA C1cLeu-sRNA 1 None 0.38 0.22 0.37 4.7 Poly U 0.23 47. 0.22 4.7 Poly UG 2.65 1.93 0.24 2 None 0.40 0.22 0.62 4.7 GpUpl 1.11 0.27 0.56 4.7 UpGpl 0.40 0.25 0.55 4.7 UpUpG 0.37 0.25 0.44 8 None 0.18 0.22 0.69 4.7 GpU 0.20 0.22 0.69 4.7 UpG 0.19 0.21 0.71 4.7 UpU 0.18 0.20 0.67 Oligonucleotide specificity in directing 6RNA binding to ribosomes. Where indicated, ¥.10 umoles C)cVal attached to 6.3 A™ unita of sSRNA. 17.0 sumoles C!<Phe attached to 0.7 A™ units of BRNA. or 23.0 pymoies C14 Leu attached to 1.5 A™ unite of BRNA were added to reaction mixtures. Vow. 52. 1964 BIOCHEMISTRY: LEDER AND NIRENBERG 423                 C7'4- WALINE- RNA 24. EUGINE: ANA 24 PHENYLALANINE - SANA 8.0 r + . . Fr 7 rr f | o | e or 64> 4 4aar ! 4 wn Y fe wo, 32h pOLY UG ae 2 {8 x 3 a ~~ Goto NO ADOKTION POLY UG Gpupl / Splipht NO ADDITION 3 oe SS —————\" (N\\\\A é ‘ j oh $e is i ce: TY < 24° 24° ase > 64F % 3 POLY UG 3 < Zaat 4 POLY UG 4 POLY UG NO) ADOITION Gpulp = NO. ADOITION Gplips 0 ea SG enruaeGnrann a o 20 0 6 50 6 0 0 oOo 0 0 0 0 © » Ww 40 50 MINUTES Fig. 2.—Effect of GpUpU and poly UG upon the rates of binding of Val-, Leu-, and Phe- 3RNA to ribosomes at 0° and 24°. The symbols represent the addition of: (A) poly UG, 4.70 mymoles of base residues; ((]) GpUpU, 2.35 mpzmoles of base residues; (©) no addition. Where indicated, 17 mumoles of C¥-Vai attached to 0.44 A units of sSRNA, 8.5 uzmoles C'*-Phe at- tached to 1.5 A™ units sRNA, or 21.0 sumoles of C'4-Leu attached to 1.5 A®™ units of sRNA were added. Samples were incubated at the temperatures and for the times indicated. amino acid incorporation into protein in E. colz extracts at 0° and 24° (also at 37° to make these data comparable to previously reported studies!* 5). As shown in Table 2, poly UG directed Phe, Val, and Leu into protein at 37° and 24°, and as re- ported previously,!* ° Leu incorporation almost equaled that of Val. Amino acid incorporation into protein was not detected at 0°. Discussion.—Previously we have shown that UpUpU, ApApA, and pCpCpC specifically induce the binding to ribosomes of Phe-, Lys-, and Pro-sRN4A, respec- tively. The present study demonstrates that the trinucleotide, GpUpU, can induce the binding of Val-sRNA to ribosomes, whereas sequence isomers such as UpGpU and UpUpG. and dinucleotides have no activity. When the specificity of poly UG was compared with that of GpUpU,. the polynucleotide was found to direct both MS 7 -STABLE 2 C'%-AmMIno Actp [NcoRPORATION INTO PROTEIN Temperature of Amino Acid Incorporated into Protein. mumoies incubation Addition Clevaline Cit phenylalanine C! leucine a° None <0.03 <0.03 <9.038 Poly UG, 33 mamoles <0 03 <0.03 <0.03 24° None <0.038 ) 04 ).04 Poly UG, 53 mumoles 0.70 1.57 0.87 37° None 0.10 0.09 0.08 Poly UG, 33 mmoles L 04 2.05 0.96 Each 0.1-mi reaction mixture contained 0.3 mg &. colt protein (DNase-treated, preincubated 3-30 fraction®), 20 mymoles of either C+ Val, C+ Phe, or CttLeu (4.8, 4.0, 4.0 ucuries, umole, respectively), in addition to components previously described. Reactions were incubated for 10, 20, or 60 min at 37°, 24°, or 0°, respectively. 424 BIOCHEMISTRY: LEDER AND NIRENBERG Proc. N. A. S.   - ° ° yp MOLES C\"'4-WALIVE SRA BOUND nN o     3.0 WO ADDITION pp MOLES C\"'4-( EUCINE -sfNA BOUND          PHENYL ALANINE pw MOLES C:'4- PHENYL ALANINE tRNA BOUND     0 ye oa O02 0040 006 08 O10 [Wott] MOLARITY Fic. 3.—Relationship between magnesium acetate concentration and C-amino-aeyl-sRNA binding to ribosomes. The symbols represent the addition of: ( 2) poly UG, 4.70 mymoles of base: (©) GpUpU, 2.35 mumoles of base; (() UpGpU, 2.35 mumoles of base, (4) UpUpG, 2.35 mumoles of base; (@)no addition. 9.10 pumoles C4-Val attached to 0.3 A™ units of BRNA. 25.0 yumoles C'-Leu attached to 1.5 A%® units of BRNA, or 10.0 yumoles C1¥-Phe attached to 0.7 A® unite of BRN A were added where indicated. The (Mg**) concentration of each reaction mixture is shown on the abscissa. Samples were washed with buffer contaiming the appropriate concentration of (Mg**). a. Phe- and Val-sRNA binding, but GpUpU induced the binding of only Val-sRNA. We conclude that the nucleotides of a valine codeword in mRNA are arranged in the sequence GpUpU. Since UpUpG does not replace GpUpU, recognition occurs with polarity. As discussed elsewhere,* the ability of tri- but not dinucleotides to induce sRNA binding demonstrates directly that triplets may code for amino acids, and agrees with conclusions derived from both genetic!\" and biochemical] studies.” Studies of the incorporation of amino acids stimulated by various polynucleotides together with the data on amino acid replacements in mutants provide a basis fv! VoL. 52, 1964 BIOCHEMISTRY: LEDER AND NIRENBERG 425 assignment of codewords to amino acids.'*~\" Unfortunately, widespread de- generacy and the difficulty of assigning words which contain three different bases prevent unique assignments. Several self-consistent schemes are possible. The additional information provided by the definite knowledge of even a single code- word such as GpUpU provides a distinction among these different possibilities. The reassignments suggested to make GpUpU the codeword for valine consistent with the other data are listed in Table 3. For example, ApUpU was assigned to isoleucine on the basis of studies employing randomly ordered polynucleotides to direct amino acid incorporation into protein, and the HNO.-induced replacement of isoleucine by valine in TMV coat protein. TABLE 3 PREDICTED NUCLEOTIDE SEQUENCES oF RNA CoDEWwoRDS Amino acid Sequence Reference Valine GpUpU Derived experimentally Isoleucine ApUpU 17, 18 Ap (UA) Tyrosine Up (AT) Alanine GpCpC 16 Gp (CA) Gp (CU) Glutamic acid GpApa* 16 Gp( Av) Glycine Gp (GU) 16 Gp (GA) Gp (GC) Note: UpGpG is ruled out Arginine ApGpa 16 CpGpC (C)Gp( A) Threonine Ap CA) 17, 18 (ACC) * The assignment of GpApA to glutamic acid is based upon limited amino acid incorporation into protein data and is. therefore, tentative.t, 24 : Nucleotides within parentheses have aot been arranged ip sequence. Amino acid replace- ments used for these predictions were ‘ound in &. colt by Yanofsky and co-workers or were induced by HNOzin TMV by Wittmann and Wittmann-Liebold\" and also by Tsugita.'* It should be noted that the predicted sequence for isoleucine, ApUpU, and also the GpUpU sequence found for valine do not agree with reported sequence deter- minations?! which, on the basis of experiments with uncharacterized polynucleo- tides, assign the sequence ApUplU to tyrosine, tentatively assign GpUpU to cysteine. and derive the polarity of mRNA for protein synthesis. Although our results show only marginal stimulation of Leu-sRNA binding by poly UG, we have consistently induced Leu- and also Cvs-sRNA binding with poly UG when reactions were incubated at 0? and at a (Mg-~) concentration of 0.03 VW. Leu-sRNA binding was readilyinduced by poly UC which confirms results reported by Kaji and Kaji? Further studies with Leu- and Cys-sRNA are in progress. We have shown that specifie binding of sRNA can be induced at 0° and have described preliminary evidence which indicates that intact amino-acyl-3sRNA can be eluted from ribosomes after incubation.’ Although these data suggest that forma- tion of peptide bonds is not required for binding to ribosomes, a requirement for the first transfer enzyme and GTP may exist (cf. refs. 1-4. and 6). Since synonym codewords corresponding to one amino acid often differ in base composition by only one nucleotide,” it is possible that bases in common may 426 BIOCHEMISTRY: LEDER AND NIRENBERG Proc. N. A. 8. occupy identical positions within each triplet. A triplet code can be constructed wherein recognition between two out of three nucleotide pairs may, in some cases, suffice for coding; or, alternatively, a base at one position in the triplet may pair optionally with more than one base.??-*4 Ambiguous and svnonvmous codewords may involve such recognitions. The development of a rapid method for measuring sRNA binding to ribosomes and the use of trinucleotides of known sequence to direct such binding should provide a general method of great simplicity to test these possibilities and also to study the genetic function of other nucleotide se- quences and interactions between specific codewords. sRN A. and ribosomes. Summary.—The binding of C’‘-valine-sRNA to ribosomes was directed both by the trinucleotide, GpUpU, and by poly UG, but not by UpGpU, UpUpsG, or di- nucleotides. GpUpU had no effect upon the binding to ribosomes of sRNA cor- responding to 17 other amino acids. The nucleotide sequence GpUpU was pro- posed for a valine RNA codeword. The implications of these findings and pre- dictions based on amino acid replacement data are discussed briefly. We are grateful to Drs. Leon Heppe! and George Rushizky for their advice on oligonucleotide separations. It is also a pleasure to thank Norma Zabriskie and Theresa Caryk for their ex- tremely skillful technical assistance. The following abbreviations are used: Val, valine; Phe, phenvlalanine: Leu, leucine: poly U, polyuridylic acid; poly C, polveytidylic acid; poly A, polvadenylie acid: poly UG, copolymer of uridylic and guanylic acids; TCA, trichlorvacetic acid; sRNA, transfer RNA; mRNA, messenger RNA. For mono- and oligonucleotides of specific structure, the ‘‘p’’ to the left of a terminal nucleoside initial indicates a 5’-terminal phosphate; the “‘p’’ to the right, a 2’-(3’)-terminal phosphate. Internal phosphates of oligonucleotides are (3’,5’+linkages. } Arlinghaus, R., G. Favelukes, and R. Schweet, Biochem. Biophys. Res. Commun., 11, 92 (1963). 2 Nakamoto, T., T. W. Conway, J. E. Allende, G. J. Spyrides, and F. Lipmann, in Synthesis and Structure of Macremolecules, Cold Spring Harbor Svmposia on Quantitative Biology, vol. 2& (1963), p. 227. 3 Kaji, A., and H. Kaji, Beochem. Res. Commun., 13, 186 (1963); Federation Proc. ( Abstr.}, 23, 478 (19645. “ Spyrides, G. J., Federation Proc. (Abstr.), 23, 318 (1964). ® Cannon, M., R. Krug, and W. Gilbert, J. Mol. Brol., 7, 360 (1963 j. 6 Nirenberg, M. W., and P. Leder, Science, in press. 7 Markham, R., and J. D. Smith, Biochem. J., 52, 55& (1952), 6 Rushizky, G. W., and H. A. Sober, J. Biol. Chem., 237, 2883 (1962). 9 Totd., 238, 371 (1963). * Tbid.., 237, 834 (1962). 1 Heppel, L. A., D. R. Harkness, and R. J. Hilmoe, J. Brel. Chem., 237, &41 (1962). 12 Bernfield, M.R., andM. W. Nirenberg, Abstracts, 148th National Meeting, American Chemical Society, Chicago, [linois, August 1964. 13 Nirenberg, M. W., in Methods in Enzymology, ed. 8. P. Colowick and N. O. Kaplan (New York: Academic Press, 1964), vol. 6, p. 17. 4 Nirenberg, M. W., J. H. Matthaei, O. W. Jones, R.G. Martin, and 8. H. Barondes, Federation Proc., 22, 55 (1963). % Singer, M. F., O. W. Jones, and M. W. Nirenberg, these Procerpines, 49, 392 (1965). 1 Yanofsky, C.. im Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 581. u Wittmann, H. G., and B. Wittmann-Liebold, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 2& : 1963), p. S8Y. % Tsugita, A., personal communication. 1° Crick, F. H. C., L. Burnett, S. Brenner, and R. J. Watts-Tobin, Naturc, 192, 1227 (196) 5. Von. 52, (964 427 0 Nirenberg, M., O. Jones, P. Leder, B. Clark, W. Sly, and 3. Pestka, Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 349. 21 Wahba, A. J., C. Basilio, J. F. Spever, P. Lengyel, R. 8. Miller, and 3. Ochoa, these ProcrED- InGs, 48, 1683 (1962). 22 Nirenberg, M. W., and ©. W. Jones. in Informational Macromolecules, ed. H. Vogel et al.. (New York: Avademic Press, 1963). 23 Jones, O. W., and M. W. Nirenberg, these ProceEpinas, 48, 2115 (1962). 24 Gardner, R. 3., A. J. Wahba, C. Basilio, R. 8. Miller, P. Lengyel, and J. F. Speyer, these PROCEEDINGS, 48, 2087 (1962). % Zubay, G., J. Mol. Biol. 4, 347 (1962).", "Nirenberg, Marshall W. ; Leder, Philip", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-i5dw_n864_8zk9", "00000000-0000-0000-C392-87D3B9D9E171", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "RNA Codewords and Protein Synthesis, VII. On the General Nature of the RNA Code", "101584910X53", null, "1965", "1965", "In this report, the template activities of twenty-six additional trinucleotides are described and related to the nature of the RNA code.  This builds on earlier work of Nirenberg and others, which describes the template activities of nineteen additional trinucleotides and nucleotide sequences suggested for RNA codons corresponding to amino acids.  While still far from an \"invariant dictionary\" at this stage in the research, a table of possible nucleotide sequences is provided.", "Articles", "Codon,Nucleotides,Base Sequence,RNA", "Translating the Code of Life and the Nobel Prize, 1962-1968", "8", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Vou. 33, 1965 BIOCHEMISTRY: NIRENBERG ET AL. 1161 RNA CODEWORDS AND PROTEIN SYNTHESIS, VII. ON THE GENERAL NATURE OF THE RNA CODE By M. Nimensere, P. Leper, M. BernFietp, R. BrowacomBe, iJ. Trupin,* F. Rorrmant, anp C. O'NEAL NATIONAL HEART INSTITUTE, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by Robert J. Huebner, March 26, 1965 Nucleotide sequences of RNA codons have been investigated recentiy by directing the binding of C'4-AA-sRNA to ribosomes with trinucleotides of defined base sequence. The template activities of 19 trinucleotides= have been described and nucleotide sequences have been suggested for RNA codons corresponding to 10 amino acids.'~* In this report, the template activities of 26 additional trinu- cleotides are described and are related to the general nature of the RN A code. Materials and Methods.—Components of reactions: E. cols W3100 ribosomes and sRNA were prepared by modifications of methods described previously. Each C!+aminoacyl-3sRNA was prepared in the presence of 19 C!%amino acids. The assay for ribosomal bound C'+AA-sRNA and components of reaction mixtures have been described.!. The characteristics and amounts of labeled AA-sRNA not described® are shown in Table 1. Synthesis and characterization of oligonucleotides: ApG and UpG were obtained from a T-1 ribonuclease digest of RNA, and ApA was prepared by chemical synthesis.» \" ApC, ApU, CpA, CpG, GpA, and GpC were obtained from Gallard Schlessinger Corp., but required extensive puri- fication prior to use. GpGpU was obtained by digesting poly UG with pancreatic RNase A: treatment with alkaline phosphatase to remove terminal phosphate groups from the degradation products; and isolation by procedures similar to those described for GpUpU.? The remaining trinucleotides were synthesized from the appropriate dinucleoside monophosphate, using either TABLE 1 RaDIOACTIVE AMINOACYL-3RNA PREPARATIONS* C% or HX AA-sRNA Added to Each Reaction Specific uumoles of C1t Origin of Radioactive radioactivity or H3-Amino sRNA E. cols anmune acidt ue’ umole A%¢ anits acid accepted straint Lys 240 4 ).25 8.5 W3100 Expt. ) 2 ).28 10.2 B Ala 88 33 8.5 W300 Expt. 5 88 0.75 32.6 W3100 Glu 205 : 0.56 14.9 B Expt. 6 {88 0.85 6.1 W3100 Gly-H3 1130 0.15 11.72 A-23§ -C% Expt. b 66 0.47 9.2 W3100 Pro 200 0.70 6.4 B Expt. 6 158 0.57 13.2 W3100 Ser 120 1.038 6.3 W3100 Expt. 6 {20 0.42 18.6 B Trypt-H? 3000 1.00 6.5 B * Other AA-sRNA ata have been described.5 + Amino acids stated were labeled with C!4 with the exception of H*-tryptophan, and H*-glycine. ¢ £. cole W3100 isa K12 strain. Aminoacy! sRNA synthetase preparations were from F&. coli W3100. « . § We thank Dr. Charles Yanofaky for this #. coli strain and Dr. Ray Byrne for the H*Gly-eRNA. A-23 sRNA and 100,000 X g supernatant fractions were used for the preparation of H?-Gly-sRNA. 1162 BIOCHEMISTRY: NIRENBERG ET AL. Proc. N. A. S. TABLE 2 CHARACTERIZATION OF TRINUCLEOTIDES 10n Digesti: Synthetic (T-2 Pei eP ane) (venom phospbodiesterune) Compound method® Producte Base ratio Products Base ratio ApCpA 1 Ap,Cp,A 1.00/0.95/1.05 A,pC,pA 1.05/0.96/1 00 ApCpC 1 Ap,Cp,C 1.05/1.00/0.95 A,pc 1.15/2.00 ApCpG? 1 Ap,Cp,G 1.00/0.95/1.05  ApC,pG 1.00/0.95/1.00 ApCpU 1 Ap,Cp,U 1.00/1.00/0.85  AjpCpUe — 100/0.90/1.10 ApGpA 1 Ap,Gp,A 1.00/1.00/0.95 A’pG/pA 1.00/1.06/1 00 ApGpC ] Ap,Gp,C 1.05/1.00/0.95 A,pG,pCe 1.00/1.00/1.15 ApGptl? 1 Ap,Gp, Us 1.10/1.09/0.95 A,pG,pU< 1.00/13 .00,1.00 ApUpG ] Ap,Up.G 1 0070\" 85 05 A,pU,pG? —-1..00/0. 96/110 CpApA 2 Cp,Ap,A 0.95/1.0571.00 Cpa 1.05/20 CpApC 2 p,Ap,C 1.00/1.10/0.95 C,pA,pC 1.00/1.06/1.00 CpApG! 2 Cp,Ap, 1.10/1.00/1.00 C,pA,pG 1.00/1.06/1.15 CpAptU 2 Cp,Ap,U 1.00/1.10/0.90  Cipa’pt 1.00/1.05/0.95 CpCpa 2e Cp,A 2.00/095 bee. CpGpat 2 Cp,Gp,A 1.10/1.00/0.95 — C,pG,pA 1.05/0.95/1.00 CpGpc 2 Cp,Gp, 1.05/1.00/0.80 C,pG.pC? —0.90/1_00/1.00 CpUpG 2 Cp,Up,G 0.95/1 00/1.10 C,pU,pG 0.95/1.00/1.10 GpApA? 1 Gp,Ap,A¢ —0.90/1.00/1.10  Gipaé 1.10/2.00 GpApCre ] Gp,Ap,C 1.05/1.06/0.90 G,pA,pC 1.05/1.00/0.90 GpApU ] Gp,Ap,U 1.00/1.00/0.95 G,pA,pU¢ 1.20/1.00/0.90 GpCpU 1 Gp,Cp, 1.05/1.00/0.90  GipC'pt 0.85/1.00/1.00 GpGpUe 3 Gp, U’ 2.00/0.95 G,pG,pUS 0.85/1 .00/1.05 UpApaA 2 Up,Ap,A¢ 1.00/13 .00/1.16 U,paA 0.95/2.00 UpApG 2 Up,Ap, 1.00/0.95/1.00 U,pA,pGe 1.00/1.15/0.90 Upc 2 Up,Cp,G 1.00/0.90/1 00 U,pC,pG 0.95/1.00/1 .05 UpGpa 2 Up,Gp,A 0.95/1.00/1.10 U,pG,pA 0.90/1.00/1.15 UpGpc* 2 Up,Gp,C 1.00/0.95/1.05 U,pG,pce 1.10/1.00/0.90 ° Methed 2: primer-requiring polynucleotide phosphorylase.* Method £: derivative of bovine pancreatic ribonuclease. Method $: isolation from a ribonuclease digest. of poly UG? + Trinucleotide contained a small amount (ea. 2%) of an unidentified contaminant. In every case, the chromato- graphic characteristics of the impurity in aoivent systems A and B precluded the pomaibility of it being an oligo- nucleotide of chain-length greater than two base residues. ¢ The di job mixture contained apether component, 3-5% of the total nucleotide content; the elevirophoretic mobility and UV spectrum of this component corresponded in each case to unreacted or partially digested oligu- nucleotide. @ As “ec” above, except that here the contaminant was only 1-3% of the total nucleotidic material. © Trinucleotide was synthesized directly from adenosine, by addition of two cytidylic acid moieties. J Trinucleotide contained 5~10% of @ contaminant which did not migrate in solvent A, but which ran with GpGpT im solvent B. The T-: digestion mixture contained 8%, and the venom digestion mixture 14%, of un- digested material: this was possibly due to aggregation of the trinucleotide. * Trinucleotide contained 10% of pC; the base ratio of the venom digestion was adjusted accordingly. primer-requiring polynucleotide phosphorylase and a nucleoside 5’-pyrophosphate,? or a deriva- tive of bovine pancreatic ribonuclease with a nucleoside 2’,3’~cyclic phosphate” (Table 2). The products were isolated by paper chromatography and electrophoresis, as previously described!—+ 12 and the purity of each preparation was assessed by two-dimensional chromatography of an aliquot (2.0 A® units) on Whatman no. 40 paper. The first dimension (solvent A) was n-pro- panol/ammonia/water, 55/10/35; the second dimension (solvent B) sras 0.10 M sodium phos- phate, pH 7.0, containing ammonium sulfate (0.4 gm/ml). Chain-length and base composition (Table 2) were determined by digestion of 2:5-A2\" unite of each trinucleotide with T-2 ribonuclease and 2.5 A™ units with venom phosphodiesterase, as previously described.+ # Results and Discussion—In Table 3 are shown the effects of 26 trinucleotides upon the binding to E. coli ribosomes of 19 C!\\\\AA-sRNA preparations, each acylated with a different C!“amino acid (C-Cys-sRNA not used). In addition, near the bottom of the table are shown the effects of 18 trinucleotides previously described!—* upon the corresponding C'-AA-sRNA (C'-Cys-sRNA and UpGpU omitted). Many of these trinucleotides have not been isolated or synthesized previously. Several factors should be mentioned which may be useful in assessing the data. (a) It is often difficult to compare directly the response of one C}/AA-sRNA preparation to a template with that of another, for Kaji and Kaji have shown that Vou. 53, 1965 BIOCHEMISTRY: NIRENBERG ET AL. 1163 both deacylated and acylated sRN. bind to ribosomes in response to polynucleotide templates. The extent of acylation of each C!*AA-sRNA preparation must be considered (see Methods and Materials) as well as the relative response of each C'-AA-sRNA to other trinucleotides. (6) A trinucleotide which stimulates the binding to ribosomes of one C'*-AA-sRNA generally decreases binding of other C'*AA-sRNA preparations.' (c) Background binding of C™-AA-sRNA to nbosomes appears to be a function of the sRNA species, the amount of sRNA added to a reaction, the proportion of sRNA acylated with a C'-amino acid, and possibly the amount ‘of template RNA on the ribosomes or in the sRNA prepara- tions. +8 (d) Reactions contained limiting concentrations of ribosomes (as determined with ApApA, UpUpU, UpUpC, or GpUpU) and therefore were satu- rated with respect to these trinucleotides and C'*AA-sRNA. Most trinucleotides markedly stimulated the binding to ribosomes of only one C'-AA-sRNA preparation; however, a number of trinucleotides displayed lower template specificity for C'*-AA-sRNA. For example, ApCpU, ApCpC, and ApCpG stimulated C'-Thr-sRNA binding to ribosomes, but did not significantly stimulate the binding of 18 other C'-AA-sRNA preparations. ApCpA also stimulated C'.ThrsRNA binding. This trinucleotide also stimulated C'LyssRNA binding. However, the template activity of ApCpA for C-Lys-sRNA was only 10 per cent that of ApApA. The disparity between the template activity of ApCpA and ApApA was even more apparent in reactions containing limiting concentrations of trinucleotides (data not shown). Such considerations suggest that the sequences ApCpG, ApCpU, ApCpC, and ApCpA correspond to threonine codons. It is possible that the template specificity of one synonym codon may differ from that of another; however, other alternatives, such as the possibility that C'-Lys-sRNA may respond to an impurity in the ApCpA preparation which we have been unable to detect, alzo must be considered. The data of Table 3 indicate that the sequence GpCpU corresponds to an RNA codon for alanine; CpCpA, CpCpU, and CpCpC correspond to proline (the tem- plate activity of CpCpA for C'*-Pro-sRNA was higher than that of pCpCpC (ef. ref. 4); UpCpG, UpCpU, and UpCpC correspond to serine (ef. ref. 4); GpApU and GpApC, to aspartic acid; GpApA, to glutamic acid; CpApU and CpapC, to histidine: CpApA and CpApG, to glutamine: CpGpC and CpGpaA, to arginine; ApUpG, to methionine; and CpUpG and UpUpG, to leucine (CpUpU and CpUpC possibly serve as internal but ngt terminal Leu-codons’). It seems clear that GpCp(*serves as a codon for alanine, for this trinucleotide stimulated only the binding of C'-alanine sRNA to ribosomes. This sequence is also in accord with predictions based upon amino acid replacement data. How- ever, the weaker response of C'*-Ala-sRNA to ApGpC, CpGpC, and UpGpC sug- gests that recognition of 2 out of 3 bases, the GpC portion only of the latter tri- nucleotides, may permit C'+-Ala-sRNA binding. Similarly, C'-GlusRNA re- sponds best to GpApA, but also responds to a weaker extent to trinucleotides con- taining GpA, such as ApGpA, CpGpA, and UpGpA. C'*-Lys-3RNA responds best to ApApA but also recognizes ApApG,* GpApA, ApCpA, CpApA, UpApaA, and CpCpA. Additional examples in Table 3 are readily apparent. These data in- dicate that one trinucleotide sometimes can direct the attachment of a limited group of C'4-AA-sRNA species to ribosomes. It is possible that correct re- 1164 BIOCHEMISTRY: NIRENBERG ET AL. Proc. N. A. §. TABLE TEMPLATE SPECIFICITY OF TRINUCLEOTIDES Oo Moles of C1 or H*Aminoacyl-sRN A Bound to cua Ce che force Cie cw Cll or He” Cw cw, Trinucleotide Ala Are Asp Asp-NH:; Gh Glu-NH: Gly His Neu ApCpU ~0 16 ~0.04 -0.06 -0.03 -602 -003 —0 27 -0.02 0 ApCpC -0.15 -0.15 0.08 0.05 -0.02 -0.29 -—0 34 0.03 0.02 ApCpA -0.04 -0.01 0.05 0.05 0.03 -003 -0.78 0 0 ApCpG -0.15 —0.37 0.02 -0.03 —0 04 003) —0. 18% 0.03 0.02 GpCpU 0.73 ~0.18 -0.08 —0.08 0.01 -0.13 -o0.13% ~9 02 0 CpCpa ~0.15 0.06 ~0.03 6 0 0.04 0.04 0.01 0.01 UpCpG —0.20 0 0 0.07 0.03 -0.23 —-0.44 0.01 0.02 GpApU —0.01% —O.14 1.29 0.33+ 005 -0.02 -0.23) ~9 93 0.0) GpApC —0.05 —9 29 1.32 a.19t gt -0 10 —6 276 0.02 -0.03 GpApA -0 07) -on) 0.01 6 062 -0.32 -0.68 -0.03 -0 6) CpAptU 0.01 -0.31 —0.0: —0.03 —®,02 0 -—0.13° 0 52 ¢ CpApc ~0.02% -025 —_6.01 © 04 oe -0.14 —0.08> 0.26 -—06.02 CpApA 0.02% 0.01 -0.06 -~0.07 -@02 2.05 —0.93> 0.02 -6.04 CpAps -0.13 ~6.01 0.02 ~0 03 G05 2.60 ~0.12 -0.03 0 UpApa -0.022 -041 -0.01  -60.07 0.02 -0.30 -0.84° —~0.08 0 UpApG —0.07 0.06 0.01 0.12 0.04 0 -0.01  —6.0} 0.02 ApGpU — 0.07% © 03 0 0.04 0 -0.03 —0.06¢ 0.03 —-0.02 ApGpC 0.43 ~0.03 6.03 010 -0.02 6 ~0.13¢ 0.03 —6.03 ApGpA —0.06 0.10 0.01 0 0.19 0 ~0.31 0 — 0.08 GpGpU -—0 022 —0.33 0.01 -0.02 0.04 -0.2) 3.04 -0.03 -0.01 CpGpc o.14 1.63 0.02 -0.13 -0.01 -0.07 -0.62 -003 —0 02 CpGpA —C.20 142 -601 -6.0; 0.07 —0.05 0.08 —~0.03 0 UpGpc 0.28 -0.18 0.05 0.12 6.04 -005 -0.55 -002 -0 02 UpGpA -012 -0.12 0.07 014 0.10 -0.13 ~0.30 —0.03 0.02 ApUpG -0.01% -0.12 ~0 06 0.02 -001 -0.14 -0.17% ~09.02 0 CpUpG -0.14 -0.11 -0.01 0.04 0.01 0.1] ~1.20 6.03 6.03 Minus trinucleotide 0.50 1 16 0.21 0.21 0.12 1.65 2.89 0.25 0.08 (uzmoles)* 0. 20 -.- .. . Q.34° ee 1.10% Le . Trinucieotides pre- a wee wee 1.19 tee a ee wee 0.72 viously Lee wee . ApApU . a a an ApUpU described Le a . 1.50 . . — oa 0.59 {4 pumolea) Loe sae -. ApApC tee . a Lee ApUpc The specificity of trinucleotides in directing the binding of C™ or H*aminoacyl-sRNA to ribosomes. Repro ducible stimulations of AA-sRN 4 binding due to the addition of trinucleotides are bold face. For comparison, the template activities of 18 trinucleotides iously described’ ~5 are shown at the bottom of the table. Reactions contained the components deacribed Materials and Methods, the amount of Cit. AA-«RNA stated previ- oustye or in Table 1, and 0.150 A unite of trinucleotide, as apecified, in a final volume of 50 al. CC Asp-NH> wes cognition of 2 out of 3 bases in a trinucleotide, in or out of phase, or 2 bases in 1 trinucleotide and 1 base in an adjacent trinucleotide, often may suffice during protein synthesis. This striking phenomenon is often observed with trinucleotides containing 2 or 3 purines. Since the stability of codon-ribosome-AA-sRNA complexes may partially depend upon interactions between bases in codons and sRNA, the affinity of sSRNA for a ribosome may be greater when each base in a codon is recognized correctly and in proper phase than when codon recognition is only partially correct. The activity of both ApGpU and ApGpC in stimulating binding of C'*Ser-sRNA may indicate that these sequences correspond to serine codons (in addition to UpCpU, UpCpc, and UpCpG). However, these assignments should be considered tentative. Although ApGpU and ApGpC have been proposed as asparagine codon sequences,'* the data of Table 3 show that they do not Significantly affect the binding of C!Asp-NH-rsRNA under the conditions employed. We have previously reported that ApApU and ApApC stimulate binding of Asp- NH-rsRNA with high specificity.® ApGpaA slightly stimulated the binding of both C’*Arg- and C'-Glu-sRN A. The sequence ApGpA was predicted for arginine on the basis of codon sequence data obtained earlier and amino acid replacement data reported by Yanofsky and Vou. 53, 1965 BIOCHEMISTRY: NIRENBERG ET AL. 1165 3 ror C'+. on H?-Aminoacyt-sRNA Ribosomes Due to Addition of Trinucleotides* cia cu S14   cua Cc cM cite cm Hu cw. ci. Leu Lys Met Phe Pro Ser Thr Trypt Tyr Val —0.12 0.01 0.03 0.02 0.02 -0.05 0.63 —90.01 0.03 0 —90.09 —0.03 0 0 —0.01 0.03 0.50 —0.03 0.03 0 —0.09 0.17 —0.04 9.01 0.05% —0.07 0.45 0.03 0.01 0.01 —0.22 0 0.02 0.07 -0.01 —0.15 0.78 —0.03 0.03 0 0.25 -0.10 —0.18 =0.04 —0.02 —0.18 0.08 0 0.02 ° 0.02 0.11 0.03 0.03 0.40 ~0.08 ~0.02 0.03 0.03 0 0 0.04 -0.11 0.03 0.06° 1.09 -0.04 0.02 0.05 0.03 -0.10 -0.12 —0.09 —0.29 —0.03 0.01% 0 04 0.04 a 0 —9.10 -0.10 —0.10 —0.24 —0.01 0.01% 0.01 0 02 0.03 0.03 -0.03 0.73 0 -0.03 -0.07° -0.23 —0.08 —-0.04 0 ~0 04 —0.03 —0.07 —0.04 —0.15 ~0.01 0.02 -9.07 -0.01 0.03 0.02 0.01 -0.13 -0.01 —0.13 0.01 ~0.03¢ 0.05 —0.03 0 04 0.03 0.03 0 10 -0 12 -0.19 -0.01 -0.02% -90.04 0 -90.0t -0 01 0.03 —0 03 -0.02 0.04 -0.01 ~0.09 -0.09 - 0.03 0.09 0.02 —0.05 0.10 -0.09 —0.38 0.02 0 02 ~0.03 0 0 OL 0.03 -0.09 —0.05 -0.02 ~0.17 0 0 ~0.05 0.03 0.03 0 -0.16 —0.18 —0.05 —0.22 —0.04 0.27 —0.02 —0 04 -0 03 0.08 -O.11 -0.12 -0.05 —0.20 -0.01 017 . —0.05 0.01 0.02 —0.09 ~0.01 ~0.06 -0.13 0.01 0.03 ~0.05 -0.05 0 02 0.02 ~0.27 -0.10 —0.08 ~0.34 -0.07 -0.07 -~O.11 ~0.04 0.93 0.03 0 03 0.168 0.01 —0.03 ~9.05% 0.12 0 -0.02 0 -0.01 -0.14 -0.11 —0.17 —0.04 ~0.12 ~0.22 -0.06 0 0.03 ~0.02 -0.07 0 -0.18 0.04 0.02% 0.02 a 0 04 0.03 0.02 -0.04 -0.11 0 ~0.27 0 0 0.08 0.03 0.03 —0.05 —0.38 —0.06 1.00 ~0.04 0 —0.08° 0 Ot —0.06 0.03 -0.10 0.30 0.03 0.10 0.05 0.03 0.03 —0.06 —0.03 0.04 0.05 0.79 0:77 0.40 0.44 0.14 0.58 0.30 0.30 0.12 0.22 _— Lee _— nn 0. 40 0.24° _— see ee Lee 0.23 1.77 Lee 1.05 0.28 1.27 --- Lee 0.31 1.84 UpUpG ApApA Lae UpUpU pCpCpC UpCpu Lee Lee TpApU GpUpU 0.04 1.00 a 171 0.08 034 Lo. Lee 0.56 Lee Cet ApApG wae UpUpc Coeee UpCpc cee Lee UpApc CpUpc i ) ay CpCpt — “ ~ “ assayed in 100-ul reactions; amounts of all components were doubled. * Background binding of C!*aminoacyl-sRN A to ribosomes in the absence of trinucleotides is expressed in samoles (shown near the bottom of the table). All other values (4 wumoles) were obtained by subtracting back- ground binding of C'aminoacyl-aRNA from binding obtained upon addition of a trinucleotide preparation. tsRNA may contain some C!-Asp-sRNA. co-workers.'4 The recognition of one triplet by sRNA corresponding to several amino acids again suggests partial codon recognition. Such observations should be considered in terms of in vivo studies, particularly those related to extragenic suppression. Possible Base Sequences of Nonsense Codons.--Since nonsense codons may per- form special functions in protein synthesis, we have been particularly interested in a small group of trinucleotides, UpApA, UpApG, UpGpA, CpUpU, CpUpC, and ApGpA, which either have little tanplate activity, or have slight activity for two or more C'4-AA-3RNA. The possilflity that CpUpU or C pUpC may serve as in- ternal, but not as terminal, codons for leucine has been discussed previously.‘ The striking results of Sarabhai, Stretton, Brenner, and Bolle indicate that certain codons in “amber” mutants of T4 phage may correspond to an amino acid in certain strains of E. cola but may specify the terminus of a protein in other E. colt strains. Further analysis of mutant phages and amino acid replacement data have led Brenner and co-workers to suggest that UpApG or UpApA may specify the end of a polypeptide chain in some &. coli strains and serine in an additional strain which contains a suppressor gene. Weigert and Garen also have found that mutations which lead to the formation of nonsense codons in the alkaline phos- phatase gene can be related to amino acid substitutions at sites corresponding to 1166 BIOCHEMISTRY: NIRENBERG ET AL, Proc. N. A. 8. nonsense codons only if the base composition of the nonsense codon is (WAG).\"* This codon also corresponds to serine in a strain containing an appropriate suppres- sor gene. We find that the sequences UpApG and UpApA have almost no template activity for C'<amino acids under the conditions employed (only very small stimulation of C1*-Asp-NH- and C-Lys-sRNA binding was observed}. These data are in full accord with the conclusions of Brenner and of Garen and their co-workers. In addition the sequences found for glutamine codons were CpApG and CpApA: the sequences found for serine codons were UpCpG. UpOpt, UpCpc, ApGpl, and ApGpC (UpCpA also predicted). These sequenges demonstrate structural rela- tionships between Terminator-, Glu-NH--, and Ser-codons and suggest mechanisms for alternate codon recognition in different strains of E. coli. The General Nature of the Code-—Thus far, the template functions of 45 of the 64 trinucleotide sequences have been investigated in this system. A summary of the data and additional codon Sequences which can be predicted from amino acid re- placement data reported for E. coli!4 and TMV mutants,’* ” are shown in Table 4. Almost all of our earlier predictions were confirmed when the appropriate tri- nucleotide was tested.2—5 Nevertheless, the summary shown in Table 4 should nat be thought of as an invariant codon dictionary, since it is clear that codon recogni- tion can be modified. Previous studies with randomly ordered polynucleotides and cell-free protein synthesizing systems showed that synonym codons often differ in composition by only one base.”% #* This suggested that bases common to synonym codons occupy identical positions and, that either 2 out of 3 bases in a triplet sometimes may be recognized, or a base may be recognized correctly in 2 or more ways.?! * On the       TABLE 4 NouciEorme Sequences or RNA Copons Upupc Phe vpepe Se EREPE. ve page Tyr UpOh ts BBE Se Uh, Net, YRM CHURC “Nonsense — BEBE Pro PGR arg Chape His ChuRG Leu Cea, Pro CREA, Arg 486 Glu-NH, ‘Ife BBE tw BEE se RE apupe. Met Acne Te AE A ae BA Live GpUpc Va Gpcpe Alt GFRPE Gy Gpape Ap Gpupa Val GpcpG “= GPERA iy Gpapa, Gly * It is possible that these sequences are readable internal-, but nonreadabile termina)]-, codons. tT Upapa a gyPArG may correspond to Terminator-, or Ser-codone in different strains of E. coli (see text or refs. and 18). Summary and predictions: The template activities of trinucleotides in BOLDFACE have been studied experi- mentally in thie system. Other Sequences are predicted. Although trinucleotides are arranged in pairs, one member a pair may have greater template activity than the other. Estimates of relative template efficiencies are not indicated. Amino acid SGflscement data used for these predictions were obtained with E. coli by Yanofsky,™ or were induced by HNO: in TMV by Wittman and Wittman-Liebold’* or Teugita.» Von. 53, 1965 BIOCHEMISTRY: NIRENBERG ET AL. 1167 basis of such data Woese suggested a code in which A, C, G and U are independently recognized at one position in the triplet, C = U at a second position, and A = C and G = U ata third position.* A modification was suggested by Eck in which U = Cand A = G at an unspecified position in a triplet.* We have previously shown that each member of a trinucleotide pair with 3’-terminal pyrimidines, such as XpYpU and XpYpC, corresponds to the same amino acid,* and each member of a codon pair with 3’-terminal purines, ApApA and ApApG, corresponds to lysine.* Several generalizations can be made concerning the nature of the code. (a) Amino acids, which are structurally or metabolically related (such as synthesized im vo from a common precursor) often have similar RNA codons. Such relation- ships would appear to reflect either the evolution of the code*‘ or direct interactions between amino acids and bases in codons.” * (6) Many codons may be recognized partially, or may contain alternate acceptable bases at certain positions. (c) Recognition of the 3’-terminal base in a trinucleotide is most variable and fits several general patterns; U = C; G= A; orG=Az=U2=C. (d) In the case of Leu- codons, U = C at the 5’-terminal position. (e) In most cases the apparent tem- plate activity of one member of a synonym codon set differs from that of another. These patterns apparently define the characteristics of several general recognition mechanisms. Although the molecular mechanisms which permit one codon to be distinguished from another are unknown, the pairing of bases in mRNA with bases in SRNA is an obvious one to consider. Enzymic modification of bases in certain doublet or triplet sequences in sRNA might affect codon recognition, as proposed by Ames and Hartman,” and also explain patterns of synonym codons sets. Inter- conversion of C and U (C = U) in an sRNA “anticodon” might resultina G = A pattern in mRNA codons and interconversion of A and I (A =I) ina U = C pat- tern in mRNA codons. However, the possibility of alternate acceptable base pairing is raised by observations which indicate that one molecule of Phe-sRNA may recognize both UpUpU and UpUpC.4 The nucleotide sequence found for an alanine codon (GpCpU found, GpCpC, GpCpA, and GpCpG predicted), together with the nucleotide sequence of an alanine sRNA isolated from yeast, reported by Holley et al.,” may provide clues to the recognition process. Two striking sequences in yeast Ala-sRNA, IpGpCpMelpy, and dihydroUpCpGpGpdihydroU, each potentially comprising a single-stranded loop at the end of a hairpin-like double-stranded segment, have been suggested as possible “‘anticodons’’.® The first sequence suggests antiparallel Watson-Criek base pairing between Ala-sRNA and a GpCpC Ala-eodon mRNA (CGI), GCC. The second sequence raises the possibility of paradlel Watson-Crick pairing with an Ala-codon of GpCpC, (CGG/GCC). Further work is necessary to determine whether one molecule of Ala-sRNA is capable of recognizing two or more alanine codons. Tt is a pleasure to thank Taysir Jaouni, Norma Zabriskie, and Theresa Caryk for invaluable assistance, * Supported by American Cancer Society postdoctoral fellowship PF 201. + Supported by USPHS postdoctoral fellowship 6 F2 AM-17, 108-O01A1, and American Cancer Society PF 244. { For brevity, trinucleoside diphosphates are referred to as trinucleotides. 1 Nirenberg, M., and P. Leder, Science, 145, 1399 (1964). 1168 BIOCHEMISTRY: NIRENBERG ET AL. Proc. N. A. §. ? Leder, P., and M. Nirenberg, these ProcEEp1NGs, 52, 420 (1964). + Leder, P., and M. W. Nirenberg, these Procrgpinas, 52, 1521 (1964). ‘ Bernfield, M. R., and M. W. Nirenberg, Science, 147, 479 (1965). 5 Trupin, J., F. Rottman, R. Brimacombe, P. Leder, M. Bernfield, and M. Nirenberg, these Proceepines, 53, 807 .1965). 6 Nirenberg, M. W., in Methods in Enzymology. ed. S. P. Colowick and X. O. Kaplan (New York: Academic Press, 1964), vol. 6. p. 17. 7 Nirenberg, M. W., J. H. Matthaei, and O. W. Jones, these ProceEpinas, 48, 104 (1962). ® Pestka, S., R. E. Marshall, and M. W. Nirenberg. these ProcrEpinGs, 53, 639 (1965). ® Lapidot, Y., and G. Khorana. J. Am. Chem. Soc., 85, 3852 (1963). 1 Chladek, 8., and J. Smri, Collection Czech. Chem. Gommeun., 28, 1301/1963 1. 4 Bernfield. M. R.. and M. W. Nirenberg. .4bstract i4Sth National Meeting. American Chem- ical Society. Chieagu. Mlinois, August 1964. 12 Leder, P., M. F. Singer, and R. L. C. Brimacombe, submitted to Brochem istry. 13 Kaji, H., and A. Kaji. these Procrgpines, 52, 1541 (1964). 14 Yanofsky, C., in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology. vol. 2& (1963), p. S&L. 1 Matthaei. J. H.. H. Kleinkauf. and G. Schramm. Angew. Chem., 76, 717 (1964); Angew. Chem. Intern. Ed. Engl., 3, 590 (1964). 16 Sarabhai, A. S.. A. O. W. Stretton, S. Brenner, and A. Bolle, \\\\ ature. 201, 13 (1964). Y Brenner, S., A. O. W. Stretton, and 8. Kaplan, Nature, in press. 1 Weigert, M. C., and A. Garen, in press. 1% Wittmann, H. G., and B. Wittmann-Liebold, in Synthesis and Structure of Macromalecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 26 (1963), p. 589. * Tsugita, A., personal communication. 1 Jones, O. W., and M. W. Nirenberg, these Prockepinas, 48, 2115 (1962). 22 Nirenberg, M. W., and O. W. Jones, in Symposium on Informational Macromolecules, ed. H. Vogel, V. Bryson, and J. Lampen (New York: Academic Press, 1963), p. 451. 23 Speyer, J. F., P. Lengyel, C. Basilio, A. J. Wahba, R. S. Gardner, and S. Ochoa, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 559. 2 Nirenberg, M. W., O. W. Jones, P. Leder, B. F. C. Clark, W.S. Sly, and 8S. Pestka, in Syntheszs and Siructure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 549. % Woese, C., Nature, 194, 1114 (1962). *® Eck, R. V., Science, 140, 477 (1963). ™ Woese, C. R., ]CSU Review of Werid Science, 5, 210 (1963). % Weinstein, I. B., in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 579. 28 Ames, B. N., and P. E. Hartman, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Svmposia on Quantitative Biology, vol. 28 (1963), p. 349. *® Holley, R. W., J. Apgar, G. A. Everett, J. T. Madison, M. Marquisee, 8. H. Merrill, J. R. Penswick, and A. Zamir, Science, 147, 1462 (1965).", "O'Neal, C. ; Nirenberg, Marshall W. ; Leder, Philip ; Bernfield, Merton ; Brimacombe, R. ; Rottman, F. ; Trupin, J.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-i2ih-ftpp-hzjw", "00000000-0000-0000-E7D9-49ED31A405CC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Regulation of Axon Formation by Clonal Lines of a Neural Tumor", "101584910X54", null, "1970", "May 1970", "This article, written by the NIH team from the laboratory of Biochemical Genetics, provides an early example of Nirenberg's transition from genetics toward neurobiological topics.  In his work on neuroblastoma cells seen here, experimental evidence suggests that axons appear to extend or retract differently depending on the history of their clonal life, suggesting a possible difference in gene expression.", "Articles", "Axons,Clone Cells,Neuroblastoma", "Neuroblastoma Research, 1967-1976", "8", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proceedings of the National Academy of Sciences Vol. 66, No. 1, pp. 160-167, May 1970 Regulation of Axon Formation by Clonal Lines of a Neural Tumor N. W. Seeds,* A. G. Gilman, T. Amano, and M. W. Nirenberg LABORATORY OF BIOCHEMICAL GENETICS, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated February 5, 1970 Abstract. Clonal lines of neuroblastoma cells were found to extend or retract axons depending upon the concentration of serum. Neurite extension was not inhibited by cycloheximide but was sensitive to colchicine or vinblastine, sug- gesting that neurite formation is dependent upon the assembly of microtubules or neurofilaments from preformed protein subunits. Clonal lines of neuroblastoma cells exhibit many properties of differentiated sympathetic neurons. Cells extend branched axons‘ up to 3000 y in length,’~‘ possess membranes that are electrically excitable,* respond to acetylcholine,’ and also contain enzymes for the synthesis and metabolism of catecholamines!: *> ® and for the hydrolysis of acetylcholine.!|7 Cultures usually contain two types of cells quite different in morphology: round cells without processes and cells with axon-like processes. Although cell shape in vitro is notoriously variable and many varieties of cells are capable of extending processes, the distinctive neuronal appearance of neuroblastoma processes agrees well with the neural characteristics demonstrated in biochemical and neurophysiological studies. The proportion of neuroblastoma cells with axons was found to vary greatly, depending upon the clone studied and the conditions of cell culture. The system is relatively simple from an experimental point of view and may be useful for studies of both the differentiation and the function of neurons. Materials and Methods. Neuroblastoma C-1300 was adapted for growth in vitro as previously described.4 Cells were first cloned as a colony from agar and were then cloned from single cells with the use of stainless steel cylinders. Cultures of neuroblastoma clone N-18 were grown in Falcon flasks or Petri dishes in Dulbecco’s modification of Eagle’s medium (DMEM) plus 10% fetal calf serum (except where indicated) at 37°C in an atmosphere of 10% CO: and 90% air. Cells were evaluated for the presence of axon-like processes from photomicrographs (data of Figs. 4 and 5) or directly under the microscope. An initial cell concentration of approximately 3000-5000/cm? proved to be convenient for counting cells and axons and was used except where indicated; 300-1200 cells were counted for each value. Repro- ducibility was approximately +15%. Trypsinized cells (500-1000) were counted with a hemocytometer and viability was estimated by exclusion of nigrosin. 3H-Proline and *H-thymidine incorporations into protein and DNA, respectively, were determined by addition of labeled and unlabeled compounds in Dulbecco’s modification of Eagle’s medium to plates to achieve the following concentrations: L-proline 10~* AM, 10 »Ci/ml, 2850 cpm/nmole; thymidine 3 X 10-° M, 5 pCi/ml, 600 epm/pmole. Cells 160 Vot. 66, 1970 BIOCHEMISTRY: SEEDS ET AL. 161 were incubated for 1 hr at 37°C in a 10% CO.-90% air atmosphere. Protein synthesis was determined by counting hot trichloroacetic acid-precipitable material on nitrocellu- lose filters. DNA synthesis was assayed by counting cold trichloroacetic acid-precipitable material on glass fiber filters (Millipore Co.). *H-Thymidine (10 Ci/mmole) and *H-proline (26.3 Ci/mmole) were obtained from Schwarz and were purified by paper chromatography prior to use. Bovine plasma protein fractions were obtained from Pentex (a:-globulins, Cohn fraction IV,; aglobulins, fraction 1V4; 8-globulins, fraction III; y-globulins, fraction II; transferrin, 67% estimated purity; bovine serum albumin, crystalline). Results. We thought it likely that the proportion of neuroblastoma cells with axons and the length of axons might be related to the rate of cell division because cells are known to retract processes prior to cell division. Since serum is required for multiplication of neuroblastoma cells, the extent of cell division was restricted by incubating cells with relatively low concentrations of serum. Few cells possess processes in 10% fetal calf serum (Fig. 14). Most cells are round and adhere to one another, forming clusters. When cells are incubated without serum, axon-like processes are rapidly extended. Within 30 to 60 min, most cells possess processes 25-100 y in length (Fig. 1B); relatively long neurites are found after one day (Fig. 1C). After four days (Fig. 1D) many cells possess axons up to 2000 » in length, and further elongation and arborization are apparent at seven days (Tig. LE). In most cases, two to four branched neurites extend from the body of a single cell. Usually a binary pattern of neurite branching was found (i.e., two neurites arise from each branch node). The relation between serum concentration and the proportion of cells with neurites is shown in Figure 2. Approximately 1-5% of the cells extend axons in 10% serum. Most of the cells extend neurites in the presence of 0 to 1% serum; however, the rate of appearance of neurites is inversely related to serum concentration. The effect of cycloheximide upon axon formation was studied to determine whether axon outgrowth and migration are dependent upon protein synthesis (Fig. 3). In addition, cells were incubated with colchicine or vinblastine to investigate the possibility that axon outgrowth is dependent upon the assembly of microtubule protein. Both alkaloids interact with microtubule protein.* ° Cycloheximide was found to have little effect on neurite outgrowth at concen- trations up to 1.8 X 10-! M. This concentration of cycloheximide inhibits the incorporation of proline into protein by more than 97%. However, vinblastine and colehicine inhibit neurite out-growth completely at 10—7 and 10-* AY, respectively. These results suggest that neurite formation does not re- quire de novo protein synthesis but is dependent upon the assembly of micro- tubules from performed protein subunits. In addition, electron microscopic observations demonstrate numerous microtubules, 240 A in diameter, and neurofilaments, 100 A in axons induced by growth in low serum. Additional factors influencing neurite outgrowth are shown in Table 1. Neurites are not extended by cells at 3° and outgrowth is greatly retarded at 24° compared to 37°. The temperature of incubation thus markedly affects neurite outgrowth. As shown in Experiment 2, the serum factor(s) affecting neurite outgrowth is not dialyzable and is active after incubation at 100° for   Proc. N. A. 8. SEEDS ET AL. BIOCHEMISTRY: 162 Conditioned media (final serum concentration, 1-2%) also inhibits neurite formation. 15 min. Cells incubated in Dulbecco’s phosphate-buffered saline s as well as those incubated in growth solution plus glucose extend processe medium (Expt. 3). Mgt*. absence of Cat+ and No processes are extended in the n gg sjuesaidat Py amos UW aul] ayy OT, ‘Apaatjoadsad ‘sfup 2 pus “p J Jaye gy pur ‘gq ‘9 sownig Ut Sa}LUNAU BAISUG)XI BOUL JO WOLWLOGV]D aaissaidoid ay} puw YZ audi UL UMOYS ST UOLVQNOUL JO UL (VG 19)FB sassa001d LOWS yo Yysod pRIUT “Wines JOYA payeqnoul puw whipeul SI? YA pasuls Wey} aiem soy[g “PY ewmiy ul uMoys SI ay PZ 40} Y)MOIT aayye ABopoydsow [ao punos jeordAy, ‘unsas jyeo Bay 24G pus was asioy 256 snd wmipau s,ayzueq jo UOTPBOYIpOW S,OVdeqTNC] WT payefd asa s]fIH (q-PV)—ToW     Vou. 66, 1970 BIOCHEMISTRY: Fig. 2.—Effect of serum concentra- tion upon rate of neurite formation. Cells were grown in Dulbecco’s modi- fication of Eagle’s medium containing the indicated total concentration of an equal mixture of horse serum and fetal calf serum, and neurites were evaluated at indicated times. A, No serum; O, 0.3% serum; X, 1.0% serum; 0, 10% serum; V, data obtained at 1, 2, and 8 hr in a separate experiment where serum was removed from cells previously grown in 10% serum for 24 hr. PERCENT CELLS WITH AXONS The effects of serum protein fractions upon neurite formation areshownin Table 2. Crystal- line bovine serum albumin, transferrin, and a bovine y-globulin fraction have little effect upon the formation of neurites. However, a-, a, and 6-globulin fractions from bovine serum are inhibitory. Partial inhibition is also observed at concentrations 10 to 100-fold lower than those shown. Chondroitin sulfate, a-lactalbumin, and g-lactoglobulin also are without effect (data not shown). An attempt was made to examine the relation between neurite extension and cell division (Tigs. 4A and B). During logarithmic growth in 10% serum, less than 3% of the cells possess neu- rites. After incubation for two days, the cul- ture media of some plates was replaced with fresh SEEDS ET AL.   163 NEURITE FORMATION v 10 20 30 40 50 60 70 80 HOURS     PERCENT CELLS WITH AXONS i 8 x? we ws MOLARITY Fic. 3.—Effect of colchicine, vinblastine, and cycloheximide on initial neurite formation. Cells were grown with 5% horse serum and 5% fetal calf serum. After 24 hr plates were rinsed with Dulbecco’s modification of Eagle’s medium and then in- cubated for 2 hr in this medium and the components indicated. media with 0.1% serum. Eighty percent of the cells extended neurites during the next 24 hours. Tasue 1. Requirements for initial neurite formation. Further incubation led to a marked increase in neurite length; Percentage of Expt. no. Conditions cells with axons 1 Minus serum, 3°C 0.3 Minus serum, 24°C 9 Minus serum, 37°C 73 10% serum, 37°C 1 2 Minus serum 70 1% serum 7 1% serum, heat for 15 min at 100°C 15 1% serum, dialyzed 12 3 Minus serum 76 Phosphate-buffered saline (minus serum and growth medium) 76 Phosphate-buffered saline without Ca++ and Mg++ (minus serum and growth medium) 8 Cells were incubated 1 day in Dulbecco’s modification of Eagle’s medium plus 10% fetal calf serum. Plates then were washed and fresh medium plus components listed ubove were added. Cultures then were incubated for 2 hr at 37°.   164 BIOCHEMISTRY: SEEDS ET AL. Proc. N. A.S. TABLE 2. Effect of serum fractions on initial neurite formation. Percentage of Additions cells with axons None 83 10% fetal calf serum 2 Albumin, crystalline 74 Transferrin 84 oy-Globulin (IV-1) 9 as-Globulin (IV-4) 4 6-Globulin (IIT) 21 y-Globulin (IT) 68 Cells, incubated for 1 day in the presence of 10% fetal calf serum, were washed with Dulbecco’s modification of Eagle’s medium and then were incubated for 2 hr with this medium plus the com- ponents indicated (serum fractions were tested at 1 mg protein/ml of media, final concentration.) however, the proportion of cells with neurites remained constant. On the fifth and seventh day, some cultures were stepped up from 0.1 to 10% serum; fewer eclls with neurites were found after incubation. Additional results obtained by time-lapse cinematography show that neurites that detach from the surface of the Petri dish are resorbed. However, many cells retain long, well-developed neu- rites. Since cells with relatively long neurites are uncommon during logarithmic growth, it seems probable that some neurites are not retracted under these con- ditions. The number of viable cells per plate is shown in Figure 4B. After a short lag, cells in 10% serum grew logarithmically; the population generation time was 16 hr. The rate of cell multiplication decreased markedly when cells were shifted down from 10 to 0.1% serum. In 0.1% serum the number of cells per plate did not increase after two days of incubation. While the addition of 10% serum on the fifth day resulted in disappearance of neurites and cell multiplication,               T T T T T T “TF T ] jo. 8 CELLS 10% 3 ' E 10% 5 (09 A AXONS SHIFT UP SHIFT UP “ a 6. F 4 ay DOr sHiFT DOWN b 4 24 [ aN 1 Zz 1 : = i — ge YF | ; HQ, a 4 x 70k ' \\\\ \\\\ 4 4 } 01% | E sis! ; 4 = 60 O41 ‘1 \\\\ \\\\ “ 3 | 3 & 50+ t \\\\ \\\\ 4 WS t a I 1 & aor | , | 4 & SHIFT | 2 UP. 2 20 10% on\\\\ - >O.2+ o i oe _ pe 20; | eo 0.4 5 lor o | 0 4 | o 0 tT 1 4 1 L 1. L 1 4 oT f¢ 3 45 6 7 8 9 b 2 3 4 5 6 7 8 DAYS DAYS Fic. 4.—(A and B) Effect of serum on axon formation and cell multiplication. On suc- cessive days the number of viable and total cells were counted and photomicrographs were obtained for axon counts as described under Materials and Methods. Symbols represent the following: O, 10% fetal calf serum; A, on the second day the cultures indicated in the figure were rinsed with Dulbecco’s modification of Eagle’s medium and then incubated in fresh medium plus 0.1% fetal calf serum; @, on the fifth day fetal calf serum was added where in- dicated (10%, final concentration); ©, on the seventh day fetal calf serum was added (10%, final concentration) to cultures as indicated. Vou. 66, 1970 BIOCHEMISTRY: SEEDS ET AL. 165 the population generation times were 45 and 29 hours for the first and second generations, compared with 16 hr found with cells in logarithmic growth. Since the generation time of individual cells during logarithmic growth was also found to be 16-18 hours (by time-lapse cinematography), division by 35% of the cells could account for the prolonged population generation time. Morphological observations of such cultures support the possibility of a population of non- dividing cells with long axons. Alternatively, all cells may be capable of dividing with a prolonged generation time. The rates of DNA and protein synthesis were also determined in the above experiment (Table 3). In general, the rate of thymidine incorporation was Tapiy 3. Effect of serum on *I-thymidine and *H-proline incor poration. Growth Percentage condition of serum Day* Logarithmic 10 1-4 Step down 0.1 3-5 Step down 0.1 6-8 Step up 10 6 Step up 10 8 *Day shown in Figs. 4A and B. Experimental conditions are given in the legend to Fig. 4. 3H-thymidine epm (X 1073) Incorporated per 10° Viable Cells tH-proline 12.541.6 3.5 + 0.8 8.384 1.1 4.3240.5 2.2 + 0.2 1940.1 19.6 3.7 13.0 4.2 3H-Thymidine incorporation into nucleic acid and *H-proline incorporation into protein were determined sis described under Materials and Methods. Average values + the standard error of the mean are shown. in triplicate each day. Assays were performed similar to the rate of cell multiplication; however, the initial rate of incorporation after cells were shifted up to 10% serum on the sixth day was high compared with the second day of the experiment. rate of proline incorporation into protein was rel- atively less affected by changes in serum con- centration, being approximately 50% after pro- longed incubation in 0.1% serum. The effect of serum upon retraction of neurites was examined in a separate experiment (Tig. 5). Cells were incubated for one day without serum; approximately 84% of the cell population then possessed short processes. The addition of serum resulted in a rapid decrease in the per- centage of cells with neurites that was dependent upon serum concentration. Colchicine and vin- blastine also induced neurite retraction (data not shown). The effect of serum on process formation by other cell lines was also investigated. In the presence of 10% serum, mouse L cells and HeLa cells flatten, spread, and form eonfluent mono- layers, in contrast to the behavior of the neuro- blastoma cells. L cells possessed processes in the It is possible that cells were syn- chronized to some extent with respect to position within the cell cycle. The NEURITE RE TRACT:ON | Z°4\" | 2 ag ne a Wy Erol ON, F Gor { 4 YOON So a 50+ | ON =~ 05% | a \\\\ eo \\\\ RH 40> \\\\ SOM i 4 a 30, 2 20- Ne oe 0s Lo o re 3. 4 HOURS Fic. 5.—Effect of serum on neurite retraction. Cells were incubated 1 day in the presence of 10% fetal calf serum and then were incubated for another day without serum; then serum was added as indicated. Symbols rep- resent the following concentra- tions of serum: CO, 10%; Y, 1%; 0,0.38%; A, no serum. 166 BIOCHEMISTRY: SEEDS ET AL. Proc. N. A. 5S. presence of 10% serum. Removal of serum from cultures of L or HeLa cells had no discernible effect upon processes. Colchicine (10-* Af) inhibited the spread- ing of L or HeLa cells and the formation of processes by L cells. Thus L cells and HeLa cells differ from N-18 neuroblastoma cells in response to shifts in serum concentration. Discussion. Separate clones derived from the same tumor differ markedly in the ability to extend neurites. For example, one neuroblastoma clone extends processes infrequently, whereas the majority of cells from another clone extend processes soon after they are plated. Neuroblastoma clone N-18 is particularly interesting because the proportion of cells with neurites was found to vary more than 100-fold depending upon environmental conditions. While Klebe and Ruddle™ have selected for populations of neuroblastoma cells with processes by elimination of dividing cells with fluorodeoxyuridine, we have devised a simple method for converting entire populations of round cells to cells with neurites that depends upon alteration of serum concentration. Serum probably affects process formation in several ways, for factors are present that influence the attachment of cells to plate, thus altering the balance be- tween process extension and retraction, in addition to stimulating cell division, nucleie acid, and protein synthesis.°—13 As Weiss'4 has emphasized, neurites migrate only on solid surfaces, thus the stability of interactions between cell and substratum is of great importance. The morphology of neuroblastoma cells is apparently derived by a process of selection. Neurites may explore an area 10,000 times that occupied by the cell body and neurites forming the most stable set of attachments relative to perturbing forces are selected. Results obtained by time-lapse cinematography show that the rate of neurite migration from neuroblastoma cells is approximately 75-125 u/hr. However, migration is discontinuous. Removal of serum from cultures results in axon outgrowth which may be re- lated, at least in part, to a more stable interaction between cells and plate. How- ever, the restriction on the rate of cell division imposed by the absence of serum probably permits the uninterrupted synthesis of relatively long neurites, since neuroblastoma cells retract processes prior to mitosis. The relation between cell division and axon or dendrite extension may be of fundamental importance. Since 70-85% of neuroblastoma cells extend pro- cesses within 60 min after removal of serum, it is clear that cells are capable of extending processes during most of the cell cycle. One may hypothesize that axons and dendrites migrate from most normal neurons of the central nervous system when the neurons are in the G-1 period of the cell cycle and are repressed with respect to cell division, since most neurons are diploid and do not divide. However, neurites may migrate from certain neurons, such as Purkinje cells, during the G-2 period, since these neurons are tetraploid. At least two modes of repressing neuron multiplication can thus be envisioned. Cycloheximide does not inhibit the initial outgrowth of neurons; thus, neurite synthesis is not dependent upon protein synthesis. Colchicine or vinblastine, which bind to microtubule protein,® ° completely inhibit neurite formation, Vou. 66, 1970 BIOCHEMISTRY: SEEDS ET AL. 167 implying that neurite synthesis is dependent upon the assembly of microtubules or neurofilaments from preformed protein subunits. The formation of relatively long neurites probably is dependent upon the synthesis of additional microtubule protein subunits. These results are in accord with observations that pertain to flagellar regeneration.’ Olmsted ef al. have shown that mouse neuroblastoma C-1300 contains micro- tubule protein in relatively high concentration and that neurites are birefringent. In addition to our observations, Schubert ef al.’ previously demonstrated the presence of microtubules and neurofilaments in neuroblastoma cells by electron microscopy. It should be noted that cell mitosis is also dependent upon the assembly of microtubule subunits and is inhibited by colchicine and vinblastine. Since the termination of neuroblast multiplication either precedes or coincides with the initiation of axon formation, one wonders whether the sequence of events may relate, at least in some cases, to a mutual requirement for micro- tubule subunit assembly. We would like to acknowledge the technical assistance of Mrs. R. Selinger and Miss E. Cutler. * Postdoctoral fellow of the National Science Foundation (48034). + Axons and neurites are used synonymously to designate any cellular extension greater than 25 » in length. 1 Augusti-Tocco, G., and G. Sato, these ProcEEpINGs, 64, 311 (1969). 2 Schubert, D., S. Humphreys, C. Baroni, and M. Cohn, these PRocEEDINGS, 64, 316 (1969). 4 Olmsted, J., K. Carlson, R. Kiebe, F, Ruddle, and J. Rosenbaum, these ProcreEDINGs, 65, 129 (1970). “Nelson, P., W. Ruffner, and M. Nirenberg, these ProceEDinGs, 64, 1004 (1969). 5 Peacock, J., and P. Nelson, manuscript in preparation. 6 Nirenberg, P., 8. Wilson, N. Seeds, and M. Nirenberg, manuscript in preparation. 7 Blume, A., F. Gilbert, T. Amano, J. Farber, 8. Wilson, R. Rosenberg, and M. Nirenberg, manuscript in preparation. 8 Weisenberg, R. C., G. G. Borisy, and E. W. Taylor, Biochemistry, 7, 4466 (1968). * Marantz, R., M. Ventilla, and M. Shelanski, Science, 165, 498 (1969). © Taylor, A. C., Exptl. Cell. Res., Suppl., 8, 154 (1961). 11 Puck, T., C. Waldren, and C. Jones, these ProceEDINGS, 59, 192 (1968). 12 Lieberman, I., and P. Ove, J. Biol. Chem., 233, 637 (1958). 13 Todaro, G., G. Lazar, and H. Green, J. Cell. Comp. Physiol., 66, 325 (1965). 4 Weiss, P. A., Int. Rev. Cytology, 7, 391 (1958). 8 Klebe, R. J., and F. H. Ruddle, J. Cell. Biol., 43, 69a (1969). 1 Rosenbaum, J., J. Moulder, and D. Ringo, J. Cell. Biol., 41, 600 (1969).", "Nirenberg, Marshall W. ; Amano, Takehiko ; Gilman, A. G. ; Seeds, Nicholas W.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-taax~63hm-sbus", "00000000-0000-0000-7464-7221A8AB117D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Dependence of Cell-Free Protein Synthesis in E. Coli Upon Naturally Occuring or Synthetic Polyribonucleotides", "101584910X50", "101584910X49", "1961", "October 1961", "In this landmark essay, published in conjunction with \"Characteristics and Stabilization of DNAase-Sensitive Protein Synthesis in E. coli Extracts,\" the authors present the same conclusions revealed in Nirenberg's address at the now famous International Congress of Biochemistry in Moscow during August 1961.  Adding an artificial form of RNA, polyuridylic acid, consisting of the base uracil, to the    E. coli extract resulted in the production of an unnatural protein composed entirely of the amino acid phenylalanine.  The experiment showed that UUU was the codon for phenylalanine and that messenger RNA, which transcribes genetic information from DNA, directs protein synthesis. This provided a route to base compositions of codons and the exploration of the general nature of the code.", "Articles", "RNA,RNA, Ribosomal,Escherichia coli,RNA, Messenger", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "15", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Reprinted trom the Proceedings of the National ACADEMY OF Scrences Vol. 47, No. 10, pp. 1588-1602. October, 1961. PHE DEPENDENCE OF CELL- FREE PROTEIN SYNTHESIS IN E. COLI UPON NATURALLY OCCURRING OR SYNTHETIC POLY RIBON UCLEOTIDES By MarsHAauu W. NinENBERG AND J. Heinricn Marruarr* NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by Joseph E. Smadel, August 3, 1961 A stable cell-free system has been obtained from E. coli which incorporates C™-valine into protein at a rapid rate. It was shown that this apparent protein synthesis was energy-dependent, was stimulated by a mixture of L-amino acids, and was markedly inhibited by RNAase, puromyein, and chloramphenicol.! The present communication describes a novel characteristic of the system, that is, a requirement for template RNA, needed for amino acid incorporation even in the Vout. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1589 presence of soluble RNA and ribosomes. It will also be shown that the amino acid incorporation stimulated by the addition of template RNA has many proper- ties expected of de novo protein synthesis. Naturally occurring RNA as well as a synthetic polynucleotide were active in this system. The synthetic polynucleo- tide appears to contain the code for the synthesis of a “protein” containing only one amino acid. Part of these data have been presented in preliminary reports.? 3 Methods and Materials ‘The preparation of enzyine extracts was modified in certain respects from the procedure previously presented.) &. cold W3100 cells harvested in early log phase were washed and were disrupted by grinding with alumina (twice the weight of washed cells) at 5° for 5 min as described previously.2. The alumina was extracted with an equivalent weight of buffer containing 0.01 Tris(hydroxy methyl jaminomethane, pH 7.8, 0.01 M Inagnesium acetate, 0.06 Af KCI, 0.006 M mercaptoethanol (stundard buffer). Alumina and intact cells were re- moved by centrifugation at 20,000 x g for 20 min. The supernatant fluid was decauted, and 3 wg DNAase per ml (Worthington Biochemical Co.) were added, rapidly reducing the viscosity of the suspension, which was then ceutriftuged again at 20,000 x g for 20 min. ‘The supernatant fluid was aspirated and was centrifuged ut 20,000 X g for 30 niin to clear the extract of remaining debris. The liquid layer was aspirated (8-30) and was centrifuged at 105,000 x gy for 2 hr to sediment the ribosomes. The supernatant. solution (8-100) was aspirated, and the solution just above the pellet was decanted and discurded. The ribosomes were washed by resuspension in the standard buffer and centrifugation again at 105,000 X g for 2 hr. Supernatant fluid was discarded and the ribosomes were suspended in standard buffer (W-Rib). Fractions S-30, 8-100, and W-Rib were dialyzed against 60 volumes of standard buffer overnight at 5° and were divided into aliquots for storage at — 15°. In some cuses, fresh S-30 was incubated for 40 min at 35°. The reaction mixture components in ymoles per mi were as follows: 80 Tris, pH 7.8: 8 magnesium acetate; 50 KCl: 9 mercapto- ethanol; 0.075 each of 20 amino acids; 2.5 ATP, K salt; 2.5 PHP, K salt; 15 «gz PEP kinase (Boehringen & Sons, Manuheim, Germany). After incubation, the reaction mixture was dialyzed at 5° for 10 hr against 60 volumes of standard buffer, changed once during the course of dialysis. The incubated S-30 fraction was stored in aliquots at —15° until needed ( Lneubated-S-30). RNA fractions were prepared by phenol extraction using freshly distilled phenol. Ribosomal RNA was prepared from fresh, washed ribosomes obtained by the method given above. In later RNA preparations, « 0.2% solution of sodium dodecyl sulfate recrystallized by the method of Crestfield ef ai.4 was udded to the suspension of ribosomes before phenol treatinent. The suspen- sion was shaken at room temperature for 5 min. Higher yields of RNA appeared to be obtained when the sodium dodecyl sulfate step was used: however, good RNA preparations were also obtained when this step was omitted. An equal volume of HoO-saturated phenol was added to ribosomes suspended in standard buffer after treatment with sodium dodecv! sulfate, and the suspension was shaken vigorously at room temperature for 8 10 tin, The aqueous phase was aspirated from the phenol phase after centrifugation at 1450 x ¢ for 15 min. The aqueous layer was extracted (wo nore times in the same manner, using ?/» volume of HyO-saturated phenol in each case. The final aqueous phase was chilled to 5° and NaCl was added to a final concentration of 0.16. Pwo volumes of ethyl alcohol at —20° were added with stirring to precipitate the RNA. The suspension was centrifuged at 20,000 X yg for 15 min and the supernatant sulution was de- canted and discarded. The RNA pellet. was dissolved in minimal concentrations of standard buffer (minus mercaptoethanol) by gentle homogenization in a glass Potter-Elvehjent homogenizer (usually the volume of buffer used was about ‘/s the volume of the original ribosome suspension), The opalescent solution of RNA was dialyzed for 18 br against. 100 volumes of standard buffer (minus mercaptoethanol) at 5°. The dialyzing buffer was changed once. After dialysis, the RNA solution was centrifuged at 20,000 X g for 15 min and the pellet was discarded. The RNA solution, which contained less than 1% protein, was divided into aliquots and was stored at ~ 15° until needed. Soluble RNA was prepared from 105,000 x g supernatant solution by the phenol extraction method described above. Soluble RNA wus ulso stored at — 15°. Atkali-degraded RNA was prepared by incubating RNA samples with 0.3 44 KOH at 35° for 18 hr. The solutions then were neutralized and dialyzed against standard buffer (minus mercaptoethanol). RNAase- digested sunples of RNA were prepared by incubating RNA with 2 pg per ml of crystalline 1590 BIOCHEMISTRY: NIRENBERG AND MATTHAEI Proc. N. A. 8. RNaAase (Worthington Biochemical Company) at 35° for 60 min. RNAase was destroyed by four phenol extractions performed as given above. After the last phenol extraction, the samples were dialyzed against standard buffer minus mercaptoethanol. RNA samples were treated with trypsin by incubation with 20 yg per ml of twice recrystallized trypsin (Worthington Biochemical Company ) at 35° for 60 min. The solution was treated four times with phenol and was dialyzed in the same manner. The radioactive amino acids used, their source, and their respective specific activities are as follows: U-C™-glycine, U-C14-L-isoleucine, U-C14-L-tyrosine, U-C14-L-leucine, U-C!L-proline, L-histidine-2(ring)-C'4, U-C14-L-phenylalanine, U-C1*-L-threonine, L-methionine (methyl-C¥) , U-C!“-L-arginine, and U-C'-L-lysine obtained from Nuclear-Chicago Corporation, 5.8, 6.2, 5.95, 6.25, 10.5, 3.96, 10.3, 3.9, 6.5, 5.8, 8.38 mC/mM, respectively; C1-L-aspartic acid, C'Lglu- tamic acid, C4-L-alanine, obtained from Volk, 1.04, 1.18, 0.75 m C/mM, respectively ; D-L-trypto- phan-3 C14\", obtained from New England Nuclear Corporation, 2.5 mC/mM; §-L-cystine obtained from the Abbott Laboratories, 2.4 mC/mM; U-C\"-L-serine obtained from the Nuclear-Chicago Corporation, 0.2 mC/mM. Other materials and methods used in this study are described in the accompanying paper.’ All assays were performed in duplicate. Results.—Stimulation by ribosomal RNA: In the previous paper,! it was shown that DNAase markedly decreased amino acid incorporation in this system after 20 min. For the purpose of this investigation, 30,000 X g supernatant fluid frac- tions previously incubated with DNAase and other components of the reaction mixtures (Incubated-S-30 fractions) were used for many of the experiments. Figure 1 shows that incorporation of C\"™-L-valine into protein by Incubated- 8-30 fraction was stimulated by the addition of purified E. coli soluble RNA. Maximal stimulation was obtained with approximately 1 mg soluble RNA. In some expcriments, increasing the concentration 5-fold did not further stimulate the system. Soluble RNA was added to all reaction mixtures unless otherwise specified. Figure 2 demonstrates that E. coli ribosomal RNA preparations markedly stimu- 200 1 1 T     200; T T T TT T T 175 oh 1 :   COUNTS /MINUTE/MG. PROTEIN   COUNTS/ MINUTE/M6, PROTEIN          100 4 75 50 J QO ° ask ! ! l 25: I o Ft ae en i 0 10 20 3.0 L l | 1 i MG. RIBOSOMAL RNA   o O5 10 16 20. 28 30 . . . Lye Fic. 2.—Stimulation of amino acid incor- MG. SOLUBLE RNA poration into protein by E. coli ribosomal Fie 1--—Stimulation of amino acid incor- poration into protein by #. cold soluble RNA. Composition of reaction mixtures is specified in Table 1. Samples were incubated at 35° for 20 min. Reaction mixtures contained 4.4 mg. of Incubated-S-30 protein. RNA in the presence of soluble RNA. Com- position of reaction mixtures is specified in Table 1. Samples were incubated at 35° for 20 min. Reaction mixtures contained 4.4 mg of Incubated-S-30 protein and 1.0 mg £. col? soluble RNA. Vou. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1591   24mg. RNA 200 0.60 mg. RNA 0.30 mg. RNA | 0.12mg RNA 4 Minus RNA | COUNTS/MINUTE /mg PROTEIN     po 0 10 20 30 40 50 60 70 80 90 MINUTES Fic. 3.—Dependence of C1-L-valine incorporation into protein upon ribosomal RNA. The composition of the reaction mixtures and the incubation conditions are presented in Table 1. Reaction mixtures contained 0.98 mg of E. cold soluble RNA and 4.4 mg of Incubated-S-30-protein. lated incorporation of C™-valine into protein even though maximally stimulating concentrations of soluble RNA were present in the reaction mixtures. A linear relationship between the concentration of ribosomal RNA and C1-valine incorpora- tion into protein was obtained when low concentrations of ribosomal RNA were used. Increasing the soluble RNA concentration up to 3-fold did not replace the effect observed when ribosomal RNA was added. The effect of ribosomal RNA in stimulating incorporation of Ch-valine into protein is presented m more detail in Figure 3. In the absence of ribosomal RNA. incorporation of C1-valine into protein by the incubated-S-30 fraction was quite low when compared with 8-30 (not incubated before storage at. — 15°) and stopped almost completely after 30 min. At low concentrations of ribosomal RNA, maxi- mum amino acid incorporation into protein was proportional to the amount of ribosomal RNA added, suggesting stoichiometric rather than catalytic action of ribosomal RNA. Total incorporation of C*-valine into protein was increased more than 3-fold by ribosomal RNA in this experiment even in the presence of maximally stimulating concentrations of soluble RNA. Ribosomal RNA may be added at any time during the course of the reaction, and, after further incubation, an increase in incorporation of C4-valine into protein will result. Characteristics of amine acid incorporation stimulated by ribosomal RNA: In Table 1 are presented the characteristics of C'-L-valine incorporation into protein 1592 BIOCHEMISTRY: NIRENBERG AND MATTHAE!S Proc. N. ALS, TABLE | CHARACTERISTICS OF C!LL-Varmng INCORPORATION INTO PROTEIN Experi- ment no, Addition Counts/min/mg protein I — Ribosomal RNA 42 + oe ad 904 + “ “+ 0.15 zmole Chloramphenicol 58 + “ “+ 0.20 umole Puromycin 7 4- “ “ deproteinized at zero time 8 2 — Ribosomal RNA 35 + a ce 10L + “ “«  — ATP, PEP, PEP kinase 7 + “ “+ 10 wg RN Aase 6 + “ \"+ 10 ng DN Aase 110 + Boiled Ribosomal RNA 127 + Ribosomal RNA, deproteinized at zero time 8 3 — Ribosomal RNA 34 _ « ¢ — 201. amino acids 21 + “ “ 99 + “ — 20-L-amino acids 52 The reaction mixtures contained the following in wmole/inl: 100 Tristhydroxymethyl) aminomethane. pH 7.8; 10 magnesium acetate: 50 KCl; 6.0 mercaptoethanol: 1.0 ATP; 5.0 phosphoenolpyruvate, K_ salt; 20 zg phosphoenolpyruvate kinase, crystalline; 0.05 each of 20 L-amino acids minns valine: 0.03 each of GTP, CTP, and UTP; 0.015 C'-L-valine (~70,000 counts); 3.1 mg. E. coli ribosomal RNA where indicated, and 1.0 mg E. coli soluble RNA; 3.2, 3.2, and 1.4 mg of incubated-S-30 protein were present in Experiments 1, 2, and 3, respectively. In addition 4.4 mg protein of W-Rib were added in Experiment 3. Total volume was 1.0 ml. Samples were incubated at 35° for 20 nin, were deproteinized with 10 per cent trichloroacetic acid, and the precipitates were washed and counted by the method of Siekevitz.2° stimulated by the addition of ribosomal RNA. Amino acid incorporation was strongly inhibited by 0.15 umoles of chloramphenicol and 0.20 umoles/ml reaction mixture of puromycin. Furthermore, the incorporation was completely dependent upon the addition of ATP and an ATP-generating system and was totally inhibited by 10 ug/ml RNAase. Equivalent amounts of DNAase had no effect upon the incorporation stimulated by the addition of ribosomal RNA. Placing a ribosomal RNA preparation in a boiling water bath for 10 min did not destroy its C'-valine Incorporation activity; instead, a slight increase in activity was consistently ob- served. However, when these RNA preparations were placed in a boiling water bath, a copious, white precipitate resulted. Upon cooling the suspension in an ice bath, the precipitate immediately dissolved. The data of Table | also demonstrate that the incorporation of amino acids into protein in the presence of ribosomal RNA was further stimulated by the addition of a mixture of 20 L-amino acids, suggesting cell-free protein synthesis. C- and N-terminal analyses of the ribosomal RNA-dependent product of the reaction were performed with carboxypeptidase and 1-fluoro-2,4-dinitrobenzene respectively (Dr. Frank Tictze kindly performed these. analyses). Four per cent of the radioactivity was released from the C-terminal end and 1% was associated with the N-terminal end. The remainder of the C'-label was internal. Similar results were obtained when reactions were performed using 8-30 enzyme fractions which had not been treated with DNAase. Protein precipitates isolated from re- action mixtures after incubation were completely hydrolyzed with HCl, and the C'Jabel incorporated into protein was demonstrated to be valine by paper chro- matography. Many of the experiments presented in this paper were performed with enzyme fractions prepared with DNAase added to reduce their viscosity. Ribosomal Vou. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1593 RNA also stimulated C-valine incorporation when enzyme extracts prepared in the absence of DNAase were used. To be effective in stimulating amino acid incorporation into protein, the ribo- somal RNA required ihe presence of washed ribosomes. The data of Table 2 show TABLE 2 ‘Tue INEFFECTIVENESS OF RiposomaL RNA In Stimunating C'}-L-VaLine INCORPORATION INTO PROTEIN IN THE PRESENCE OF RIBosomEs oR 105,000 X g SUPERNATANT SoLUTIONS ALONE Additions Counts/min Complete 51 « + 2.1 mg Ribosomal RNA 202 “ — Ribosomes 17 “ — Ribosomes + 2.1 mg Ribosomal RNA 20 “ — Supernatant solution 36 “ — Supernatant solution + 2.1 mg Ribosomal RNA 45 “ Deproteinized at zero time 25 The components of the reaction mixtures and the incubation conditions are presented in Table 1. 0.86 and 3,: mg protein were present in the ribosome (W-Rib) and 105,000 & g supernatant (8-100) fractions, respectively. that both ribosomes and 105,000 X g supernatant solution were necessary foi ribosomal RNA-dependent amino acid incorporation. No incorporation of aminc acids into protein occurred when the 105,000 X g supernatant solution alone was added to ribosomal RNA preparations, demonstrating that ribosomal RNA prepara- tions were not contaminated with intact ribosomes. This conclusion also was substantiated by showing that the activities of ribosomal RNA preparations were not destroyed by boiling, although the activities of the ribosomes were destroyed by such treatment. The effect of ribosomal RNA upon the incorporation of seven different amino acids is presented in Table 3. The addition of ribosomal RNA increased the incorporation of every amino acid tested. The effect shown by ribosomal RNA was not observed when other polyanions were used, such as polyadenylic acid, highly polymerized salmon sperm DNA, or a high-molecular-weight polymer of glucose carboxylic acid (Table 4). Pretreat- ment of ribosomal RNA with trypsin did not affect its biological activity. How- ever, treatment of the ribosomal RNA with either RNAase or alkali resulted in a complete loss of stimulating activity. The active principle, therefore, appears to be RNA. The sedimentation characteristics of the ribosomal RNA preparations were examined in the Spinco Model E ultracentrifuge (Fig. 44). Particles having the characteristics of S-30, S-50, or S-70 ribosomes were not observed in these prepara- tions. The 8.7 of the first peak was 23, that of the second peak 16, and that of the third, small peak, 4. Pretreatment with trypsin did not affect the S¥ values of the peaks appreciably (Fig. 4C); however, treatment with RNAase completely destroyed the peaks (Fig. 4B), confirming the ancillary evidence which had suggested that the major component was high-molecular-weight RNA. Preliminary attempts at fractionation of the ribosomal RNA were performed by means of density-gradient centrifugation employing a linear sucrose gradient. The results of one such experiment are presented in Figure 5. Amino acid in- corporation activity of the RNA did not follow absorbancy at 260 my; instead, the activity seemed to be concentrated around fraction No. 5, which was approxi- mately one-third of the distance from the bottom of the tube. These results again 1594 BIOCHEMISTRY: NIRENBERG AND MATTHAEIL Proc, N. ALS, TABLE 3 SPECIFICITY oF Amino Actp LNCORPORATION STIMULATED BY Rinosomat RNA CtM-Amino Acid Addition Counts/min/mg protein C'*L-Valine Complete 25 “ “ + Ribosomal RNA 137 (OLL-Threonine “ 31 “ “ + Ribosomal RNA 121 C!L-Methionine “ 12] “ “ + Ribosomal RNA 177 C'-T-Arginine “ 49 “ “ + Ribosomal RNA 224 C'~L.-Phenylalanine “ 7 “ “ + Ribosomal RNA 147 (+DL-Lysine “ 36 “ “= + Ribosomal RNA 175 CUAL-Leucine “ 134 “ “ + Ribosomal RNA 272 « “ Deproteinized at. zero time 6 The composition of the reaction mixtures are presented in Table 1. The mixture of 20 L-amino acids ineluded all amino acids except the C!4amino acid added to one reaction mixture. Reaction mixtures contained 4.4 mg Incubated-8-30 protein. Famples were incubated at 35° for 60 min, 2.1 ing ribosomal RNA were added where indicated. TABLE 4 Riposomau RNA Conrrou EXPERIMENTS DESCRIBED IN TExt Iexperi- ment No. Addition Counts/min/mg protein I Complete 54 “ + 2.4 mg Ribosomal RNA 144 “ + 2.0 mg Polyadenylic acid 10 7 + 2.0 mg Salmon sperm DNA 4 “ + 2.0 mg Polyglucose carboxylic acid 49 “ + 2.4 mg Ribosomal RNA, deproteinized at zero time 7 2 Complete 39 “ + 2.0 mg Ribosomal RNA* 150 “ + 2.1 mg Ribosomal RNA preincubated with trypsin*® 166 “ + 2.0 mg Ribosomal RNA preincubated with RNAase*,} 47 “ Deproteinized at. zero time 8 3 Coniplete 20 ” + 1.2 mg Ribosomal RNA 82 ‘ + 1.2 mg Alkali degraded ribosomal RNA t 2) . Deproteinized at zero time 7 The composition of the reaetion mixtures and the incubation conditions are given in Table L. 44, 5.2, and 4.4 mg Incubated-8-30 protein were present in Experiments 1, 2, and 3, respectively. 2.4. 0.98. and 0 me &. col? soluble RNA were present in Experiments 1, 2, and 3, respectively. * Ribosomal RNA preparations were deproteinized by phenol extraction after enzymatic digestion as specified under Methods and Materials. ft mg Ribosomal RNA refers to RNA concentration hefore digestion. demonstrate that the activity was not associated with a soluble RNA fraction, present in maximum concentration in fraction No. 11, near the top of the tube. In addition, all amino acid incorporation analyses were performed in the presence of added soluble RNA, and the addition of more soluble RNA would not stimulate C™-L-valine incorporation into protein. Effects of RN A obtained from different species: The data of Table 5 demonstrate that; RNA from different. sources stimulates C™-valine incorporation into protein. Yeast ribosomal RNA prepared by the method of Crestfield et al.4 was considerably more effective in stimulating incorporation than equivalent. amounts of EF. coli ribosomal RNA. Yeast ribosomal RNA prepared hy this method has little or no amino acid acceptor activity and has a molecular weight of about 29,000.7 Tobacco mosai¢ virus RNA prepared by phenol extraction and having a molecular weight of Vou. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAKEI 1595 TABLE 5 STIMULATION oF AMINO Acrp IncoRPORATION BY RNA FRACTIONS PREPARED FROM DIFFERENT SPECIES Additions Counts/min/mg protein None 42 + 0.5 mg E. coli ribosomal RNA 75 + 0.5 mg Yeast ribosomal RNA 430 + 0.5 mg Tobacco mosaic virus RNA 872 + 0.5 mg Ehrlich ascites tumor microsomal RNA 65 The components of the reaction mixtures and the inenbation conditions are presented in Table 1. Reaction samples cantained 1.9 mg Incuhated-S-30 protein. approximately 1,700,000} stimulated amino acid incorporation strongly. Marked stimulation due to tobacco mosaic virus RNA was observed also with HE. cola en- zyme extracts which had not been treated with DNAase. More complete details of this work will be presented in a later publication. Stimulation of amino acid incorporation by synthetic polynucleotides: The data of Figure 6 show that the addition of 10 ug of polyuridylic acidt per ml of reaction mixture resulted in a remarkable stimulation of C'-L-phenylalanine incorporation. Phenylalanine incorporation was almost completely dependent upon the addition of polyuridylic acid, and incorporation proceeded, after a slight lag period, at a linear rate for approximately 30 min. The data of Table 6 demonstrate that no other polynucleotide tested could re- place polyuridylic acid. The absolute specificity of polyuridylic acid was con- TABLE 6 POLYNUCLEOTIDE SPECIFICITY FOR PHENYLALANINE INCORPORATION Experi- ment no. Additions Counts/min/mg protein 1 None 44 + 10 wg Polyuridylic acid 39, 800 + 10 ug Polyadenylic acid 50 + 10 pg Polycytidylic acid 38 + 10 pg Polyinosinic acid 57 + 10 wg Polyadenylic-uridylic acid (2/1 ratio) 53 + 10 yg Polyuridylic acid + 20 ug polyadenylic acid 60 Deproteinized at zero time 17 2 None 75 + 10 we UMP 81 + 10 wg UDP 77 4 10 pe UTP 72 Deproteinized at zero time 6 Components of the reaction mixtures are presented iu Table 1, Reaction mixtures contained 2.3 mg Incubated- $-30 protein. 0.02 zmoles U-C!-L-phenylalanine (~125,000 counts/minute) was added to each reaetion mixture. Samples were incubated at 35° for 60 min. firmed by demonstrating that randomly mixed polymers of adenylic and uridylic acidt (Poly A-U, 2/1 ratio and 4/1 ratio) were inactive in this system. A solution of polyuridylic acid and polyadenylic acid (which forms triple-stranded helices) had no activity whatsoever, suggesting that single-strandedness is a necessary requisite for activity. Experiment 2 in Table 6 demonstrates that UMP, UDP, or UTP were unable to stimulate phenylalanine incorporation. The data of Table 7 demonstrate that both ribosomes and 100,000 X g super- natant solution, as well as ATP and an ATP-generating system, were required for the polyuridylic acid~dependent incorporation of phenylalanine. Incorpora- tion was inhibited by puromycin, chloramphenicol, and RNAase. The incorpora- 1596 BIOCHEMISTRY: NIRENBERG AND MATTHAEI Proc. N. ALS.   Fig. 4.—-E. coli ribosomal RNA preparations. (4) Untreated (above). (2) digested with RNA- aase; (C’) digested with trypsin. Preparation and digestion of samples presented under Methods and Materials. 9.8 and 10.5 mg/ml RNA were present in Aand C. 1t.5mg/ml RNA was present in B (Continued on facing page) tion was not inhibited by addition of DNAase. Omitting a mixture of 19 L-amino acids did not, inhibit’ phenylalanine incorporation, suggesting that polyuridylic acid stimulated the incorporation of L-phenylalanine alone. This conclusion was substantiated by the data presented in Table 8. Polyuridylic acid had little effect in stimulating the incorporation of 17 other radioactive amino‘acids. Each labeled amino acid was tested individually, and these data, corroborating the results given in Table 8, will be presented in a subsequent publication.     Vou. 47, 196] BIOCHEMISTRY: NIRENBERG AND MATTHAEI 1597   (Fig. 4—continued) before digestion. Photographs were taken in a model KE Spinco ultracentrifuge equipped with schlieren optics. The product. of the reaction was partially characterized and the results are pre- sented in Table 9. The physical characteristics of the product of the reaction resembled those of authentic poly-L-phenylalanine, for, unlike many other poly- peptides and proteins, both the product of the reaction and the polymer were resistant to hydrolysis by 6N HCl at 100° for 8 hr but were completely hydrolyzed by 12N HCl at 120-130° for 48 hr. Poly-L-phenylalanine is insoluble in most solvents” but is soluble in 33 per cent 1598 BIOCHEMISTRY: NIRENBERG AND MATVTHAEI Proc. N. ALS.         TOT T T ] T TE A260 60 ba + 80 140 ~ + 70 QO 1.20 +60 2 2a © 1.00 + 50 G ~ < 800 140 = Zz 600 4305 mM 400 + 20 200 + 10 Oo 0 1 2 3 4 5 6 7 8 9 10 It 12 13 BOTTOM FRACTION NUMBER TOP Fic. 5.—Sucrose density-gradient centrifugation of ribosomal RNA. A linear gradient of su- crose concentration ranging from 20 per cent at the bottom to 5 per cent at the top of the tube was prepared.*5 The sucrose solutions (4.4 ml total volume) contained 0.01 1 Tris, pH 7.8, 0.01 .M Mg acetate and 0.06 17 KCI. 0.4 ml of ribosomal RNA (4.6 mg) was layered on top of each tube which was centrifuged at 38,000 & y for 4.5 hours at 3° in a swinging bucket rotor, Spinco type SW-39, using a Spinco Model J. ultracentrifuge. 0.30 ml fractions were collected after piercing the bottom of the tube.*4 0.025 ml aliquots diluted to 0.3 ml with H2O were used for A2® measurements. 0.25 ml aliquots were used for amino acid incorporation assays. Reaction mixtures contained the components pre- sented in Table 1. 0.7 mg of &. coli soluble RNA and 2.2 mg Incubated-8-30 protein were added. Control assays plus 0.25 ml 12.5 per cent sucrose in place of fractions gave 79 counts/min. This figure was subtracted from each value. Total volume was 0.7 ml. Samples were incubated at 35° for 20 min. 64,320 5 1 oper epee ep + POLY UY 56,000 - 4 48000 - 24,000 COUNTS/MINUTE/mg. PROTEIN 6,000     ° 0 ~* Is 30 45 60 75 30 MINUTES Fic. 6.—Stimulation of U-C!«-L-phenylalanine incorporation by polyuridylic acid. @ without polyuridylic acid; 410 ug polyuridylic acid added. The components of the reac- tion mixtures and the incubation conditions are given in Table 1. 0.024 umole U-C!“L phenylalanine (~600,000 counts/min) and 2.3 mg Incubated-S-30 protein were added /m\\\\ of reaction mixture. Vou. 47, 1961 BIOCHEMISTRY: NIRENBERG AND MATTHAEL 1599 TABLE 7 CHARACTERISTICS OF POLYURIDYLIC Acip-DEPENDENT PHENYLALANINE INCORPORATION Additions Counts/min/mg protein Minus polyuridylie acid 70 None 29,500 Minus 100,000 * y supernatant solution 106 Minus ribosomes 52 Minus ATP, PEP, and PEP kinase 83 + 0.02 umoles puromycin 7,100 + 0.31 wmoles chloramphenicol 12,550 + 6 pg RN Aase 120 + 6 ue DNAase 27,600 Minus amino acid mixture 31,700 Deproteinized at zero time 30 The components of the reaction mixtures are presented in Table 1. 10 ug of polyuridylic acid were added to all samples except the specified one. 2.3 mg of Incubated-S-30 protein were added to each reaction mixture except those in which ribosomes alone and 100,000 X g supernatant solution alone were tested. 0.7 mg W-Rib protein and 1.3 mg 5-100 protein were used respectively. 0.02 pmoles U-C!*L-phenylalanine, Sp. Act. = 10.3 mC/mM (~125,000 counts/minute) were added ta each reaction mixture. Samples were incubated at 35° for 60 min. TABLE 8 SpecIFICITY OF AMINO ACID INCORPORATION STIMULATED BY PoLyURipyLic ACID Experi- ment Counts/min/mg no. C!Lamino acids present Additions protein ] Phenylalanine Deproteinized at zero time 25 None 68 + 10 wg polyuridylic acid 38,300 2 Glycine, alanine, serine, Deproteinized at zero time 17 aspartic acid, glutamic None 20 acid + 10 wg polyuridylic acid 33 3 Leucine, isoleucine, threonine, Deproteinized at zero time 73 methionine, arginine, histidine, None 276 lysine, tyrosine, tryptophan, + 10 ug polvuridylic acid 899 proline, valine 4 Sa cysteine Deproteinized at zero time 6 None 95 + 10 wg polyuridylic acid 113 Components of the reaction mixtures are presented in Table 1. The unlabeled amino acid mixture was omitted. 0.015 4M of each labeled amino acid was used. The specific activities of the labeled amino acids are present in the Methods and Materials section, 2.3 mg of protein of preincubated 8-30 enzyme fraction were added to each reaction mixture. All samples were incubated at 35° for 30 min. TABLE 9 ComPaRISON OF CHARACTERISTICS OF Propuct oF REAcTION AND Poiy-L-PHENYLALANINE Treatment Product of reaction Poly-L-phenylalanine 6 N HCI for 8 hours at 100° Partially hydrolyzed Partially hydrolyzed 12 N HCI for 48 hours at 120-130° Completely hydrolyzed Completely hydrolyzed Extraction with 336, HBr in glacial acetic acid Soluble Soluble Extraction* with the following  sol- vents: HO, benzene, nitrobenzene, chloroform, N,N-dimethylform- amide, ethanol, petroleum ether, concentrated phosphoric acid, gla- cial acetic acid, dioxane, phenol, acetone, ethyl acetate, pyridine, acetophenone, formic acid Insoluble Insoluble * The product was said to be insoluble if <0.002 gm of product was suluble in 100 ml of solvent at 24°. Extrae- tions were performed by adding 0.5 mg of authentic poly-L-phenylalanine and the C)+-product of a reaction mixture (1800 counts/min) to 5.0 ml of solvent. The suspensions were vigorously shaken for 30 min at 24° and were centrifuged. The precipitates were plated and their radioactivity was determined, HBr in glacial acetic acid.§ The product of the reaction had the same apparent solubility as authentic poly-L-phenylalanine. The product of the reaction was purified by means of its unusual solubility behavior. Reaction mixtures were de- proteinized after incubation, and precipitated proteins were washed in the usual 1600 BIOCHEMISTRY: NIRENBERG AND MATTHAEI Proc. N. ALS. manner according to the method of Siekevitz.22_ Dried protein pellets containing added carrier poly-L-phenylalanine were then extracted with 33 per cent HBr in glacial acetic acid, and the large amount of insoluble material was discarded.\\", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j87y~w9tz~hjsk", "00000000-0000-0000-9BAE-1B2360A8E85F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "A Neuroblastoma x Glioma Hybrid Cell Line with Morphine Receptors", "101584910X56", null, "1974", "September 1974", "This article is one of the first in a series devoted to hybrid cells and morphine receptors.  Experimental evidence from this article reveals that hybrid cell lines have the ability to bind analgesics whereas parental cells do not.  The mechanism of morphine uptake, dependence, and tolerance is explored in this and subsequent articles.", "Articles", "Hybrid Cells,Neuroblastoma,Morphine,Receptors, Drug ; Metabolism", "Neuroblastoma Research, 1967-1976", "4", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Nat. Acad. Sci. USA Vol. 71, No. 9, pp. 3474-3477, September 1974 WERNER A. KLEE AND MARSHALL NIRENBERG “A Neuroblastoma x Glioma Hybrid Cell Line with Morphine Receptors , (cell membranes/ narcotics /neurobiology/tissue culture) Laboratory of General and Comparative Biochemistry, National Institute of Mental Health and the Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, June 12, 1974 ABSTRACT A neuroblastoma X glioma hybrid cell line with well-developed neural properties was found that has high-affinity morphine receptors. The average cell contains approximately 3 X 10° receptors. In contrast, parent cells and other neuroblastoma or hybrid cell lines tested had few or no morphine receptors.   Membrane preparations that bind narcotic analgesics such as morphine and its congeners have been described by a number of laboratories (1-9). Such binding satisfies the criteria first enunciated by Goldstein et al. (10) for a narcotic receptor in that it is specific for the pharmacologically active D (—) en- antiomers of the narcotics. Furthermore, there is a generally satisfactory correlation between binding affinities measured for various opiates and their potencies as analgesics in vivo (5) as well as a somewhat more satisfactory correlation be- tween binding strength and narcotic agonist potency mea- sured with isolated intestinal preparations (11). The mem- brane preparations studied to date were isolated from brain or intestine and, therefore, are derived from mixtures of cell types. We hoped to obtain a relatively homogenous cell population for our studies of opiate action by examining clonal lines of neuroblastoma cells (12) for morphine receptors. Somatic cell hybrids also were studied, since previous results showed that genes for neural properties can be activated (13) or deactivated (14) in hybrids formed by the fusion of neuro- blastoma cells with other cell types. New phenotypes thus generated can be inherited and thereby perpetuated in a fairly stable fashion. In this report, we wish to describe cell lines with and with- out morphine receptors. MATERIALS AND METHODS Chemicals were obtained from the following sources: (?H |di- hydromorphine (51.6 Ci/ mmole) from New England Nuclear Corp.; morphine sulfate from Merck and Co.; naloxone hydrochloride was donated by Endo Laboratories, Inc.; the other narcotics used were kindly supplied by Dr. Everette May; fetal bovine serum was from Colorado Serum Co.; DMEM (Dulbecco-Vogt modification of Eagle’s minimal es- sential medium) was from GIBCO (Catalogue no. H21). Other chemicals were of reagent grade purity. Cell Lines. The neuroblastoma cell lines used were derived from the mouse C-1300 tumor; their properties have been described previously (12). Neuroblastoma x glioma hybrids, NG108-5 and NG108-15, were obtained* by Sendai virus-in-   * B. Hamprecht, T. Amano, and M. Nirenberg, in preparation. duced fusion of neuroblastoma clone N18TG-2, resistant to 6- thioguanine (14), and C6Bu-1, a 5-bromodeoxyuridine-re- sistant clone of the rat glioma C6 (15). Clone NLIF is a hy- brid formed by fusion of neuroblastoma clone N4TG1 and mouse L cell clone B82 (14). Neuroblastoma and C6Bu-1 cells were grown in petri dishes or flasks in 90% DMEM, 10% fetal bovine serum, in a humidi- fied atmosphere of 90% air, 10% CO:, and 37° as described (12). Hybrid cells were grown in the same medium supple- mented with 0.1 mM hypoxanthine, 1 »M aminopterin, and 12 uM thymidine. For drug binding experiments, the cells us- ually were grown to confluency and harvested by scraping (from petri dishes) or shaking (from flasks) in their growth medium. The cells were centrifuged at approximately 500 X< ¢ for 5 min at room temperature and the pellets (generally less than 1 ml) were washed two times with 50 ml of D1 [137 mM NaCl, 5.4 mM KCl, 0.17 mM NasHPO,, 0.22 mM KH:PO,, 5.5 mM glucose (16)] and suspended in a solution of 0.32 M sucrose, 0.01 M Tris: HCI (pH 8) at 3° so that the concentra- tion of protein was close to 0.5 mg/ml. Uniform cell suspen- sions were achieved by aspirating the suspension in a 10-ml plastic pipet three times. Cell suspensions were kept at 3° and usually used immediately for binding studies. Cell sus- pensions that were frozen and stored at —60° retained the specific binding of [8H dihydromorphine. Routine Assay Method. A cell suspension prepared as just described (900 ul) was added to a plastic test tube containing either 10 wl of water or 10 ul of 0.1 mM morphine sulfate. A solution, 100 ul, containing [*H Jdihydromorphine (10 nM, 51.2 Ci/mmole) dissolved in 0.32 M. sucrose was added to each tube in a darkened room and the samples were mixed with a vortex mixer. After incubation at 37° for 10 min, tubes were centrifuged at 19,000 rpm (45,000 x g) for 10 min in a Sorvall RC2B centrifuge at 0° and supernatant fluids were removed with a 5-ml syringe equipped with a blunt 19-gauge needle and then discarded. Each tube and surface of the pellet was washed with 1 ml of 0.32 M sucrose and the washed pellet was suspended in 1 ml of 1% Triton X-100 and assayed for radioactivity after mixing with 7.5 ml of Triton-toluene scintillation fluid (17). All operations involving dihydromor- phine were performed in very dim, indirect light. Each sample was assayed in triplicate or quadruplicate. Neuroblastoma cell lines used were subcultured 5 to 30 times; hybrid cells were subcultured 10 to 20 times. In most cases a number of different subcultures were tested. Specific _ binding is defined as fmoles of {*H]dihydromorphine bound per mg of protein (upon incubation of cells with nM ('H \\\\di-   Proc. Nat. Acad. Sct. USA 71 (1974) hydromorphine) that are displaced by a 1000-fold, or higher, excess of unlabeled morphine. Binding affinities of morphine and of other narcotics were estimated from experiments in which a range of concentrations of unlabeled opiates was tested for its ability to displace bound [*H]dihydromorphine as described previously (9). RESULTS The specific binding of (?H dihydromorphine was tested with various cell lines derived from the nervous system as shown in Table 1. Little or no-specific binding of dihydromorphine was detected with neuroblastoma clone N18TG-2 or rat glioma clone C6BU-1. However, specific binding sites for [*H]dihy- dromorphine were found with a hybrid cell line, NG108-15, originating from the fusion of the neuroblastoma N18TG-2 and glioma C6Bu-1*. Relatively few specific narcotic binding sites were detected, under the conditions used, with a sister neuroblastoma X glioma hybrid cell line, NG108-5, neuro- blastoma clones $20 and S20Y, and a neuroblastoma X L cell hybrid, NLIF. The low values may indicate the presence of a reduced number of receptors or receptors with a reduced affinity for morphine. Narcotic receptors were not detected with neuroblastoma lines NIE, NJE-115, N4, N18, or N18- TG-2, or with C6BU-1. Thus, two classes of cells were found: cells with morphine receptors, and cells that do not have these receptors in quantities sufficient to detect with the assay used. Characterization of the Narcotic Receptors of NG108-16 Hybrid Cells: A portion, only, of the binding of [?H|dihydro- morphine is saturable in that it is displaced by nonradioactive morphine (Fig: 1), and indeed, this phenomenon i is the basis of our assay procedure. Nonsaturable (that is nondisplaceable) binding of {*H]dihydromorphine has been found in all tissues tested and is thought to be nonspecific binding. Only the saturable component ef the binding shown in Fig. 1 is con- sidered to be specific binding. Morphine Receptors in Neural Cell Lines 3475         pF T 7 T T s00r- 4 ; Z op “Ae a& ° S~ 600+ a” g » = soot 4 a 3 Ly pe L 1 1 l 1 10°* io 107 io* 10° MORPHINE (M) Fig. 1. The displacement of bound [*H]dihydromorphine by different concentrations of unlabeled morphine from a suspension of NG108-15 cells (subculture 10, 468 ug of protein per tube). Each tube contained 1 nM {*H]dihydromorphine and the indi- cated concentration of morphine. The specific binding sites discriminate between (+-) and (—) enantiomers of the narcotics as shown for the pair dextror- phan and levorphanol in Fig. 2. The latter compound is seen to displace specifically bound dihydromorphine at concentra- tions more than two orders of magnitude lower than does its analgesically inactive enantiomer, dextrorphan (18). A similar result has been obtained with the (—) and (++) enantiomers of metazocine, the latter of which is inactive as an analgesic (19) and as a displacer of dihydromorphine, at least at con- centrations of 4M and below. The number of binding sites present in suspensions of NG108-15 can be estimated by measuring the amount of specific binding as a function of [*H]dihydromorphine con- centration. We find that, at saturation, approximately 0.6 pmoles of dihydromorphine are bound specifically per mg of cell protein (Fig. 3). The insert to the Figure shows the data     TaBie 1. Specific binding of (*H dihydromorphine to neuroblastoma and hybrid cell lines fmoles of (*H]- No. of dihydromorphine experi- Neurotransmitter Cell lines bound /mg of protein* ments synthesis Ref. A. Parents Mouse neuroblastoma N18TG-2 O+1 2 None 6 Rat glioma Cé6BU-1 1+1 5 None (15) B. NisTG2 x C6BU-1 hybrids NG108-15 17 +6 13 Acetylcholine 5 NG108-5 442 11 Acetylcholine 5 C. Neuroblastoma NS20 342 3 Acetylcholine (12) NS20Y 4+2 7 Acetylcholine (12) NIE 0 1 Catechols (12) NIE-115 221 9 Catechols (12) N4 0 1 None (12) N18 221 6 None (12) D. Neuroblastoma X L cell hybrid (N4TG-1) X (B82) NLIF 6+3 3 None (14)   * Specific binding of [*H]dihydromorphine is that amount of the bound radioactivity which is displaced by 1 «M unlabeled morphine (see the Materials and Methods section for details). ® B. Hamprecht, T. Amano, and M. Nirenberg, in preparation. 3476 —_- Biochemistry: Klee and Nirenberg   8 8          DIHYDROMORPHINE BOUND (cpm)     900! 4 SO ° “Oo. 0 800! © 24-8 A a TOOL, L 1 1 1 L 1 0 jo\" 10% 10° +107 16% 10° MOLARITY OF ADDED DRUG Fig. 2. The displacement of bound [*H]dihydromorphine by levorphanol (©), [(— )3-hydroxy-N-methylmorphinan] and dex- trorphan(A){(+ )3-hydroxy-N-methylmorphinan] at the concen- trations shown from a suspension of NG108-15 cells (subculture 15). plotted according to Scatchard (20) and is consistent with there being only one class of dihydromorphine binding site with a dissociation constant of 20-30 nM. We have examined the ability of a number of other narcotic analgesics as well as of the specific antagonist, naloxone, to bind to the receptors found in NG108-15 suspensions, as measured by their ability to displace specifically bound (8H ]dihydromorphine. In Table 2 the binding affinities mea- sured in this way with NG108-15 suspensions and also with a crude mitochondrial fraction of rat brain as the source of receptors are shown. Note that the binding affinities of the NG108-15 preparations are generally somewhat lower than those of the brain preparation, but that the relative binding strengths are similar in the two preparations. The order of binding affinities is seen to be well correlated with the affinities found by Kosterlitz and Watt (11) for the pharmacological action of these agents on segments of guinea pig ileum. An important question concerns the location of the narcotic receptor sites. Trypsin is often used as an aid in detaching TABLE 2. Binding affinities of a series of narcotics to \" suspensions of NG108-16 and to brain homogenates   Apparent dissociation constant? (nM)   Activity in Compound NG108-15 Ratbrain? guinea pig ileum* Levorphanol 0.3 0.8 7 Morphine 20 3 88 Pentazocine 50 10 150 Methadone 80 20 — Codeine 1000 800 8800 Naloxone 10 1 1.2 Dextrorphan 150 100 _—   « Binding was measured as the concentration required to dis- place 1/2 of the specifically bound [*H}dihydromorphine in our usual assay system. Under our conditions of assay this number should be close to a true dissociation constant. > Measured as described in Materials and Methods and (9). (R. A. Streaty and W. A. Klee, unpublished observations. ) ¢ These are apparent dissociation constants (nM) for the antagonist activity of these compounds in the isolated ileum (11). Proc. Nat. Acad. Sct. USA 71 (1974)         T 1 T 4 T g 3 °o 2 0.4- 4 a LG] 2 0 1 8 0.3 +4 a Oo oO oO Le = o2b 4 a % ° O.1F 4 1 | 1 1 L 0 1 2 3 DIHYDROMORPHINE ( Mx 10*) Fic. 3. The specific binding of [*H]dihydromorphine to a suspension of NG108-15, measured as the difference between the amount of binding found in the absence and presence of 10 uM unlabeled morphine, as a function of [*H]dihydromorphine con- centration. The cells used were subculture 12 and were grown in the presence of dibutyryl cyclic AMP. cells from surfaces and is believed not to penetrate the cell membrane. Treatment of a cell suspension of NG108-15 with 0.05% trypsin for 10 min at 37° resulted in a decrease of the specific binding of [*H dihydromorphine from 26 to 8 fmoles/ mg of protein under standard conditions. Although most of our experiments were performed with cell suspensions, we found that the specific binding of (H]dihydromorphine to homogenates of NG108-15, prepared in 0.32 M sucrose, was unchanged when compared with whole cells. When a homog- enate of NG108-15 was centrifuged to equilibrium in a 58- 20% w/w continuous sucrose gradient, the bulk of the mor- phine receptors were found at a sucrose concentration (mea- sured by refractometry) equivalent to a density of 1.16 g/ml. These results are consistent with the working hypothesis that the receptor is localized on the plasma membrane. DISCUSSION The results show that a neuroblastoma X glioma hybrid cell line (NG108-15) has the ability to bind narcotic analgesics in a stereospecific manner and with high affinity, whereas little or no specific binding was observed with parental cells and other cell lines tested. The relative binding affinities of narcotic analogues to NG108-15 cells were approximately the same as those found with rat brain homogenates and match well the pharmacological potency of the narcotics reported for isolated intestinal segments. Thus, there is good reason to believe that the neuroblastoma X glioma hybrid cells have morphine receptors similar to those found in brain and guinea pig ileum. In addition to morphine receptors, NG108-15 cells also have other neuronal characteristics that are not found with parental cells, such as choline acetyltransferase*, intracellular acetylcholine, and clear and dense core vesicles, 500 and 1500 A in diameter (21). In addition, the cells have long neurites, electrically excitable membranes, and nicotinic acetylcholine receptors.t   +B. Hamprecht, T. Amano, W. Kemper, and M. Nirenberg; unpublished observations.   Proc. Nat. Acad. Sct. USA 71 (1974) Many properties of neuronal cell lines in culture have been shown to be regulated (22), Thus, the specific activities of choline acetyl] transferase and cholinesterase* as well as the extent of morphological differentiation of NG108-15 cells can be increased many-fold in cultures which are grown in the presence of dibutyryl cAMP or are maintained in stationary phase (21). We did not detect regulation of the number of morphine receptor sites, since logarithmically dividing NG- 108-15 cells had the same number of morphine receptors as sister cultures maintained in the presence of dibutyryl cAMP that were highly differentiated with respect to cell mor- phology. On the other hand, we observed with one popula- tion of NG108-15 that the cells lost. much of their ability to bind dihydromorphine in a specific manner after 19 to 20 subcultures. Thus, it should be possible to obtain defective cell lines with respect to the synthesis, structure, or function of morphine receptors. At saturation of dihydromorphine we estimate that there are 0.6 pmoles of bound morphine per mg of cell protein. This is approximately twice the specific activity of rat brain homogenates measured under identical conditions. Since 1 mg of protein corresponds to about 1.2 X 10° cells, there are approximately 300,000 receptor sites per cell. This is a large number and is similar to the number of nicotinic acetylcholine receptors of cultured sympathetic ganglion neurons of the chick (23). One mechanism that has been proposed for the action of morphine is to inhibit the release of acetylcholine (24). Since NG108-15 cells both synthesize acetylcholine and release this compound into the medium, it should be possible to test this hypothesis. In addition, the recent demonstration by Collier and Roy of the specific reversal by narcotic analgesics of prostaglandin E-stimulated cAMP formation in rat brain homogenates (25) provides a possible mechanism for the action of these drugs. In collaboration with Shail K. Sharma, we have confirmed these findings with homogenates prepared from NG108-15, in that morphine was found to inhibit adenyl- ate cyclase activity of NG108-15. The availability of relatively homogeneous populations of cells with and without morphine receptors and with other neuronal properties provides opportunities to explore ques- tions relating to the mechanism of action of morphine and, Morphine Receptors in Neural Cell Lines 3477 possibly, phenomena such as morphine dependence and tolerance. We thank Mr. Doyle Mullinex and Mrs. Theresa M. Caryk for growing cells and Dr. Everette May for samples of the narcotics tested. : Pert, C. B. & Snyder, S. H. (1973) Science 179, 1011-1014. Terenius, L. (1973) Acta Pharmacol. Toxic. 32, 317-320. Simon, E. J., Hiller, J. M. & Edelman, I. (1973) Proc. Nat. Acad. Sct. USA 70, 1947-1949. Pert, C. B. & Snyder, 8. H. (1973) Proc. Nat. Acad. Sci. USA 70, 2243-2247, Wong, D. T. & Horng, J. S. (1973) Life Sci. 13, 1543-1556. Kuhar, M. J., Pert, C. B. & Snyder, 8. H. (1973) Nature 245, 447-450. Hiller, J. M., Pearson, J. & Simon, E. J. (1973) Res. Com- mun. Chem. Pathol. Pharmacol. 6, 1052-1062. Pert, C. B., Pasternak, G. & Snyder, S. H. (1973) Science 182, 1359-1361. Klee, W. A. & Streaty, R. (1974) Nature 248, 61-63. Goldstein, A., Lowney, L. I. & Pal, B. K. (1971) Proc. Nat. Acad. Sci. USA 68, 1742-1747. 11. Kosterlitz, H. W. & Watt, A. J. (1968) Brit. J. Pharmacol. Chemother. 33, 266-276. 12. Amano, T., Richelson, E. & Nirenberg, M. (1972) Proc. Nat. Acad. Sci. USA 69, 258-263. 13. Minna, J., Nelson, P., Peacock, J., Glazer, D. & Nirenberg, M. (1971) Proc. Nat. Acad. Sci. USA 68, 234-239. 14, Minna, J., Glazer, D. & Nirenberg, M. (1972) Nature New Biol. 238, 225-231. 15. Amano, T., Hamprecht, B. & Kemper, W. (1974) Exp. Cell Res., in press. 16. Ham, R. G. & Puck, T. T. (1962) in Methods in Enzymology, eds. Colowick, 8. P. & Kaplan, N. O. (Academic Press, New York), Vol. 5, pp. 90-119. 17. Patterson, M. S. & Greene, R. C. (1965) Anal. Chem. 37, 854-857. 18. Eddy, N. B., Halbach, H. & Braenden, O. J. (1957) Bull. W.H.O. 17, 569-863. 19. May, E. L. & Eddy, N. B. (1959) J. Org. Chem. 24, 1435- 1437. 20. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. 21. Daniels, M. & Hamprecht, B. (1974) J. Cell Biol., in press. 22. McMorris, F. A., Nelson, P. G. & Ruddle, F. H. (1973) Neurosci. Res. Progr. Bull. 412-536. 23. Greene, L. A., Sytkowski, A. J., Vogel, Z. & Nirenberg, - -M. W. (1973) Nature 243, 163-166. 24. Smith, C. B. (1972) in Chemical and Biological Aspects of Drug Dependence, eds. Mule, S. J. & Brill, H. (CRC Press, Cleveland, Ohio), pp. 495-504. 25. Collier, H. O. J. & Roy, A. C. (1974) Nature 248, 24-27. Pr SNE So 2 N SH _", "Nirenberg, Marshall W. ; Klee, Werner A.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2tx3_d93x.raaw", "00000000-0000-0000-39AC-812A6412E2CF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Receptor-Mediated Shifts in cGMP and cAMP Levels in Neuroblastoma Cells", "101584910X57", null, "1975", "September 1975", "Nirenberg's interest in neuroblastoma research defined his work by the early 1970s.  In this article, neuroblastoma cells are used to examine the various responses of neurons to neurotransmitters based on differences in tissue and cell type.  The experimental strategy is to use the relatively homogeneous clonal neuroblasts, which have excitable membranes and respond well to chemical and electrical stimuli, to define receptor mediated responses more clearly.", "Articles", "Neuroblastoma,Receptors, Drug ; Receptors, Cholinergic ; Cyclic AMP,Cyclic GMP", "Neuroblastoma Research, 1967-1976", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3472-3476, September 1975 Biochemistry Receptor-mediated shifts in cGMP and cAMP levels in neuroblastoma cells {acetylcholine receptor/prostaglandin Ey receptor /adenosine receptor /synapse /neurotransmitter) HIROSHI MATSUZAWA AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, July 10, 1975 ABSTRACT 1,5'-eGMP levels of neuroblastoma NIE-115 cells increase as much as 200-fold upon activation of muscar- inic acetylcholine receptors, resulting in intracellular cGMP concentrations >600 pmol/m of protein. The cells also have receptors for adenosine which mediate an increase in 3/:5'- cAMP levels. Unexpectedly, prostaglandin E, was found to increase the concentrations of both cGMP and cAMP. Car- bamylcholine, adenosine, and PGE) were added to cells sep- arately and in pairs to determine the effect of one compound on cell responses to another. Reciprocal inhibition, unilateral inhibition, additive, and nonadditive responses were ob- served with respect to cGMP and cAMP levels when differ- ent pairs of receptors were activated simultaneously.     The response of neurons to transmitters frequently can be modified by other species of [transmitter-receptor] interac- tions; thus, two types of synaptic stimuli may potentiate or inhibit one another. The pharmacology and electrophysiolo- gy of such phenomena have been studied extensively but much remains to be learned about the mechanisms which couple the activities of different species of receptors. In part, the difficulty in defining the reactions associated with re- ceptor activation stems from the heterogeneity of neural tissues with regard to cell type. One approach to this prob- lem is to define receptor-mediated responses of relatively homogeneous, clonal neuroblastoma cells which have excit- able membranes and respond to chemical and electrical stimuli. In this report the effects of activators of inhibitory mus- carinic acetylcholine receptors, adenosine receptors, and prostaglandin E; (PGE) receptors (1-5) on 37:5’-cGMP and 3/-5’-cAMP levels of neuroblastoma cells are described. Mus- carinic acetylcholine receptors frequently mediate slow, prolonged responses which may be excitatory or inhibitory; in contrast to the nicotinic acetylcholine receptor, which in higher organisms mediates fast excitatory responses. Activa- tion of the inhibitory or excitatory muscarinic acetylcholine receptors of cardiac (6, 7) and smooth muscle (7) results in an increase in cGMP concentration. We find that cGMP lev- els of neuroblastoma NIE-115 cells are elevated as much as 200-fold upon activation of muscarinic acetylcholine recep- tors. When different pairs of receptors were activated simul- taneously, reciprocal inhibition, unilateral inhibition, addi- tive, and nonadditive responses were observed with respect to cGMP and cAMP levels. MATERIALS AND METHODS Chemicals were obtained from the follawing sources: car- bamylcholine chloride, atropine sulfate, and d-tubocurarine Oe Abbreviations: cGMP, guanosine 3/:5'-cyclic monophosphate; cAMP, adenosine 3':5’-cyclic monophosphate; PGE, prostaglandin E,; IBMX, 3-isobutyl-1-methy]-xanthine, Hepes, N-2-hydroxy- ethylpiperazine-N 1.9.ethanesulfonic acid; DMEM, Dulbecco-Vogt modification of Eagle’s minimal essential medium. chloride from Sigma; N -2-hydroxyethylpiperazine-N’-2- ethanesulfonic acid (Hepes), Calbiochem; 3-isobuty]-1- methyl-xanthine (IBMX) from Aldrich Chem. Co.; PGE), a gift from Dr. John Pike, the Upjohn Co.; adenosine, Nutri- tional Biochem. Corp.; cAMP, Schwarz/Mann; cGMP, JEM Research Products; [G-7H]cAMP (22.1 Ci/mmol), New En- gland Nuclear; [8-3H|eGMP (15 Ci/mmol), Amersham/ Searle Corp.; cAMP-dependent protein kinase, Sigma; cGMP antiserum and succinyl-cGMP 125]-tyrosine methy] ester, Collaborative Research, Inc.; fetal bovine serum, Colo- rado Serum Co.; and DMEM (the Dulbecco-Vogt modifica- tion of Eagle’s minimal essential medium), GIBCO, Cat. no. H_-21. Other chemicals were of reagent grade purity. Cell Culture. The following cell lines were used: mouse neuroblastoma C-1300 clones NI1E-115 and N18 (8), mouse neuroblastoma X mouse L cell hybrid clone NLIF (9), and rat glioma clone C6BU-1 (10). Neuroblastoma cells were grown in 100 mm petri dishes (Falcon, 55 cm? surface area) in 15 ml of 90% DMEM-10% fetal-bovine serum (340 mOsm/kg) in a humidified atmosphere of 10% CO2-90% air at 37°. Hybrid cells were grown in the same medium sup- plemented with 0.1 mM hypoxanthine, 1 ~M aminopterin, and 16 uM thymidine (HAT). Cells usually were grown to confluency (1 to 3 X 10° cells, 0.1 to 0.3 mg of protein per cm2) prior to use. Incubation of Cells. Each plate was washed three times with 7 ml portions of solution A (DMEM minus NaHCOs with 25 mM Hepes adjusted to pH 7.4 with NaOH and to 340 mOsm/kg with NaCl). Unless stated otherwise, 10 ml of solution A supplemented with 0.5 mM IBMX were added to each plate and cells were equilibrated for 30 min at 37°. Re- actions were initiated by the addition of 0.1 ml of a solution of the compound to be tested dissolved in 0.17 M NaCl, or 0.1 ml of a PGE; solution (1 mM PGE), 0.153 M NaCl, and 10% ethanol). The final concentration of ethanol in the petri dish, 0.1%, did not alter cGMP or cAMP levels of cells. Cells were incubated at 37° for the times specified, then the me- dium was removed by aspiration (discarded unless specified otherwise) and 4 ml of cold 5% trichloroacetic acid was added to the monolayer to terminate reactions and extract nucleotides. A solution (0.05 ml) containing 10,000 cpm of (SH|cGMP (0.67 pmol) and/or [SH|cAMP (0.45 pmol) was added to each dish and cells were detached by scraping. The suspension was transferred to a tube, the dish was washed with 1 ml of cold 5% trichloroacetic acid, and the combined suspension and wash was centrifuged for 15 min at 20,000 x gat 3°. Purification of cGMP and cAMP. A rapid method was devised for separating cGMP from cAMP and removing tri- chloroacetic acid and endogenous interfering compounds from cyclic nucleotide fractions. The supernatant solution © the deproteinized sample in 5% trichloroacetic acid was ap plied to an ion exchange column (0.7 X 8 cm) of AG50W-X4 3472 Biochemistry: Matsuzawa and Nirenberg (200-400 mesh, hydrogen form, Bio-Rad) previously washed and equilibrated with H2O. The column was washed with 2 ml of H2O (eluates discarded) and then with 2 additional ml of HzO. The latter eluate (2.0 ml) which contains cGMP was collected on a neutral alumina (WN-3, Sigma) column (0.7 X 3 cm) previously washed with 20 ml of 200 mM sodium acetate, pH 6.2, and then with 10 ml of 5 mM sodium ace- tate, pH 6.2. The eluate was discarded, and the column was washed with 3 ml of 5 mM sodium acetate buffer, pH 6.2, and then with 1 ml of 200 mM sodium acetate buffer, pH 6.2 (eluates discarded). Then cGMP was eluted with 2 ml of 200 mM sodium acetate, pH 6.2, and portions of the eluate were assayed for cGMP concentration and for recovery. The average recovery of cGMP was about 50%; <1% of the cAMP of the sample was present in the cGMP fraction. Cyclic AMP fractions were obtained as follows: after cGMP was eluted from the AG50W-X4 column, the column was washed with 2 ml of HgO (eluate discarded) and cAMP was eluted with 3 additional ml of H2O. Portions of the el- uate were assayed for cAMP concentration and recovery. The cAMP fraction contained <0.2% of the cGMP of the sample prior to fractionation. The average recovery of (3HIcAMP was 70%. Each AG50W-X4 column was regenerated by washing with 8 ml of 1 N HCl and, prior to use, with 30 ml of water. Assay of cGMP and cAMP. The cGMP concentrations of purified alumina column eluates were determined by the ra- dioimmune method of Steiner et al. (11) modified as fol- lows: each 0.5 ml reaction mixture contained 200 mM (in some experiments 140 mM) sodium acetate, pH 6.2 (solution B); 17 fmol of succinyl-eGMP !**I-tyrosine methyl ester {10,000-20,000 cpm); cGMP antibody; and sample (0-20 pmol of cGMP). After incubation (14-18 hr, 3°) each reac- tion mixture was diluted with 2 ml of solution B at 3°; 10 min later the sample was passed through a Millipore filter (0.45 um pore size, 25 mm diameter, HAWP 02500) pre- viously rinsed with solution B and washed three times with cold solution B (4 ml each wash). Each filter was placed in a scintillation vial with 1 ml of 2-methoxyethanol; after filters dissolved (0.5-1 hr) 15 ml of Aquasol (New England Nucle- ar) were added. Radioactivity was determined 12-24 hr later in a Beckman liquid scintillation counter (3H plus !4C isoset). Cyclic AMP concentrations of samples purified as de- scribed above were determined by the method of Gilman (12). Each cGMP or cAMP sample was assayed at three or four concentrations. Each value shown corresponds to 100% recovery of cyclic nucleotide and is the mean of duplicate dishes. Protein was determined by a modification of the method of Lowry et al. (13). RESULTS A rapid method was devised for separating cGMP from cAMP and removing trichloroacetic acid and endogenous factors which interfere with the cGMP assay. The proce- dure, based upon the Materials and Methods section, is based upon the methods of Salomon, Londos, and Rodbell (14) and White and Zenser (15); CGMP and cAMP are recov- ered in separate fractions in high yield and in sufficiently high concentration to assay directly without lyophilization. One hundred samples can be purified in 2-3 hr. The effect of carbamylcholine, a relatively stable analog of acetylcholine, upon intracellular cGMP levels of neuro- blastoma and other cell lines in the absence of a 3/:5’-cyclic nucleotide phosphodiesterase inhibitor is shown in Fig. 1. Cyclic GMP levels of neuroblastoma clones NI8 and N1E- Proc. Nat. Acad. Sci. USA 72 (1975) 3473   TOOR —   pmol cGMP/mg protein       MINUTES Fic. 1. Effect of 1 mM carbamylcholine on intracellular levels of cGMP in neuroblastoma clones NIE-115 and N18, neuroblasto- ma X L cell hybrid clone NL1F, and glioma clone C6BU-1. Cul- tures were confluent; the average protein content was: 11.5, 16.4, 7.6 and 6.9 mg of protein per 100 mm petri dish for N1E-115, N18, NLIF, and C6BU-1, respectively. A 3’:5’-cyclic nucleotide phos- phodiesterase inhibitor was not present. 115 rose 40- and 210-fold, respectively, upon incubation with carbamylcholine for 15 sec, and then fell rapidly to normal levels (50% decrease in cGMP at 45 sec). Carbamyl- choline also elicited a 6-fold increase in the cGMP concen- tration of the neuroblastoma X L cell hybrid, NL-1F, but had no effect upon cGMP levels of rat glioma clone C6BU-1. Thus, two types of cell lines were found with respect to ace- tylcholine-receptor-mediated reactions which increase cGMP levels: cell lines which are sensitive to carbamylcho- line and a line which is insensitive. The effect of IBMX, a 3’:5’-cyclic nucleotide phosphodies- terase inhibitor, upon the rate of disappearance of intracel- lular cGMP after stimulation with carbamylcholine is shown in Fig. 2A. In the absence of IBMX, maximum cGMP levels were attained 15 sec after the addition of carbamylcholine, and then rapidly decreased (50% decrease at 45 sec; Figs. 1 and 2A). In the presence of 0.5 mM IBMX, maximum cGMP levels were attained at 30 sec and cGMP levels then de- creased at a slower rate (50% decrease at 3 min). The effect of IBMX upon carbamylcholine dependent cGMP accumulation also was studied with NIE-115 cells adapted to grow in suspension culture. The assay was modi- fied so that both intra- and extracellular cGMP was deter- mined. As shown in Fig. 2B, carbamylcholine elevated cGMP levels of suspension cells and in the presence of IBMX cGMP levels remained constant during the 10 min incuba- tion period. These results suggest that part of the cGMP formed by cells may be excreted into the medium. Other 3':5’-cyclic nucleotide phosphodiesterase inhibitors such as 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone — (Ro-20- 1724) and papaverine were not as effective as IBMX in maintaining cGMP levels of N18 cells. The relation between carbamylcholine concentration and eGMP accumulation in N1E-115 cells is shown in Fig. 3. The maximum increase in cGMP concentration was ob- tained with 1073 M carbamylcholine; 50% of the maximum increase in cGMP concentration was evoked by 1074 M car- bamylcholine. The effects of atropine and d-tubocurarine, selective in- hibitors of muscarinic and nicotinic acetylcholine receptors, 3474 Biochemistry: Matsuzawa and Nirenberg   pmo! cGMP or cAMP/mg protein           ett SUSPENSION CELLS AND MEDIUM 200k c [ B 100 9 , & 50 ao £ = 20 8 2 woke ° & ef a 5 a_i 4 1 1 1 1 L L 1 oT 23 4 5 6 7 8 9 10 MINUTES Fic. 2. Effect of 1 mM carbamylcholine in the presence of 0.5 mM IBMX, a 3:5’-cyclic nucleotide phosphodiesterase inhibitor, on cGMP levels of neuroblastoma NI1E-115 cells. (A) Cells were grown for 9 days to confluence; the average cellular protein was 12.2 mg/100 mm dish. The broken line corresponds to the data for N1E-115 shown in Fig. 1 (cells were incubated in the absence of IBMX). (B) Cells were grown in suspension culture in DMEM with 10 mM NaHCOs, 25 mM Hepes, and 2% fetal bovine serum (pH 7.3, 340 mOsm/kg). Cells were washed and 1 ml portions (4 X 10 cells) were preincubated in the presence of 0.5 mM IBMX for 15 min with gentle shaking. Two experiments are shown. Cells were incubated for 3 min or less (aA) with 1 mM carbamylcholine; or (4) without this compound. In another experiment (m) cells were incubated for 0 to 10 min with 1 mM carbamylcholine. In both experiments reactions were terminated by the addition of 4 ml of cold 6% trichloroacetic acid to each tube. Hence the com- bined intra- and extracellular cGMP concentration was deter- mined. respectively, upon carbamylcholine-dependent accumula- tion of cGMP in N1E-115 cells are shown in Fig. 4. Atro- pine, at 1 X 1077 M, inhibited the carbamylcholine-depen- dent increase in cGMP concentration by 50%; 97% inhibi- tion was obtained with 1 X 1076 M atropine. In contrast, d- tubocurarine did not inhibit appreciably even at 1 X 1074 M: in fact, in other experiments d-tubocurarine potentiated the carbamylcholine-dependent elevation of cGMP. The in- hibition by atropine suggests that the increase in cGMP elic- ited by carbamylcholine is mediated by the muscarinic ace- tylcholine receptors. The effects of PGE; upon cAMP and cGMP levels of NIE-LI5 cells are shown in Fig. 5. Cyclic AMP levels were found to increase markedly upon the addition of 10 4M PGE). Unexpectedly, the level of cGMP also increased 6- fold in the presence of PGE). The maximum concentration of cAMP attained in this experiment was 9-fold higher than that of cGMP; however, in other experiments, the concen- tration of cGMP dependent on PGE, increased to 250 pmol Proc. Nat. Acad. Sci. USA 72 (1975) 250 as 70           . 200 s rr a i S & 50 -TUBOCURARINE © g 6 g 5 150 os js £ Ww g & 30 ATROPINE | x & 100 6 © Fi e 2 1 9 3 20 - € E z 4 | t \\\\°        ° ie oo ie MOLARITY io io Ww 1 IC MOLARITY CARBAMYLCHOUINE Fic. 3 (left). Relation between carbamylcholine concentration and cGMP levels of neuroblastoma N1E-115 cells. Cultures were grown for 9 days to confluence; the average cellular protein was 13.5 mg/100 mm dish. Cells were incubated for 30 sec in the pres- ence of the indicated concentrations of carbamylcholine and IBMX. Fic. 4 (right). Effect of atropine and d-tubocurarine concen- tration on the carbamylcholine dependent increase in cGMP level of N1E-115 cells. Cultures were grown for 9 days to confluence; the average cellular protein was 14.6 mg/100 mm dish. Cells were incu- bated for 30 sec in the presence of 1 mM carbamylcholine and the indicated concentrations of atropine or d-tubocurarine. of cGMP per mg of protein, approximately 60% of the con- centration attained by cAMP in the presence of PGE. In other experiments not shown here, the increase in cGMP concentration elicited by PGE; was shown to be insensitive to atropine. Thus, the effect of PGE; upon eGMP is not me- diated by the muscarinic acetylcholine receptor. The maxi- mum cGMP concentration was achieved 30 sec after the ad- dition of PGE,; cGMP levels decreased during subsequent incubation. In contrast, the maximum cAMP level was achieved 4 min after the addition of PGE). The effects of carbamylcholine, PGE), and adenosine upon cGMP and cAMP levels of N1E-115 cells plotted on a logarithmic scale to illustrate inhibitory as well as stimulato- ry effects are shown in Fig. 6A and B, respectively. In the presence of carbamylcholine or PGE;, cGMP levels in- creased and reached maximum levels within 1 min. The rates of disappearance of cGMP during the subsequent incu- bation period were linear. In contrast, the basal level of cGMP was reduced approximately 50% in the presence of 0.1 mM adenosine. As shown in Fig. 6B, cAMP levels in- creased markedly in the presence of PGE; or adenosine, but decreased 30% in the presence of carbamy|choline. Various combinations of carbamylcholine, PGE), and adenosine were added simultaneously to determine the ef- fect of one compound upon cell responses to another (Fig. b OQ pmol cGMP “mg protein nN Oo NM ° 3 pmol cAMP/mg protein     Oo-Tss4567 8 MINUTES Fic. 5. Effect of PGE, on intracellular levels of cGMP and cAMP in neuroblastoma N1E-115 cells. Cultures were grown for © days; the average cellular protein was 6.8 mg/100 mm dish. Close and open circles correspond to cGMP levels with and without 10 uM PGE), respectively; closed and open triangles correspond t¢ cAMP levels with and without 10 uM PGE), respectively. Biochemistry: Matsuzawa and Nirenberg          200 pea FO cGMP 1 ® cAMP loo E, 4 F CARBACHOL 4 50 + pce, 4500                     Cc a & 6 2 a 9 F 20 Poe, 4200 § a t 1 £ S + swe 4 a Qo 10 + ADENOSINE 11909 5S 2 ¥ contro f { s ° + 4 —_ 4 3 B 7 7°90 CONTROL a oO ‘ ¥ 7 ADENOSINE 2b ai 4 20 E rt ° CARBACHOL J I 1 4 1 1 C 4 1 4 4 10 Oo t 23 4 Oo |! 2 3 4 MINUTES FIG, 6. Effect of 1 mM carbamylcholine (carbachol), 0.1 mM adenosine, or 10 uM PGE, on intracellular levels of cGMP (A) and cAMP (B) of neuroblastoma N1E-115 cells. Culture conditions were identical to those described in the legend to Fig. 5. 7). The effect of exposing cells to both carbamylcholine and adenosine upon cGMP and cAMP levels is shown in Fig. 7A and B, respectively. Adenosine inhibited the carbamylcho- line-dependent increase in cGMP concentration 25 and 60% when incubations were 30 and 60 sec, respectively. In Fig. 7B, the effect of carbamylcholine upon the adeno- sine-dependent increase in cAMP concentration is shown. Carbamylcholine did not inhibit the adenosine-stimulated increase in cAMP during the first 30 sec of incubation even though the maximum increase in cGMP concentration evoked by carbamylcholine was achieved at 30 sec. How- ever, during further incubation, carbamylcholine inhibited almost completely the adenosine-dependent elevation in cAMP concentration. Carbamylcholine and PGE, added separately increased cGMP levels as indicated by the broken lines in Fig. 7C. The effects of carbamylcholine and PGE, added simultaneously to cultures were additive or more than additive. However, carbamylcholine inhibited the PGE,-induced increase in cAMP concentration, particularly during the latter part of the incubation period (Fig. 7D). Thus, carbamylcholine may have a delayed inhibitory effect upon both adenosine- and PGE\\\\-dependent increases in cAMP concentrations. Atro- pine completely blocked the inhibitory effect of carbamyl- choline upon adenosine- or PGE)-dependent increases in cAMP levels (data not shown). The effect of activating receptors for adenosine and PGE; separately and simultaneously upon cGMP and cAMP levels is shown in Fig. 7E and F, respectively. Adenosine inhibited slightly the rise in CGMP levels elicited by PGE). The results show that the increases in cAMP levels elicited either by adenosine or PGE, are not additive. In other experiments, the sequence of addition of carbamylcholine and either adenosine or PGE, was varied, but the results did not differ significantly from those found when two compounds were added simultaneously. DISCUSSION Two types of cells were found with respect to muscarinic acetylcholine receptor activity: cells sensitive to carbamyl- choline and cells insensitive to this compound. The degree of response also varied widely from one clone to another. Acti- vation of the muscarinic acetylcholine receptors of neuro- Proc. Nat. Acad. Sci. USA 72 (1975) 3475               150 Ff T r T ‘TT v ¥ T (@) cGMP {® cAMP oO Pry 4 l0Ors \\\\\\\\ CARBACHOL + ADENOSINE ew & c 2 3 ° 3 a a £ 2 a a = = 20 g : 6 3 & 0) E =--- ===>. A bo Oo O-1 234 01234 MINUTES Fic. 7. Effect of various combinations of 1 mM carbamylcho- line, 0.1 mM adenosine, and 10 uM PGE, on intracellular levels of cGMP (panels A, C, and E) and cAMP (panels B, D, and F) of neu- roblastoma N1E-115 cells. Culture conditions were identical to those described in the legend to Fig. 5. The broken lines corre- spond to the effect of each compound added separately (data shown in Fig. 6); C, A, and P represent carbamylcholine, adeno- sine, and PGE, respectively. blastoma N1E-115 cells evoked up to a 200-fold increase in cGMP concentration, yielding levels in excess of 600 pmol of cGMP per mg of protein. These cells have high tyrosine hy- droxylase activity (8), generate action potentials in response to electrical stimuli (8), have neurosecretory vesicles, and also possess PGE, receptors and adenosine receptors which upon activation lead to elevated cAMP levels (2, 3). Adenosine, carbamylcholine, and PGE, were added to cells separately and in pairs to determine the effect of one compound upon cell responses to another. The results, sum- marized in Table 1, show that in the presence of adenosine cAMP levels increase and cGMP levels decrease; whereas, in Table 1. Summary of receptor-mediated shifts in cGMP and cAMP levels in neuroblastoma cells     cGMP cAMP Addition percent None 100 100 Adenosine 40 500 Carbamylcholine 1500 70 PGE, 500 1250 Adenosine + carbamylcholine 650 250 PGE, + carbamylcholine 2050 1000 PGE, + adenosine 300 1100   100% corresponds to 7.5 pmol of cGMP/mg of protein or 27.5 pmol of cAMP/mg of protein. The data are trom the experiments shown in Figs. 6 and 7. 3476 Biochemistry: Matsuzawa and Nirenberg the presence of carbamylcholine, cGMP levels increase and cAMP levels decrease. In addition to the inverse relationship between cGMP and cAMP concentrations, a novel phenom- enon was observed; that is, in the presence of PGE; the lev- els of both cGMP and cAMP increase. The increase in cGMP elicited by carbamylcholine was not inhibited by PGE; in spite of the fact that PGE; elicits a marked increase in cAMP levels. Prostaglandin-E)- and carbamylcholine-de- pendent increases in cGMP were fully additive, and in some experiments, were greater than additive. In contrast, the ele- vated cAMP concentrations induced either by PGE or by adenosine were not additive when both compounds were added simultaneously to cells. We conclude that activation of a species of muscarinic acetylcholine receptor inhibits adenosine- and PGE)-recep- tor-mediated reactions which elevate cAMP levels, whereas, activation of adenosine receptors inhibits reactions mediated by muscarinic acetylcholine receptors and PGE, receptors which elevate cGMP levels. In contrast to these results, acti- vation of PGE, receptors did not affect acetylcholine- or adenosine-receptor-mediated reactions which affect cGMP or cAMP levels. Thus, reciprocal inhibition, unilateral inhi- bition, additive, and nonadditive responses were observed when different pairs of receptors were activated simulta- neously. Relatively little is known at the molecular level about the mechanisms which couple one receptor with another. The observed phenomena are obviously complex and may reflect changes in membrane permeability, metabolism of cyclic nucleotides, and so forth. Other examples of reciprocal cou- pling of cGMP and cAMP levels and possible mechanisms of coupling have been discussed by Goldberg et al. (22, 23). Blume et al. (5) have reported that acetylcholine inhibits the increase in cAMP levels induced by PGE, or adenosine in mouse neuroblastoma cells. Acetylcholine also has been shown to inhibit the PGE)-dependent elevations of cAMP levels, the inhibition mediated by excitatory muscarinic ace- tylcholine receptors in neuroblastoma X glioma hybrid cells (16). Acetylcholine and carbamylcholine also have been re- ported to stimulate mouse neuroblastoma adenylate cyclase activity (17). . The inhibitory effect of carbamylcholine on reactions me- diated by other species of receptors shows that some N1E- 115 cells with muscarinic acetylcholine receptors also have adenosine or PGE) receptors. Some cells also have both adenosine receptors and PGE receptors. Although N1E-115 is a clonal cell line, single cell analysis by electrophysiologic methods reveals two acetylcholine receptor phenotypes; cells sensitive and cells insensitive to carbamylcholine. All NIE- 115 cells sensitive to acetylcholine or carbamylcholine exhib- it hyperpolarizing responses to these compounds, frequently —25 mV in magnitude and 10-15 sec in duration. Hence, N1E-115 cells have inhibitory muscarinic acetylcholine re- ceptors. The activity of PGE, in eliciting increases in both cGMP and cAMP levels should be considered in context with the different actions of PGE; on various tissues which have been reported. For example, PGE, elevates cAMP levels in some tissues and reduces hormone-dependent elevation of cAMP levels in others (18), activates adenylate cyclase activity in Proc. Nat. Acad. Sci. USA 72 (1975} homogenates (19), including those prepared from neuroblas- toma X glioma hybrid cells (20), and inhibits the vasopres- sin-dependent activation of adenylate cyclase (21). Prostaglandin E, elicits a transitory, 30 sec, increase in cGMP accumulation and a prolonged increase in cAMP ac- cumulation. The difference in the duration of cGMP and cAMP accumulations may reflect the activities of different enzymes which catalyze the synthesis and perhaps the hy- drolysis of the nucleotides, or alternatively, may indicate the presence of two species of PGE, receptor, one mediating an increase in cAMP concentration, the other, an increase in cGMP. Preliminary results which support the latter hypoth- esis will be described elsewhere. We thank Doyle Mullinax, Deborah Carper, and Linda Lee for their excellent assistance, and Mary Ellen Miller for typing the manuscript. 1. Gilman, A. G. & Nirenberg, M. (1971) Nature 234, 356-358. Blume, A. J., Dalton, C. & Sheppard, H. (1973) Proc. Nat. Acad. Sci. USA 70, 3099-3102. Hamprecht, B. & Schultz, J. (1973) FEBS Lett. 34, 85-89. 4. Schultz, J. & Hamprecht, B. (1973) Naunyn-Schmiedeberg's Arch. Pharmacol. 278, 215-225. 5. Blume, A. J., Foster, C. & Karp, G. (1974) Trans. Amer. Soc. Neurochem. 5, 150. 6. George, W. J., Polson, J. B., O'Toole, A. G. & Goldberg, N. D. (1970) Proc. Nat. Acad. Sci. USA 66, 398-403. 7. Lee, T., Kuo, J. F. & Greengard, P. (1972) Proc. Nat. Acad. Sci. USA 69, 3287-3291. 8. Amano, T., Richelson, E. & Nirenberg, M. (1972) Proc. Nat. Acad. Sci. USA 69, 258-263. : 9. Minna, J., Glazer, D. & Nirenberg, M. (1972) Nature New Biol. 235, 225-231. 10. Amano, T., Hamprecht, B. & Kemper, W. (1974) Exp. Cell Res. 85, 399-408. 11. Steiner, A. L., Parker, C. W. & Kipnis, D. M. (1972) J. Biol Chem. 247, 1106-1113. 12. Gilman, A. G. (1970) Proc. Nat. Acad. Sci. USA 67, 305-312. 13. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J (1951) J. Biol. Chem. 193, 265-275. 14. Salomon, Y., Londos, C. & Rodbell, M. (1974) Anal. Biochem. 58, 541-548. 15. White, A. A. & Zenser, T. V. (1971) Anal. Biochem. 41, 372- 396. 16. Traber, J., Fischer, K., Buchen, C. & Hamprecht, B. (1975) Nature 255, 558-560. 17. Prasad, K. N., Gilmer, Kk. N. & Sahu, S. K. (1974) Nature 249, 765-767. 18. Butcher, R. W. & Baird, C. E. (1968) J. Biol. Chem. 243, 1718-1717. 19. Wolfe, S. M. & Shulman, N. R. (1969) Biochem. Biophys. Res. Commun. 35, 265-272. 20. Sharma, S. K., Nirenberg, M. & Klee, W. A. (1975) Proc. Nat. Acad. Sci. USA 72, 590-594. 21. Marumo, F. & Edelman, I. S. (1971) J. Clin. Invest. 50, 1613-1620. 22, Goldberg, N. D., O'Dea, R. F. & Haddox, M. K. (1973) in Ad- vances in Cyclic Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, Publishers, New York), Vol. 3. pp. 155-223. 23. Goldberg, N. D., Haddox, M. K., Dunham, E., Lopez, Gc & Hadden, J. W. (1974) in Control of Proliferation in Animal Cells, eds. Clarkson, B. & Baserga, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 609-625. e", "Nirenberg, Marshall W. ; Matsuzawa, Hiroshi", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-96u2.8g9s~g7sb", "00000000-0000-0000-27F8-0AFE7CEE6ADA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Synapse Formation Between Clonal Neuroblastoma x Glioma Hybrid Cells and Striated Muscle Cells", "101584910X58", null, "1976", "January 1976", "As part of a series of articles in this collection covering Nirenberg's work on hybrid cells, the authors in this report show that clonal hybrid cells, which are known to synthesize, store and excrete acetylcholine, form synapses with striated muscle cells in vitro.  The authors conclude that the ability of hybrid cells to form synapses provides opportunities to correlate aspects of synaptogenesis with the molecular basis of developmental and electrophysiological characteristics of cells.", "Articles", "Neuroblastoma,Hybrid Cells,Neuromuscular Junction,Synapses,Action Potentials,Membrane Potentials", "Neuroblastoma Research, 1967-1976", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Nat. Acad. Sci. USA Vol. 73, No. 1, pp. 123-127, January 1976 Biochemistry Synapse formation between clonal neuroblastoma X glioma hybrid cells and striated muscle cells (cell recognition/acetylcholine receptor /transmitter storage and release/regulation of synapse formation) PHILLIP NELSON, CLIFFORD CHRISTIAN, AND MARSHALL NIRENBERG National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, November 10, 1975 ABSTRACT Clonal neuroblastoma X glioma hybrid cells were shown to form synapses with cultured, striated muscle cells. The properties of the synapses between hybrid and muscle cells were similar to those of the normal, neuromus- cular synapse at an early stage of development. The number of synapses formed and the efficiency of transmission across synapses were found to be regulated, apparently indepen- dently, by components in the culture medium. Under appro- priate conditions synapses were found with 20% of the hy- brid-muscle cell pairs examined; thus, the hybrid cells form synapses with relatively high frequency.   Little is known at the molecular level about the process of synapse formation and the basis for the specificity of synap- tic connections between cells. Some of the difficulties in studying this problem stem from the many cell types that are present in the nervous system and the lack of adequate criteria that can be used to distinguish one type of neuron from another. Clonal lines of neuroblastoma cells (1, 2) and somatic cell hybrids (3-6) derived from neuroblastoma cells have proved experimentally advantageous for the study of some neural properties. Such cells continue to proliferate; thus populations of cells that appear to be fairly homoge- neous can be obtained, yet cells retain the ability to express various properties of differentiated neurons. One of the pri- mary objectives in generating and characterizing these cell lines has been to obtain cell lines that are able to form sy- napses in vitro with high frequency. In this report, we show that clonal neuroblastoma X glioma hybrid cells which are known to synthesize, store, and excrete acetylcholine* form synapses with striated muscle cells in vitro. MATERIALS AND METHODS Cell Cultures. NG108-15 hybrid cells were derived by fu- sion of mouse neuroblastoma clone N18TG-2 resistant to 6- thioguanine (4), with rat glioma clone C6BU-1, resistant to 5-bromodeoxyuridine (5). NG108-15 cells routinely were cultured at 37° in medium D (90% Dulbecco-Vogt modifi- cation of Eagle’s minimal essential medium, GIBCO H-21, 10% fetal bovine serum; | X 1074 M hypoxanthine; 1 X 1076 M aminopterin; and 1.6 X 1075 M thymidine) in a humidi- fied atmosphere of 10% CO2-90% air. NG108-15 cells were cultured for 1-3 weeks in the presence of 1 mM N®, 0?-di- butyryl-adenosine-3’:5’-eyclic monophosphate (dibutyryl cAMP) to shift the hybrid cells to a more differentiated state*. All experiments were performed with NG108-15 cells that had been subcultured 14 to 20 times.   Abbreviations: DMEM, the Dulbecco-Vogt modification of Eagle's minimal essential medium: MEM, Eagle's minimal essential medi- um; dibutyryl cAMP, N®,O?-dibutyryl-adenosine-3”:5’-cyclic mo- nophosphate; dibutyryl cGMP, N?,0?-dibutyryl guanosine-3’:5’- cyclic monophosphate. 123 It was necessary to remove butyric acid and perhaps other contaminants from dibutyryl cAMP prior to use. The fol- lowing method was used. Dibutyryl cAMP dissolved in HzO (1 g/20 ml) was adjusted to pH 3.0 with 0.1 M HCl and then extracted vigorously (and sometimes repeatedly) with 10 volumes of diethylether. The aqueous layer was recovered, adjusted to pH 6.5 with 0.1 M NaOH, and lyophilized. Cultures of striated muscle cells were prepared by disso- ciation of cells from the hindlimbs of 18 to 21-day-old C57BL/6N mouse embryos with 0.25% trypsin for 40 min at 37°. The concentration of fibroblasts was reduced by incu- bating cell suspensions for 20 min in Falcon plastic petri dishes resulting in the preferential attachment of fibroblasts. A suspension of 3.5 X 10° single cells that did not attach to the petri dish was plated in a collagen-coated 35 mm plastic culture dish containing either 1.5 ml of 80% DMEM, 10% fetal bovine serum, 10% horse serum; or 40% DMEM, 40% MEM (adjusted to 33 mM dextrose and 44 mM sodium bi- carbonate), 10% fetal bovine serum, and 10% horse serum. Cells were grown at 36° in a humidified atmosphere of 10% CO2-90% air. The medium was replaced 24 and 48 hr later; thereafter, cultures were fed twice weekly with 90% DMEM and 10% horse serum, except where noted. Usually on the ninth day of incubation of muscle cells 3 X 104 NG108-15 hybrid cells were added to each muscle culture. NG108-15 and muscle cells usually were cocultured for an additional 1-2 weeks in 90% DMEM, 10% horse serum; 1 mM dibutyr- yl cAMP; 0.1 mM hypoxanthine; and 0.016 mM thymidine before cells were assayed for synapses. Electrophysiology. Intracellular recording and stimula- tion techniques were essentially as described (7). d-Tubocu- rarine chloride solutions (0.6 to 6 X 1075 M) in pipettes with tips 5-20 um in diameter were applied to muscle cells by al- lowing the solution to diffuse from the pipette tip. Usually d-tubocurarine chloride was dissolved in the same medium that the cells were exposed to; to avoid nonspecific effects, the pH was maintained at 7.0-7.5. The medium bathing cells during the electrophysiologic experiments was either DMEM or modified MEM without serum usually adjusted as follows (except where stated): 3.8 mM CaCly; 20 uM eserine sulfate (freshly prepared); 1 mM dibutyryl cAMP, purified as described above; 0.1 mM hypo- xanthine; and 0.016 mM thymidine. In some experiments cells were treated with 1 mM N?,O?-dibutyryl-guanosine- 3/:5’-cyclic monophosphate (dibutyryl cGMP) for 1 day prior to the assay in place of dibutyryl cAMP. This substitu- tion had no apparent effect on synapse formation. Materials. The following materials were used: aminopter-   * B. Hamprecht, T. Amano, P. Simpson, and M. Nirenberg, unpub- lished observations. 124 Biochemistry: Nelson et al. in, choline chloride (Nutritional Biochem. Corp.); hypoxan- thine (General Biochem, Inc.); d-tubocurarine chloride (Ab- bott or Sigma Chem. Co.); eserine sulfate, dibutyryl cGMP, sodium salt (Sigma Chem. Co.), thymidine (Worthington Biochem. Corp.); and dibutyryl cAMP, sodium salt (JEM Research). RESULTS Neuroblastoma X glioma NG108-15 hybrid cells usually were grown for 1-3 weeks in the presence of 1 mM dibutyr- yl cAMP, and then were dissociated and plated on a mono- layer of mouse striated muscle cells formed by fusion of myoblasts in vitro during the previous 1-2 weeks. The elec- trical excitability of the cells was improved greatly when contaminants were removed from dibutyry] cAMP by ex- traction with diethylether prior to use as described in Mate- rials and Methods. The hybrid cells attached readily to the muscle monolayer and exterided long branched processes with varicosities. Processes with terminal swellings were found which were attached to muscle cells (Fig. 1A). Pairs of interacting hybrid and muscle cells that were well differen- tiated morphologically and were not obscured by other cells were selected for electrophysiological study to determine whether synapses were present. The muscle and hybrid cells were impaled with separate microelectrodes, the resting membrane potentials were determined; then the hybrid cell was stimulated electrically to elicit action potentials, and muscle responses were recorded. Nearly all adequately pen- etrated NG108-15 cells exhibited action potentials in re- sponse to electrical stimulation, and spontaneous repetitive action potentials were found with 5-10% of the hybrid cells tested. As shown in Fig. 1C, about 50% of the action potentials elicited in the NG108-15 hybrid cell by repetitive electrical stimulation resulted in transient depolarizing shifts in the muscle membrane potential which varied in amplitude. The proportion of hybrid action potentials that evoked muscle responses varied with different cell pairs, but usually was <50%. As shown in Fig. 1B, electrical stimulation of an NG108- 15 cell (trace 6) elicited an action potential in the hybrid cell (trace 5) which was followed after a delay of several msec by a muscle response. Four examples of muscle responses evoked by hybrid action potentials are shown (traces 1-4). Some variation was observed in the delay and in the ampli- tude and duration of muscle responses to different hybrid action potentials. These muscle responses clearly were not due to direct electrical coupling between hybrid and muscle cells since hyperpolarizing currents passed across the hybrid or the muscle cell membrane did not change the membrane potential of the other cell. Electrical coupling between NG108-15 and muscle cells were observed infrequently (<1% of the cell pairs examined). The action potential of a muscle cell (Fig. 1C) elicited by electrical stimulation of the muscle cell characteristically had a faster rate of rise than that of NG108-15 and a time course and amplitude that was easily distinguished from muscle responses to NG108-15 action potential. In Fig. 2 the amplitudes of muscle responses are shown as a function of response frequency. The response amplitudes of the muscle cells varied from 0.4 mV, just above the noise level, to 14 mV. Most muscle responses involved relatively small shifts in membrane potential; the mean response am- plitude was 4.3 mV. Analysis of other synapses revealed am- Proc. Nat. Acad. Sci. USA 73 (1976)   Dd, mv[_ [| 20 nv ce zi LJ 10 sec FIG. 1. (A) Phase contrast photomicrograph of a culture con- taining neuroblastoma X glioma NG108-15 hybrid cells and nor- mal mouse muscle cells. NG108-15 cells, grown in the presence of 1 mM dibutyryl cAMP for 10 days, were dissociated and added toa culture of muscle cells that had been cultured for 20 days. The me- dium used to assay cells for synapses consisted of MEM minus so- dium bicarbonate, 15 mM N-2-hydroxyethylpiperazine-N’-2- ethanesulfonic acid (Hepes), pH 7.1, 8.0 mM CaCl, 260 uM choline chloride, 1 mM dibutyryl cAMP, 0.1 mM hypoxanthine, 16 uM thymidine, and 33 mM dextrose in a humidified atmosphere of 5% COo-95% air. (B) Intracellularly recorded responses of the muscle cell labeled M shown in panel A to action potentials electrically elicited in the NG108-15 hybrid cell labeled N. The resting mem- brane potential of the muscle cells was ~50 mV. Traces 1-4: Each high gain trace shown represents 5-10 superimposed oscilloscope sweeps showing 1 muscle response per set of sweeps. Trace 5: An example of an NG108-15 action potential that elicited a response such as those shown in traces 1-4. Traces 1-5 are aligned so that temporal relationships between the neuron action potential and muscle response are shown. Trace 6: Current pulse used to elicit an NG108-15 action potential. The hybrid cell was stimulated three times per sec. The resting membrane potential of the hybrid cell was ~30 mV; the membrane potential was adjusted to —75 mV with constant current. (C) Action potential of a muscle cell elicited by electrical stimulation of the muscle cell (0.4 nA, 40 msec). Rest- ing membrane potential was —50 mV. (D) Penwriter recordings from a muscle cell obtained with intracellular electrodes (upper record) and from an NG108-15 cell with an intracellular electrode (lower record) showing spontaneous muscle events and muscle re- sponses to NG108-15 action potentials elicited by repetitive elec- trical stimulation of the hybrid cell. The largest muscle responses in the second set of evoked responses are action potentials atten- uated by the penwriter frequency response. plitude distribution similarly skewed towards the lower am- plitudes, but with a wide range of response amplitudes. d-Tubocurarine inhibits cell responses to acetylcholine that are mediated by nicotinic acetylcholine receptors and thus would be expected to inhibit muscle responses to acetyl- choline released by NG108-15 cells. The effect of d-tubocu- rarine is shown in Fig. 8. When the tip of a pipette 10 um in   Biochemistry: Nelson et al. ft Dp N o a oO a NUMBER OF RESPONSES a     ° 2 467-8 101214 RESPONSE AMPLITUDE (amv) Fic. 2. Muscle response amplitude (A mV) to NG108-15 ac- tion potentials plotted as a function of the number of muscle re- sponses observed. A total of 456 action potentials were evoked in NG108-15 by electrical stimulation; 215 muscle responses were ob- served. The mean response amplitude (x) is 4.3 mV. The resting membrane potential of the muscle cell was —44 mV. diameter that contained 6 X 10-5 M d-tubocurarine dis- solved in DMEM was positioned near the site of the synapse, the frequency and amplitudes of muscle responses to NG108-15 action potentials were reduced greatly, whereas little or no change (<2 mV) was observed in the membrane potentials of muscle and hybrid cell or in NG108-15 action potentials. Muscle responses returned when the pipette was withdrawn; thus the inhibition was reversible. As shown in Fig. 3B, the muscle response rate was not affected when the tip of a control pipette that contained only DMEM was placed near the site of the synapse. In Fig. 3C a graded inhi- bition of muscle response was obtained by varying the dis- tance between the tip of the d-tubocurarine pipette and the site of the synapse. Perfusion studies (10~6 M) also show that d-tubocurarine inhibits muscle responses evoked by NG108-15 action potentials as well as spontaneous muscle L__STC PIPETTE j Lh i\" ; | nan | Ti |     edu aTC PIPETTE CONTROL PIPETTE j L l l 10 mV GRADUAL INTERMEDIATE CLOSE WITHDRAWN ant APPROACH —- POSITION APPROACH Fic. 3. Inhibition by d-tubocurarine of muscle responses evoked by NG108-15 action potentials. (A) The tip of the pipette containing 6 X 10-5 M d-tubocurarine chloride (dTC) dissolved in DMEM (the cells were also in DMEM) was placed near the site of the NG108-15 synapse with the muscle cell shown in Fig. 1A while the hybrid cell was stimulated three times/sec to evoke action po- tentials. The duration of the application of d-tubocurarine is indi- cated by the bar above the tracing. The muscle resting membrane potential was -50 mV. (B) Recording obtained from the same muscle cell as in panel A after recovery from exposure to d-tubocu- rarine was almost complete. A control pipette containing medium without d-tubocurarine was placed in the same position as the d- tubocurarine pipette of panel A. The amplitudes of the muscle events were essentially normal. (C) Experiment with a different NG108-15 muscle cell showing a graded reversible inhibition of muscle responses by d-tubocurarine chloride which depended on the proximity of the tip of the pipette containing the d-tubocurar- ine solution to the site of synapse between the hybrid and muscle cells. The position of the d-tubocurarine pipette tip is indicated below the tracing. The muscle resting membrane potential was —48 mV. Proc. Nat. Acad. Sci. USA 73 (1976) 125 events, and that the inhibition by d-tubocurarine is revers- ible. The inhibition of spontaneous muscle events by d-tubo- curarine indicates that spontaneous events were of synaptic origin. To test the relation between the amplitude of muscle re- sponses and the membrane potential of the muscle cell, con- stant current was passed across the muscle cell membrane through the recording microelectrode The amplitudes of muscle responses evoked by hybrid cell action potentials were increased by steady polarizing currents which in- creased the muscle membrane potential, and were decreased by steady depolarizing currents. The muscle responses changed in the directions expected for membrane polariza- tion reactions mediated by nicotinic acetylcholine receptors; however, the nonlinearity of the muscle membrane resis- tance precluded a meaningful estimate of the reversal po- tential for the muscle response. Further experiments are needed to clarify this important point. During the course of these studies, exploratory experi- ments were performed to determine the effect of culture and assay conditions upon the abundance of synapses (Table 1). Increasing the CaCl, concentration from 2.7 to 8 mM did not appreciably affect the frequency with which synapses were detected. Synapses were found with 20% of the cell pairs tested when the following conditions were used. NG108-15 cells were grown in the presence of 1 mM dibuty- ryl cAMP, purified as described in Materials and Methods, for 2 weeks, and muscle cells were grown for approximately 9 days as described in Materials and Methods. Hybrid and muscle cells were then cocultured for an additional 8-15 days in 90% DMEM, 10% horse serum, 1 mM purified di- butyryl cAMP, 0.1 mM hypoxanthine, 0.016 mM thymidine and were assayed for synapse formation in this medium without serum modified as follows: 3.8 mM CaCle, 270 uM choline chloride, and 20 4M eserine sulfate. Increasing the choline concentration to 270 uM (124 uM in more recent ex- periments) when assaying for synapses may be helpful since the intracellular choline concentration may limit the rate of acetylcholine synthesis, whereas growth of cells in the pres- Table 1. Effect of growth and assay conditions on synapse frequency     Cell * * airs growth Synapse assay Synap- Cell with Choline Choline CaCl, ses pairs synapse uM uM mM found tested (%) 6t 6T 1.8 0 54 0 24 6t 1.8 1 12 8 24 6t 3.8 4 45 9 24 6t 8.0 1 15 7 24 260t 8.0 2 8 25 24 24 3.8 1 1 24 270 3.8 21 104 20 270 24 3.8 0 15 0 270 270 3.8 1 35 3 Total 31 289 11%   *The medium was DMEM, except where specified, plus the com- ponents described under Materials and Methods. + MEM medium adjusted to 33 mM dextrose and 44 mM sodium bicarbonate plus the components described under Materials and Methods. {MEM with 15 mM Hepes, pH 7.1, and 33 mM dextrose without sodium bicarbonate plus the components described under Mate- rials and Methods. 126 Biochemistry: Nelson et al. ence of 270 uM choline may decrease the number of synaps- es that are present. These results suggest that both synapse formation and the efficiency of transmission across the sy- napse are regulated by choline or other media components and that synapse frequency and efficiency are regulated by different processes. DISCUSSION In previous studies, neuroblastoma X glioma NG108-15 hy- brid cells were shown to synthesize, store, and excrete ace- tylcholine and to generate action potentials in response to electrical stimulation*, whereas striated muscle cells have abundant nicotinic acetylcholine receptors. Thus, the com- ponents that are known to be required for synaptic commu- nication between the two cell types can be expressed in vitro. In this report, we show that clonal NG108-15 cells do, indeed, form chemical synapses with striated muscle cells. The evidence for synapse formation is based upon the fol- lowing observations: (a) action potentials in NG108-15 hy- brid cells elicit muscle responses after a variable delay; (b) d-tubocurarine, an inhibitor of the nicotinic acetylcholine receptor, inhibits muscle responses reversibly; and (c) muscle response amplitudes increased when the muscle membrane potential was adjusted with constant hyperpolarizing cur- rent, and decreased, when adjusted with constant depolariz- ing current, as expected for reactions that are mediated by the nicotinic acetylcholine receptor. Muscle responses to NG108-15 action potentials of the type shown in Fig. 1 were shown not to be due to direct electrical coupling between muscle and hybrid cells. We have considered a more complex electrical circuit involving a second muscle cell electrically coupled to both the NG108-15 and the muscle cell recorded from but reject this interpretation because it is not in accord with the findings that the incidence of direct electrical coupling between hy- brid and muscle cells is low (<1% of the cell pairs tested) compared to the higher incidence of synapse formation (11% of the 276 cell pairs tested and, in some cultures, 20- 25% of the cell pairs examined). In addition, the inhibition of muscle responses by d-tubocurarine agrees well with re- ceptor-mediated synaptic communication, but not with an electrical coupling mechanism of communication. Electrical connections between NG108-15 and muscle cells, although infrequent, represent a second mode of communication be- tween cells and deserve further study. According to the quantal hypothesis (8), a neuron action potential may induce the release of acetylcholine in packets of transmitter which may correspond to one or more storage vesicles and thereby simultaneously activate many muscle acetylcholine receptors. If the release mechanism obeys Poisson statistics, the mean number of vesicles that release their contents per action potential (the mean quantal re- sponse) can be calculated from either the number of neuro- nal action potentials that fail to elicit muscle responses or the coefficient of variation of muscle response amplitudes (9- 11). There was excellent agreement between the values ob- tained by the two methods of calculation with the four NG108-15 synapses analyzed in this way, with the mean number of vesicles released per NG108-15 action potential ranging from approximately 0.1 to 0.5. The low quantum content indicates that most of the muscle responses observed were due to the release of acetylcholine from a single vesi- cle. The mean amplitude of the muscle responses ranged from 0.5 to 5.0 mV; in all cases studied the range of ampli- Proc. Nat. Acad. Sci. USA 73 (1976) tudes was large. These observations may be due to the pres- ence in NG108-15 cells of both clear vesicles 600 A in diam- eter and dense core neurosecretory vesicles 1800 A in diame- ter (6). Whether one or both types of vesicles contains ace- tylcholine and releases this compound following an NG108- 15 action potential remains to be determined. Early in the development of synapses between normal mammalian motor neurons and striated muscle cells, a single muscle cell is innervated by more than one neuron (12, 13), whereas at a later developmental stage, only one neuron sy- napses with a single muscle cell. The efficiency of synaptic communication during the early stage in synapse formation is low, and most muscle responses are below the threshold for activation of action potentials (12-14). At a later stage in the development of the synapse the efficiency of transsynap- tic communication becomes 100% since every neuronal ac- tion potential elicits muscle cell contractions. The synapses formed between NG108-15 hybrid cells and striated muscle cells closely resemble those formed between normal motor neurons and striated muscle cells during the early stage of synapse development. The frequency of synapse formation between hybrid and muscle cells equals or exceeds the sy- napse frequency reported for normal dissociated spinal cord neurons and striated muscle cells in vitro (15). The efficien- cy of synaptic transmission might be affected by many fac- tors. For example, high concentrations of nicotinic acetyl- choline receptors have been found at points of contact be- tween neuroblastoma and striated muscle cells (16); how- ever, similar receptor “hot spots” are found on muscle cells in the absence of neuroblastoma cells (17, 18). Establishment of synapses between nerve and muscle poses a paradox, for on the one hand, muscle movements are highly coordinated, which suggests that neuromuscular sy- napses and other synapses in the neural circuits are assem- bled with high precision, whereas the demonstrated ability of autonomic neurons of the vagus (19), sympathetic gangli- on neurons (20), and clonal NG108-15 hybrid cells to sy- napse with striated muscle cells suggests that functional sy- napses can form that may not be dependent upon highly specific cell recognition molecules. Although most striated muscle cells in mammals are innervated by spinal motor neurons, it is interesting to note that the esophagus contains striated muscle cells that normally are innervated by auto- nomic neurons of the vagus. The molecular nature of the cell interactions that lead to synapse formation is not known. It is clear that NG108-15 cells adhere firmly to muscle cells; however, the hybrid cells also adhere to one another, to fibroblasts, to other cell types, and to the polystyrene petri dish. Whether NG108-15 hy- brid cells have specific cell recognition molecules that are required for synapse formation remains to be determined. Since NG108-15 hybrid cells are clonal cells and under ap- propriate culture conditions synapses formed between 20 and 25% of the hybrid-muscle cell pairs examined, the sim- plest hypothesis is that NG108-15 may constitute one class of cells with respect to synapse formation, and muscle cells, an- other class, and that any sufficiently well differentiated NG108-15 cell may be able to form synapses with any mus- cle cell in the culture. If specific cell recognition molecules are required to establish synapses between NG108-15 hybrid cells and mouse striated muscle cells, it seems likely that few kinds of recognition molecules may be required to establish synaptic connections during the early stage of development. Synapses between NG108-15 and muscle cells resemble in various ways the early form of the normal neuromuscular Biochemistry: Nelson et al. synapse. The results suggest that synapse formation and the efficiency of transmission are regulated in vitro, apparently by independent processes. It may be possible to find condi- tions for the conversion of the early form of synapse to the late, mature form. It also seems probable that NG108-15 cells may synapse with clonal muscle cells, since normal spi- nal cord neurons reportedly synapse with clonal muscle cells (21). Clearly, the ability of NG108-15 hybrid cells to form synapses affords many opportunities to correlate biochemi- cal aspects of synaptogenesis with developmental and elec- trophysiologic aspects and to explore the molecular basis for the specificity of synaptic connections between cells. We thank Doyle Mullinex and Phyllis Bullock for expert assis- tance with the cell cultures. 1. Augusti-Tocco, G. & Sato, G. (1969) Proc. Nat. Acad. Sci. USA 64, 311-315. 2. Schubert, D., Humphreys, S., Baroni, C. & Cohn, M. (1969) Proc. Nat. Acad. Sct. USA 64, 316-323. 3. Minna, J., Nelson, P., Peacock, J., Glazer, D. & Nirenberg, M. (1971) Proc. Nat. Acad. Sci. USA 68, 234-239. 4. Minna, J., Glazer, D. & Nirenberg, M. (1972) Nature New Biol. 235, 225-231. 10. ll. 12. 18. 14. 15. 16. 17. 18. 19. 20. 21. Proc. Nat. Acad. Sci. USA 73 (1976) 127 Amano, T., Hamprecht, B. & Kemper, W. (1974) Exp. Cell Res. 85, 399-408. Daniels, M. P. & Hamprecht, B. (1974) J. Cell Biol. 63, 691- 699. Nelson, P. G., Peacock, J. H., Amano, T. & Minna, J. (1971) J. Cell. Physiol. 77, 337-352. Katz, B. (1969) The Release of Neural Transmitters (C. C Thomas, Springfield, IIl.). del Castillo, J. & Katz, B. (1954) J. Physiol. (London) 124, 560-573. Martin, R. (1966) Physiol. Rev. 46, 51-66. Robbins, N. & Yonezawa, T. (1971) J. Gen. Physiol. 58, 467- 481. Redfern, P. A. (1970) J. Physiol. (London) 209, 701-709. Bennett, M. R. & Pettigrew, A. G. (1974) J. Physiol. (London) 241, 515-545. Diamond, J. & Miledi, R. (1962) J. Physiol. (London) 162, 393-408. Fischbach, G. (1972) Dev. Biol. 28, 407-429. Steinbach, J. H., Harris, A. J., Patrick, J., Schubert, D. & Heinemann, S. (1973) J. Gen. Physiol. 62, 255-270. Sytkowski, A. J., Vogel, Z. & Nirenberg, M. W. (1973) Proc. Nat. Acad. Sci. USA 70, 270-274. Fischbach, G. D. & Cohen, S. A. (1973) Dev. Biol. 31, 147- 162. Landmesser, L. (1971) J. Physiol. (London) 213, 707-725. Nurse, C. A. & O’Lague, P. H. (1975) Proc. Nat. Acad. Sci. USA 72, 1955-1959. Kidokoro, Y. & Heinemann, S. (1974) Nature 252, 593-594.", "Nirenberg, Marshall W. ; Nelson, Phillip G. ; Christian, Clifford", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-vbj9~rhdv_6qq3", "00000000-0000-0000-FCC3-75C98934BD66", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Localization of Acetylcholine Receptors During Synaptogenesis in Retina", "101584910X59", null, "1976", "June 1976", "Working with neuroreceptors synthesized in chick embryo retina before synapses appear, this article presents findings that dissociated retina cells have the ability to synthesize receptors and extend neurites from cell bodies.  These cultured retina cells were extremely valuable to researchers, as in subsequent studies they were used as a model system for explaining the process of synapse formations.", "Articles", "Receptors, Cholinergic ; Synapses", "Neuroblastoma Research, 1967-1976", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 1806-1810, June 1976 Biochemistry Localization of acetylcholine receptors during synaptogenesis in retina (a-bungarotoxin/neuron development/cultured neurons) ZVI VOGEL AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, January 9, 1976 ABSTRACT __ Nicotinic acetylcholine receptors are synthe- sized in chick embryo retina before synapses appear. Most of the receptors are found associated with neurites in the inner synaptic layer of the retina; later in development the receptors appear in the outer synaptic layer. . Cells dissociated from retina and cultured in vitro form ag- gregates and also sort out into regions comprised either of neurites or cell bodies. Most of the nicotinic acetylcholine re- ceptors of aggregates are found associated with neurite regions. The receptor distribution of cultured retina cells thus resembles that of intact retina.   125]-Labeled a-bungarotoxin, a specific ligand of the nicotinic acetylcholine (AcCh) receptor, was used to detect AcCh re- ceptors in the developing chick embryo retina. This species of AcCh receptor is a synaptic marker, since the AcCh receptors of striated muscle cells (1, 2) and certain parasympathetic neurons (3) are found in abundance only at the site of the sy- napse. After denervation, the receptors are found over the entire surface of the cell. Most of the recent information regarding the nicotinic AcCh receptor has been obtained with the receptors of the electric organ of fish, and striated muscle (for reviews see refs. 4-6). a-Bungarotoxin has been used to detect AcCh receptors in sympathetic ganglia (7) and brain (8-10), but the nicotinic AcCh receptors of retina have not been studied in this manner. The vertebrate retina is a model system for studies on synaptic communication, since relatively few types of neurons are present and cells dissociated from retina form synapses in vitro (11, 12). The retina contains acetylcholine and enzymes that catalyze the synthesis and the hydrolysis of this compound (13-17). In addition, electrophysiologic studies show that some neurons in retina respond to acetylcholine and that both nico- tinic and muscarinic AcCh receptors are present in the retina of certain organisms (18-22). In this report, the concentration of nicotinic AcCh receptors and their location in chick embryo retina are described as a function of the developmental age of the retina. MATERIALS AND METHODS Assay of Homogenates for !25I-Labeled a-Bungarotoxin Binding. Neural retinas from white leghorn chick embryos were dissected in cold Dulbecco’s phosphate-buffered saline (Gibco) and then homogenized in 0.05 M Tris-HCl, pH 7.4, in a ground glass Duall homogenizer (Kontes) using 0.25-1.25 ml of buffer per retina so that the final concentration of protein was 3-6 mg/ml. Homogenates were frozen immediately and stored at —191°; binding sites for a-bungarotoxin (aBT) were stable for at least 1 year. a-Bungarotoxin labeled with 2 atoms of }25] per molecule of toxin (!>I-aBT) was prepared as de-   Abbreviations: aBT, a-bungarotoxin; !251-aBT, a-bungarotoxin labeled with 2 atoms of !25I per toxin molecule; AcCh, acetylcholine. 1806 scribed previously (23). Stock solutions of !I-aBT contained 0.25-0.5 uM !5]-aBT; 3 mM sodium phosphate buffer, pH 7.5; 150 mM NaCl; and 2 mg of crystalline bovine serum albumin per ml. Each binding reaction mixture contained the following components in a final volume of 0.1 ml: 10 nM !*]-aBT (2-4 ul of a stock solution); 0.05 M Tris-HCl, pH 7.4; 0.2 mg of serum albumin; and 0-180 ug of retina homogenate protein. Reaction mixtures were incubated for 30 min at 37°, diluted with 3 ml of solution C (0.05 M Tris-HCl, pH 7.4, and 2 mg of serum al- bumin per ml) at 8°, and immediately filtered (vacuum) through a wet cellulose acetate filter (Millipore, EGWP 25 mm in diameter, 0.2 um pore size). The filter was washed four times with 3 ml portions of solution C, the filter was dissolved in 10 ml of Instabray (Yorktown Research), and radioactivity was determined. Zero time values (100-200 cpm, approximately 0.25 fmol of }25J-aBT) were subtracted from the values shown. The amount of !2]-aBT bound was proportional to protein concentration in the range studied (0-180 yg of protein). Pro- tein was determined by modification of the method of Lowry et al. (24). Autoradiography of '%5J-aBT Bound to Intact Retina. Neural retina was dissected in cold solution A [the Dulbecco- Vogt modification of Eagle’s medium (Gibco Catalog no. H-21) with 20 mM Hepes (N-2-hydroxyethylpiperazine-N’-2-eth- anesulfonic acid), pH 7.4, instead of NaHCOs, and 2 mg of serum albumin per ml of medium]. Pieces of retina were in- cubated for 60 min at 37° in 0.5-1.0 ml of solution A with 10 nM !25]-aBT and then were washed five times with 5 ml of cold solution A and twice with solution A without serum albumin. The tissue was fixed with 10% (vol/vol) neutral formalin or 2.5% (wt/vol) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and embedded in paraffin. Serial sections, 8 um thick, were mounted on glass slides, and the paraffin was removed; the slides were coated with Kodak NTB-2 emulsion diluted 1:1 with water. Slides were cooled to gel the emulsion and stored at 4° in the dark. Autoradiographs were developed for 4 min at 20° with Kodak D-19 developer diluted 1:1 with water and fixed for 5 min with Kodak F5. The slides were rinsed with water; some were stained with 0.05% toluidine blue in 0.66 M borax. Retina Cell Cultures. Cells were dissociated from retinas of 8-day-old chick embryos and aggregated in vitro by a modi- fication of the method described by Sheffield and Moscona (11). Single cells, dissociated from retina with 0.25% trypsin (three times crystallized, Worthington) and 50 ug/ml of DNase I (Worthington), were cultured in rotating flasks (80 rpm) in a 37° incubator in a humidified atmosphere of 5% CO2-95% air. Each flask contained 9 X 108 cells and 1.5 ml of medium (80% Eagle's Basal Medium and 20% fetal bovine serum). The me- dium was changed every other day. After 7 days 10 nM !?*I- aBT was added to the aggregates in the rotating flasks. One hour later the aggregates were collected and washed four times   Biochemistry: Vogel and Nirenberg           Q = 5 5 a Z 25+ Pa 2 © 20F x 5 9 15} 2 a & 10 ° Bs o Oo bee 87] 2 0 6% 30 ~~«90 = MINUTES Fic. 1. The rate of !25I-aBT binding. Portions of a 2-week-old chick retina homogenate (62.2 ug of protein per reaction mixture) were incubated with 1 nM !25]-aBT with or without 2 »M unlabeled aBT at the temperatures and times specified. with growth medium by centrifugation, and three times with growth medium without serum. The aggregates were fixed with glutaraldehyde, embedded in paraffin, sectioned, and subjected to autoradiography as described above. RESULTS AND DISCUSSION 125].oBT Binding. The rate of binding of !*I-aBT to re- ceptors in a homogenate of chick retina is shown in Fig. 1. Maximum !5]-aBT binding was obtained after 15 min of in- cubation at 37°; rates of binding were lower at 19° and 0°. Nonspecific binding of !?5-aBT in the presence of a large ex- cess of unlabeled aBT (2 uM) was relatively low. The relation between !*I-aBT concentration and the amount of labeled toxin bound is shown in Fig. 2. The amount of 125].¢BT bound increased sharply as the concentration of 1235]_aBT was elevated from 1 to 7.5 nM. Higher concentrations of 125]-@BT (10-40 nM) increased binding of labeled aBT only slightly. Little nonspecific binding of !*I-aBT was observed in the presence of 2 uM unlabeled aBT. The number of specific aBT binding sites in chick retina, 2 weeks after hatching, esti-             oO 02 03       0 fmol DIIODO-a-BUNGAROTOXIN BOUND 10 20 30 40 DIIODO-e-BUNGAROTOXIN (nM) Fic. 2. Relation between 125{-diiodo-aBT concentration and the amount of labeled toxin bound. Portions of a 2-week-old chick retina homogenate (62.2 ug of protein per reaction mixture) were incubated for 5 min at 20°, in the absence (O), or presence (@), of 2 uM unlabeled aBT. !25]-Labeled a-bungarotoxin then was added at the concen- trations specified and the reaction mixtures were incubated and as- sayed for bound radioactivity as described in Materials and Methods. A Scatchard plot of specific !25I-aBT binding; i-e., binding obtained after subtracting the radioactivity bound in the presence of 2 uM unlabeled toxin, is shown in the insert; the ordinate refers to the bound/free }25I-aBT ratio; the abscissa corresponds to nM toxin bound per 62.2 ug of protein. Proc. Natl. Acad. Sci. USA 73 (1976) 1807         x 1004 Zz x< © got O ow OOF Zz Z 40 & ?. Py oq 20+       0-9 6-8 7 -6 -§ -4 -3 LOG MOLARITY Fic. 3. Inhibitors of }25I-aBT binding. Portions of a 2-week-old chick retina homogenate (50 wg of protein per 80 ul of reaction mix- ture) were incubated for 5 min at 20° in the presence of the com- pounds indicated. }75I-aBT (10 nM) then was added (the final volume was 100 ul) and tubes were incubated for an additional 10 min at 20°. The final concentration of each compound is indicated on the abscissa. Tubes with acetylcholine also contained 3 uM eserine sulfate. One hundred percent on the ordinate corresponds to 6.25 fmol of !I-oBT specifically bound in the absence of inhibitor. This value was 35% of that obtained after 30 min of incubation at 37°. Abbreviations rep- resent the following: a-BT, unlabeled a-bungarotoxin; NIC, nicotine; d-TC, d-tubocurarine; ACH, acetylcholine plus 3 1M eserine sulfate; CARB, carbamylcholine; ATR, atropine; ESE, eserine sulfate; PILO, pilocarpine. mated from a Scatchard plot (Fig. 2, insert), is 525 fmol/mg of protein. The (!251-aBT-receptor) complex dissociates in a bi- phasic manner in the presence of 2 uM unlabeled aBT (50% dissociation in 30 min at 37°). Slower rates of dissociation were observed at 20° and 0° (data not shown). Receptor Specificity. Nicotinic and muscarinic AcCh re- ceptors can be distinguished by differences in affinity for li- gands. In Fig. 3, the effects of various ligands upon !*I-aBT binding are shown. Binding was inhibited 50% by 1 nM unla- beled aBT, 0.3 uM nicotine, and 1 »M d-tubocurarine, each compound known to have a high affinity for nicotinic AcCh receptors. Fifty percent inhibition of toxin binding was obtained with 6 uM acetylcholine (3 uM eserine sulfate also was present, a concentration without effect on aBT binding), and with 100 uM carbamylcholine. Compounds that interact preferentially with muscarinic AcCh receptors, such as pilocarpine and at- ropine, either did not affect }25I-aBT binding or inhibited binding only at relatively high concentrations. Putative neu- rotransmitters such as L-glutamic acid, glycine, y-aminobutyric acid, dopamine, and [-norepinephrine did not affect «BT binding at 10-1000 uM; however, serotonin stimulated aBT binding 15%. Choline (100 4M) inhibited !>I-aBT binding (data not shown). AcCh Receptors During Embryonic Development. The amount of bound !25]-q@BT is shown in Fig. 4 as a function of the developmental age of chick retina. Under the conditions used (10 nM !25]-@BT, 30 min incubation at 37°) approximately 80% of the receptors bind aBT. Specific binding sites for aBT are present in low concentration (7.5 fmol/mg of protein) in chick retina on the 6th embryonic day. The concentration of receptors increases 10-fold between the 6th and the 13th em- bryonic day; however, >80% of the receptors are synthesized between the 13th and last embryonic day during the period of retina synapse formation. These results show that the receptors 1808 Biochemistry: Vogel and Nirenberg   —           06+ 3 ? | zit g é 5 oO 4 & od 2 ~ z 5 1s 5 | 2 o z a Z Z 0 <x O2t ae o 3 e Ee E a a a 4 0 - 250° IN OVO DAYS POST HATCHING Fic. 4. Amount of !251-aBT bound as a function of the develop- mental age of chick retina. Filled symbols (@) represent pmol of toxin bound specifically per mg of protein; open symbols (©) represent pmol of toxin bound specifically per retina. Each point is the mean of 18 to 60 values (3 to 10 retina homogenates; six concentrations of protein were assayed from each homogenate). Specific toxin binding is shown; nonspecific binding has been subtracted. are synthesized before synapses appear and suggest that ces- sation of both receptor accumulation and synapse formation may be coupled. The maximum receptor concentration is at- tained at the time of hatching (400 fmol of }25I-aBT bound per mg of protein). Almost the same receptor concentration is found in adult retina; however, the number of specific binding sites for aBT per adult retina (2700 fmol per retina) is twice that of the newly hatched chick (1350 fmol per retina). Thus, adult retina either has more synapses, more receptors per synapse, or more extrajunctional receptors than the retina of the newly hatched chick. Adult rabbit retina bound 107 fmol of }5]-aBT per mg of protein under the same conditions. Receptor Distribution in Retina. The distribution of nico- tinic AcCh receptors in retina was determined by autoradi- ography. Adult retina was incubated with '**I-aBT, fixed, sectioned, and either stained with toluidine blue or subjected to autoradiography as shown in Fig. 5A and B, respectively. Cell bodies and neurites rich in synaptic connections are found in separate layers (Fig. 5A). The upper layer consists of photore- ceptor cells; next is the outer plexiform layer which consists of neurites and synapses; next is a layer of cell bodies, the inner nuclear layer; and then there is a wide layer of synaptic con- nections and neurites, the inner plexiform layer. The lowest layer of cell bodies is composed of ganglion neurons; the last layer consists of ganglion neuron axons. The autoradiographs show that most of the binding sites for 125]_qBT are located in the inner plexiform layer, which consists primarily of synaptic connections between processes of bipolar, amacrine, and ganglion neurons. Four horizontal bands with relatively high concentrations of silver grains can be distin- guished within the inner plexiform layer. Silver grains also are found in the outer plexiform layer, which contains synapses between photoreceptor, horizontal, and bipolar neurons. Rel- atively few grains are associated with cell body layers, or with Proc. Natl. Acad. Sci. USA 73 (1976)   Fic. 5. Section of an 8-month-old chicken retina. (A) Tolui- dine-blue-stained section, not subjected to autoradiography. (B) The corresponding field in an unstained serial section, subjected to au- toradiography for 110 days. The initial specific activity '*]-aBT was 335 Ci/mmol of toxin. Bright field views are shown in both panels; the bar represents 25 um. Abbreviations of retina layers are as follows: NF, nerve fiber layer, GC, ganglion cell layer, IP, inner plexiform layer (i.e., inner synaptic layer); IN, inner nuclear layer, OP, outer plexiform layer (i.e., outer synaptic layer); IS and OS, inner and outer segments of photoreceptor cell layer, respectively. s axons of ganglion neurons. }25]-«-Bungarotoxin does not bind to pigment cells, choroid, pecten, or sclera. Autoradiographs of sections of rat and rabbit retina are shown in Fig. 6. Again, most of the silver grains are associated with the inner plexiform layer. Some putative ganglion and amacrine neurons are labeled in rat retina (Fig. 6A); however, the outer plexiform layer of the rat is essentially unlabeled. In rabbit retina (Fig. 6B and C) silver grains are found in both the inner and outer plexiform layer. Approximately 10-20% of the rabbit ganglion cell bodies and some cell bodies in the lower portion of the inner nuclear layer, presumably amacrine neurons, are   Fic. 6. Autoradiography of sections of adult rat and rabbit retina labeled with !25I-aBT. (A) Bright field view of a stained section of Fisher rat retina subjected to autoradiography for 23 days. The initial specific activity of !251-aBT was 260 Ci/mmol of toxin. (B) and (C) Phase contrast views of stained sections of New Zealand white rabbit retina subjected to autoradiography for 27 days; the initial specific activity of 1251-aBT was 220 Ci/mmol of toxin. The bar represents 25 pm. Biochemistry: Vogel and Nirenberg   Fic. 7. Autoradiography of sections of 6- to 17-day-old chick embryo retina, labeled with }75I-aBT. Retina sections were subjected to autoradiography for 29 days. The initial specific activity of 51-aBT was 485 Ci/mmol of toxin. The same magnification was used for each photomicrograph; the bars represent 25 um. (A) Phase contrast view of a stained autoradiograph of 6-day-old embryo retina. (B) Bright field view of the area shown in panel A before staining. (C) Phase contrast view of a stained autoradiograph of 9-day-old embryo retina. (D) Bright field view of the same area shown in panel C before stain- ing. (E) Bright field view of a stained section of 13-day-old embryo retina not subjected to autoradiography. (F) Bright field view of the corresponding area of an unstained autoradiograph of an adjacent section. (G) and (H) 17-Day-old embryo retina sections, treated as in panels FE and F, respectively. labeled. Some cells in the outer synaptic layer, thought to be horizontal neurons, also are labeled. The concentration of ganglion neurons is considerably higher in the center of the eye compared to the periphery; however, the concentration of silver grains in the inner plexiform layer does not change markedly (<2-fold). Three types of control experiments were performed. First, incubation of chicken retina with 200 uM d-tubocurarine or 1 uM unlabeled aBT reduced the amount of !25]-aBT bound to retina by 85% and 95%, respectively, and the few silver grains found after autoradiography were randomly distributed. Sec- ond, autoradiography of 0.5 xm thick, Epon-embedded sections of chick retina revealed the same silver grain distribution as that of 8 um paraffin sections described above. Since Epon sections have an essentially flat surface coated with the autoradiograph emulsion, the grain distribution does not result from surface irregularities of sections after paraffin has been removed. Third, the concentration of specific !1-aBT binding sites in intact retina is approximately the same as that of homogenates pre- pared from sister retinas, which suggests that most binding sites in intact retina are accessible in the labeled toxin. Embryonic chick retinas at various stages of development were labeled with !25I-aBT and sections were subjected to autoradiography. Pairs of photomicrographs, one emphasizing retina structure, the other silver grain distribution, are shown in Fig. 7 for the 6th, 9th, 13th, and 17th embryonic days. The first neurons to appear are ganglion neurons which, by the 6th embryonic day, are found ina layer next to the margin of the retina (25, 26). Autoradiographs of retina on the 6th embryonic day (Figs. 7A and B) show that !*5]-aBT binds to ganglion neurons but not to most neuroblasts. Coulombre (25) has re- ported that the inner and outer plexiform layers of chick retina appear on the 8th and 9th embryonic days, respectively. 125}_«-Bungarotoxin binds predominantly to the inner plexi- form layer of 9-, 13-, and 17-day-old chick embryo retina. The receptors were not found in appreciable numbers in the outer synaptic layer until the 17th embryonic day. Proc. Natl. Acad. Sci. USA 73 (1976) 1809   FIG. 8. Sections of an aggregate formed from dissociated 8- day-old chick embryo retina cells, cultured in vitro for 7 days. (A) Toluidine-blue-stained section, not subjected to autoradiography, phase contrast view. (B) Bright field view of a stained autoradiograph exposed for 36 days. The initial specific activity of !*I-aBT was 300 Ci/mmol of toxin. The bar represents 50 um. The AcCh receptors are distributed fairly uniformly throughout the inner synaptic layer on the 9th and 11th em- bryonic days; by the 17th embryonic day 2 horizontal bands of receptors were observed in some autoradiographs, and in the adult, four diffuse bands of receptors can be seen within the inner synaptic layer. Acetylcholinesterase activity first appears in the 4-day-old chick embryo retina associated with ganglion neurons and later with amacrine neurons (13, 17, 18); after hatching four horizontal bands of acetylcholinesterase activity also are present in the inner synaptic layer of chick retina (17, 18). On the 13th embryonic day retina cells with abundant AcCh receptors were found which resemble amacrine neurons. Synaptic connections first appear in chick retina on the 13th embryonic day (27-29). Thus genes for nicotinic AcCh recep- tors are expressed in some retina cells at least 7 days before sy- napses appear. Striated muscle cells also synthesize nicotinic AcCh receptors in the absence of neurons (28, 30, 31) and after denervation (2, 32, 33). Both rod and cone photoreceptor cells of the turtle synthesize acetylcholine (16). In chick retina, nicotinic AcCh receptors are associated with dendrites of ganglion neurons and cells which resemble amacrine neurons. Our working hypothesis is that acetylcholine is a transmitter of bipolar neurons, and that some amacrine and ganglion neuron responses to bipolar neurons are mediated by nicotinic AcCh receptors. Further work also is needed to determine whether nicotinic AcCh receptors in the outer plexiform layer of chick and rabbit, but not rat, retina are synthesized by certain horizontal and/or bipolar neurons. The relatively high concentrations of nicotinic AcCh re- ceptors in the synaptic layers of chick retina and the low con- centrations of receptors associated with cell bodies and axons of ganglion neurons suggest that nicotinic AcCh receptors of retina may be localized at certain synaptic sites much as AcCh receptors are localized at the neuromuscular synapse (1, 2) and at certain synapses of parasympathetic neurons (3). Preliminary results with an immunochemical assay for detecting nicotinic AcCh receptors at the electron microscope level (34) suggest that most of the receptors are not randomly distributed in the inner synaptic layer but instead are found in small areas which contain relatively high concentrations of receptors. Retina Cell Cultures. Neural retina tissue from 8-day-old chick embryos was dissociated into single cells and the cells then were cultured in rotating flasks under conditions that favor aggregation as described by Sheffield and Moscona (11). A section of a cell aggregate cultured for 10 days is shown in Fig. 8A. Cell bodies and processes were found to sort out into discrete regions which resemble the layers of cell bodies and processes of the intact retina. A similar section of an aggregate which was incubated with !251-aBT and subjected to autoradiography is shown in Fig. 8B. Labeled toxin bound primarily to the neu- 1810 Biochemistry: Vogel and Nirenberg rite-rich regions; considerably less toxin was associated with cell bodies, These results show that dissociated retina cells form aggregates, synthesize nicotinic AcCh receptors, and extend neurites which together with AcCh receptors sort out from cell bodies. Cultured retina cells can be used as a model system for studies related to the process of synapse formation. At least three types of synapses form in vitro (11, 12), and the concentration of synaptic connections synthesized in vitro almost equals that found in the intact retina (35). eonr 10. 11. 12. 13. Miledi, R. (1960) J. Physiol. 151, 24-30. Axelsson, J. & Thesleff, S. (1959) J. Physiol. 149, 179-198. Kuffler, S. W., Dennis, M. J. & Harris, A. J. (1971) Proc. R. Soc. London Ser. B. 177, 555-568. Cohen, J. B. & Changeaux, J. P. (1975) Annu. Rev. Pharmacol. 15, 83-103. de Robertis, E. & Schacht, J., eds. (1974) Neurochemistry of Cholinergic Receptors (Raven Press, New York). Bennett, M. V. L., ed. (1974) “Synaptic transmission and neuronal interaction,” Society of General Physiologists Series (Raven Press, New York.), Vol. 28. Greene, L. A., Sytkowski, A. J., Vogel, Z. & Nirenberg, M. W. (1973) Nature 243, 163-166. Moore, W. J. & Loy, N. J. (1972) Biochem. Biophys. Res. Com- mun. 46, 2093-2099. Eterovic, V. A. & Bennett, E. L. (1974) Biochim. Biophys. Acta 362, 346-355. Polz-Tejera, G., Schmidt, J. & Karten, H. J. (1975) Nature 258, 349-351. Sheffield, J. B. & Moscona, A. A. (1970) Dev. Biol. 23, 36-61. Stefanelli, A., Zacchei, A. M., Caravita, S., Catoldi, A. & leradi, L. A. (1967) Experientia 23, 199-200. Stell, W. K. (1972) in Handbook of Sensory Physiology, ed. 14. 15. 17. 18. 19. 20. 21. 22. 24. 26. 28. 29. 30. 31. Proc. Natl. Acad. Sci. USA 73 (1976) Fourtes, M. G. F. (Springer-Verlag, Berlin, Heidelberg and New York), Vol. 7, part 2, pp. 111-213. Gouras, P. (1976) in Pharmacology and Toxicology of the Eye, ed. Dikstein, 5. (Charles C Thomas, Springfield, Illinois), in press. Lindeman, V. F. (1947) Am. J. Physiol. 148, 40-44. Lam, D. M. (1972) Proce. Nail. Acad. Sci. USA 69, 1987-1991. Shen, S. C., Greenberg, P. & Boel, E. J. (1956) J. Comp. Neurol. 106, 433-461. Nichols, C. W. & Koelle, G. B. (1969) J. Comp. Neurol. 133, 1-16. Noell, W. K. & Lansansky, A. (1959) Fed. Proc. 18, 115. Masland, R. H., Ames, A., II & Livingstone, C. J. (1975) Neurosct. Abst. 5th Meeting of the Soc. for Neuroscience, New York, Vol. 1, page 110, Abstract 173. Ames, A. & Pollen, D. A. (1969) J. Neurophysiol. 32, 424-442. Straschill, M. & Perwein, J. (1973) Pfligers Arch. 339, 289-298. Vogel, Z., Sytkowski, A. J. & Nirenberg, M. W. (1972) Proc. Natl. Acad. Sci. USA 69, 3180-3184. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Coulombre, A. J. (1965) Am. J. Anat. 96, 153-189. Kahn, A. J. (1973) Brain Res. 63, 285-290. Meller, K. (1968) in Veroffentlichungen aus der Morpholog- ischen Pathologie, eds. Buchner, F. & Giese, W. (Gustav Fischer-Verlag, Stuttgart), Vol. 77, pp. 1-77. Sheffield, J. B. & Fischman, D. A. (1970) Z. Zellforsch. M ikrosk. Anat. 104, 405-418. Hughes, W. F. & LaVelle, A. (1974) Anat. Rec. 179, 297-302. Fambrough, D. & Rash, J. E. (1971) Dev. Biol. 26, 55-68. Sytkowski, A. J., Vogel, Z. & Nirenberg, M. W. (1973) Proc. Nail. Acad. Sci. USA 70, 270-274. Miledi, R. & Potter, L. T. (1971) Nature 233, 599-603. Hartzell, H. C. & Fambrough, D. M. (1972) J. Gen. Physiol. 60, 248-262. Daniels, M. & Vogel, Z. (1975) Nature 253, 339-341. Vogel, Z., Daniels, M. & Nirenberg, M. W. (1974) Fed. Proc. 33, 1476.", "Vogel, Zvi ; Nirenberg, Marshall W.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8nkz~8qat~wvxg", "00000000-0000-0000-4217-60A2B01B469A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Synapse and Acetylcholine Receptor Synthesis by Neurons Dissociated From Retina", "101584910X60", null, "1976", "July 1976", "In this report on Nirenberg's retina studies, the formation of synapses by cultured neurons that were dissociated from chick embryo retina is correlated with the synthesis of neuroreceptors found in earlier studies.  Descriptions of the developmental sequence of events leading to synaptogenesis in vitro, along with the type and number of synapses, correspond well with processes in the intact retina.", "Articles", "Neurons,Acetylcholine,Receptors, Cholinergic ; Synapses", "Neuroblastoma Research, 1967-1976", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 73, No. 7, pp. 2370-2374, July 1976 Biochemistry Synapse and acetylcholine receptor synthesis by neurons dissociated from retina (5]-Jabeled a-bungarotoxin/autoradiography/cultured embryonic cell aggregates/electron microscopy) ZVI VOGEL*, MATHEW P. DANIELS, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, May 10, 1976 ABSTRACT Neurons dissociated from chick embryo retina and cultured form more than 1 X 108 synapses per mg of protein. At least three types of synapses are synthesized which resemble those of the intact retina. In addition, two populations of neu- rons were found, those with nicotinic acetylcholine receptors and those without the receptors.   Sheffield and Moscona (1) and Stefanelli et al. (2) have shown that cultured neurons dissociated from chick embryo retina form synapses in vitro. Since only five types of neurons are present in the vertebrate retina and both the structure and function of the neurons and synaptic circuits of the retina have been studied extensively (3-5), chick embryo retina would appear to be an excellent cell system for studying the formation of synapses. Avian retina and retina from several mammalian species contain relatively high concentrations of nicotinic acetylcholine (ACh) receptors, that are primarily associated with the synaptic layers of the retina (6, 7). Acetylcholine receptor synthesis is an early event in chick embryo retina when compared with the appearance of synaptic connections. In this report, the forma- tion of synapses by cultured neurons that were dissociated from chick embryo retina is correlated with the synthesis of nicotinic ACh receptors. METHODS Retina Dissociation. Eight-day-old chick embryo retinas were dissociated into single cells essentially as described by Sheffield and Moscona (1). Retinas were dissected in Eagle’s basal medium (BME, Microbiological Associates), washed, cut into pieces, and incubated for 15 min at 37° in Tyrode’s solution without Ca**+ or Mgt* in an atmosphere of 5% CO2-95% air. Trypsin (crystallized three times) and DNase I (crystallized, Worthington Cat. no. 2058) were added so that the final con- centrations were 2.5 and 0.05 mg of protein per ml, respectively (about 0.3 ml per retina). The tissue was incubated for an ad- ditional 15 min, then 0.5 ml of cold growth medium (80% BME with 2 mM glutamine and 20% fetal bovine serum) was added per retina and the partially disrupted tissue was sedimented by low-speed centrifugation. The pellet was mixed with 0.3 ml of culture medium per retina and dispersed into single cells by pipetting in and out of a pasteur pipette with a fire-polished tip. Each retina yielded 30 to 40 X 10° cells. At least 95% of the cells were single cells; a few clusters comprised of two to three cells also were present. Cell viability, determined by dye exclusion, was >95%. Monolayer Cultures. A cell suspension (9 X 10° cells in 1.5 ml of growth medium) was added to each 35 mm collagen-   Abbreviations: a-BT, a-bungarotoxin; !25]-labeled aBT, a-bungaro- toxin labeled with 2 atoms of !251 per toxin molecule; ACh, acetyl- choline. * Present address: Neurobiology Unit, The Weizmann Institute of Science, Rehovot, Israel. coated tissue culture petri dish (Falcon Plastics). Cultures were incubated at 37° in a humidified atmosphere of 95% air-5% COs. The culture medium was changed every other day. Aggregate Cultures. A cell suspension (9 X 108 cells in 1.5 ml of growth medium) was added to each 35 mm petri dish (bacteriological, rather than tissue culture grade, Cat. no. 1008, Falcon Plastics) and the dishes were swirled on a gyratory shaker (Bellco) with an excursion of approximately 2.6 cm at 80 rpm in a 87° incubator with a humidified atmosphere of 95% air-5% COx. The cells adhere to one another and form aggre- gates, Two-thirds of the culture medium (1.0 ml) was changed every other day. Assay of Homogenates for !*°I-Labeled a-Bungarotoxin Binding. Aggregates from one to three dishes were collected by gentle centrifugation, washed three times with Dulbecco's phosphate buffered saline (8), and homogenized in 0.05 M Tris-HCl, pH 7.4. Monolayer cultures of retina cells were washed with Dulbecco’s phosphate-buffered saline, scraped from the dish in that buffer, sedimented in a Duall ground glass conical homogenizer (Kontes), and homogenized in 0.05 M Tris-HCl, pH 7.4. The homogenates were incubated with 10 nM 125] diiodo-labeled a-bungarotoxin, henceforth termed 125]_labeled aBT, for 30 min at 37° and the amount of !%51- labeled aBT bound was determined by filtration through a cellulose acetate filter (EGWP; Millipore Co.) 25 mm in di- ameter with a 0.2-um pore size (7). !>1-labeled aBT was pre- pared as described previously (9). Protein was determined by a modification of the method of Lowry et al. (10). Autoradiography of Cultured Cells. Cultures were incu- bated with 10 nM !75]-labeled eBT in growth medium for 1 hr and then cooled on ice. Monolayer cultures were washed five times with cold growth medium (2 ml each wash) and twice with cold growth medium without serum. The total wash time was 15 min. Aggregates were transferred to conical siliconized (Siliclad) glass tubes and washed.as first described except that aggregates were recovered by centrifugation. All cultured material was fixed for 1 hr or longer with cold 2.5% glutaral- dehyde in 0.1 M sodium cacodylate buffer, pH 7.4, containing 2 mM CaCl, and rinsed six times for 30 min to 12 hr (total) with 0.1 M sodium cacodylate, pH 7.4 containing 2 mM CaCl. Monolayer culture dishes then were washed briefly with water, excess fluid was removed, and the cultures were coated with Kodak NTB-2 emulsion. Autoradiographs were exposed and developed as previously described (11). Aggregates fixed and washed as described above were dehydrated, cleared, and embedded in paraffin by standard methods. Sections 6 4m thick were mounted on glass slides, deparaffinized, subjected to au- toradiography, and stained with toluidine blue as described previously (7). Some cultured aggregates were dehydrated in ethanol and propylene oxide and embedded in Epon. Sections 0.5 wm thick were mounted on glass slides, subjected to auto- radiography, and stained with toluidine blue. Electron Microscopy. Aggregates fixed and washed as de- Biochemistry: Vogel et al.     z NE ee ee ae wi Aggregates ° cc 125 a O = 100 £ 2 5% 9 Monolayers Z 50 x 2 4 25 o = - Fa OT nav I ee 0 3 6 9 12 15 18 DAYS IN CULTURE Fic. 1. Binding of }25I-labeled aBT to cultured cells dissociated from retina and grown in vitro for different periods. Cells from 8- day-old chick embryo retina were dissociated and cultured as cell aggregates in rotating petri dishes or as monolayers in stationary petri dishes. Each petri dish was inoculated with 9 X 10° cells. Cells were harvested, homogenized, and assayed for binding of }25]-labeled aBT as described under Methods. Symbols represent the following: fmol of toxin bound per mg of protein with cell aggregates (@) and with stationary cell monolayer cultures (0). scribed above, were postfixed for 1 hr in 1% OsO, in 0.1 M so- dium cacodylate, pH 7.4, containing 2 mM CaCl, and dehy- drated and embedded in Epon. Ultrathin sections were stained with urany] acetate and lead citrate. RESULTS Activity and distribution of ACh receptors Cells dissociated from chick embryo retina on the 8th embry- onic day, were cultured for 1-10 days, either in stationary or rotating petri dishes to obtain cell monolayers or aggregates, respectively. Homogenates were prepared at different times as indicated in Fig. 1 and the amount of !5]-labeled aBT bound specifically was determined. With cell aggregates, specific binding of !25-labeled «BT increased 5-fold between the 1st     Proc. Natl. Acad. Sci. USA 73 (1976) 2871 and 7th day in vitro and the number of aBT binding sites in aggregates cultured for 5 days was equal to that of the intact retina of the same age (13th embryonic day). The maximum concentration of receptors was attained between 7 and 10 days in vitro and then gradually declined. The decrease in receptors may result from cell death, for necrotic areas were found in some of the larger aggregates. With stationary cell monolayer cultures, receptor concentration increased 9-fold between the Ist and 3rd culture days and did not change thereafter. The maximum concentrations of aBT binding sites found with cell aggregates, monolayers, and intact retina of newly hatched chicks were 125, 75, and 400 fmol, respectively, of toxin bound specifically per of mg of protein (80. min incubation with 10 nM 125]_labeled aBT at 37°). Autoradiographs of sections of cell aggregates cultured for 1-7 days and then incubated with I-labeled aBT are shown in Fig. 2. As shown in panel A, the cell bodies and ACh receptors within the 1-day-old aggregate are distributed more uniformly than in older aggregates (panels B and C), but small areas with higher levels of !I-labeled aBT binding are present after 1 day of incubation. Larger aggregates are present after 7 days of culture (panels B and C) and cell bodies and processes (neurites) have sorted out into discrete regions which are equivalent to the layers of cell bodies and processes in intact retina. The neurite rich regions are heavily labeled with 125]-labeled aBT. Little binding of }251-labeled «BT was observed in the presence of d-tubocurarine (Fig. 2C) which competes with oBT for binding sites on the nicotinic ACh receptor. Previous studies with d-tubocurarine and other ligands of the nicotinic ACh receptor have shown that the specificity of aBT binding sites of chick embryo retina for various ligands is similar to that of the nicotinic ACh receptor (7). The sorting out of neurites and ACh receptors from cell bodies was observed consistently in aggregates cultured for 3 or more days. Photomicrographs at higher magnification of 0.5 um Epon sections of retina cell aggregates cultured for 7 days are shown in Fig. 3. The use of Epon sections results in improved autora- diographic resolution as well as superior cell structure as com- pared to the paraffin sections. In Fig. 3A a section which was not subjected to autoradiography is shown in order to show cell a\" .” wo er A FIG. 2. Autoradiographs of 6 um thick sections of retina cell aggregates cultured for 1 or 7 days, to illustrate the progressive sorting out of neurites with nicotinic ACh receptors from cell bodies. The cell aggregates were incubated with 125] labeled aBT (300 Ci/mmol of toxin), sectioned, subjected to autoradiography for 36 days, and stained with toluidine blue. A, Cell aggregates cultured for 1 day. B, A cell aggregate cultured for 7 days. C, A cell aggregate cultured for 7 days and incubated for 10 min with 0.5 mM d-tubocurarine before the addition of 125]-labeled aBT. ‘The bars represent 50 um. The areas packed with neurites appear black in panel B due to the relatively high concentration of silver grains resulting from 1251-labeled aBT bound to receptors on neurites. In panel C these regions appear to be relatively devoid of silver grains due to inhibition of aBT binding by d-tubocurarine. 2372 Biochemistry: Vogel et al. vA wor, ES vd oe ‘ ee Oe cE “ant spe   Fic. 3. Distribution of bound }75I-labeled aBT in neurite-rich regions compared with cell body regions. Phase contrast views of to- luidine blue stained 0.5 4m thick Epon sections of retina cell aggre- gates cultured for 7 days. A (left), A section not subjected to autora- diography. B (right) A section of an aggregate which was incubated with }25]-labeled aBT (250 Ci/mmol of toxin) and subjected to au- toradiography for 50 days. The microscope was focused on the silver grains. The letter N corresponds to areas packed with neurites. The bar represents 25 ym. body and neurite structure more clearly. A similar section subjected to autoradiography is shown in Fig. 3B. Most of the silver grains are associated with neurites rather than cell bodies. Ultrastructure of the neurite regions Aggregates of cells dissociated from 8-day-old chick embryo retinas were fixed for electron microscopy after 1-21 days of culture. The retinal neurons of 1-day-old aggregates had ex- tended many neurites (Fig. 4A) which were loosely packed in the cell aggregates. No synapses were seen at 1 or 2 days al- though a few synaptic lamellae (ribbons) and small electron lucid vesicles were present. However, other types of inter-   Proc. Natl. Acad. Sci. USA 73 (1976) cellular junctions such as macula adhaerens diminuta and zonula adhaerens were present, as reported by Sheffield and Moscona (1). At 5 days the neurite regions were larger and neurites were packed closer together. A few, immature synaptic connections were seen at this time. By 7 days (Fig. 4B), the in- tercellular gap between closely packed neurites was reduced to a width of about 200 A at most points. Synapses now were very abundant; the average thin section of neurites sorted out from cell bodies contained approximately 30 synapses per 100 um?. The maximum number of synapses (38 synapses per 100 um?) was achieved by the 14th day in vitro. Similar counts with electron micrographs of inner synaptic layer of adult chick retina revealed about 50 synapses per 100 um?. Assuming that the diameter of the average synapse is 1 um, the number of synapses found in a thin section 0.05 4m in thickness, corre- spond to a tissue section with a maximum thickness of 2 um. Thus, a thin section 100 um? is equivalent to 200 zm? of tissue with respect to the detection of synapses. Assuming that neurite regions comprise about 40% of the cell aggregates, and protein about 10% of the cell aggregate wet weight, the number of sy- napses formed by cultured retina cells is 1.5 to 2 X 10°/mg of protein; this is a remarkably high value and is in the range of that found in the intact retina (12). The minimum number of synapses formed in vitro is, by conservative estimate, >1 X 10°/mg of protein. Many synapses in 14- to 21-day-old aggregates appeared more mature than those of 7-day-old aggregates. For example, the submembrane densities were more extensive and the elec- tron lucid (synaptic) vesicles more closely packed. Between 14 and 21 days, necrotic areas appeared in the larger aggregates, sy eee rca 4 Lan Slat Se ra Z Bae | Zs er, r Fic. 4. Neurite-rich regions of aggregates of 8-day-old chick embryo retina cells. A (left), Cells cultured 1 day. The neurites are loosely packed. The arrows refer to macula adhaerens junctions. Synaptic junctions have not yet formed. B (right), Cells cultured 7 days. The neurites are closely packed and many synapses (arrows) are present. Bar representa 1 ym. Biochemistry: Vogel et al.      FiG. 5. Synapses in neurite-rich regions of aggregates of 8-day-old chick embryo retina cells. A (upper panels), Cultured 18 days. Several synapses of the amacrine-amacrine type are present. Neurite labeled (2) appears to be postsynaptic to neurites (1) and (3) whereas (3) is postsynapt’+ to (4). Note presynaptic tufts of electron dense material (arrows). B (middle panel), Cultured 14 days. A bipolar ribbon sy- napse with presumed ganglion and/or amacrine neurons is present. Note the synaptic ribbon, dense cap, and synaptic vesicles in the bi- polar axon terminal and the densities under the postsynaptic mem- branes. A small symmetrical junction also is present (arrow). C (lower panel) Cultured 10 days. Two photoreceptor cell processes in synaptic contact with several neurite endings. The photoreceptor processes contain many synaptic ribbons, electron lucid vesicles, and a few dense-core vesicles (d). Regions of presumed synaptic contact, with submembrane densities are indicated (arrows). The alignment and opposition of synaptic ribbons of two different photoreceptor cell processes seen here was unusual, and was not observed in the intact chicken retina. Bar represents 0.5 um. and some neurites appeared to be replaced by Miller cell processes. Proc. Natl. Acad. Sci. USA 73 (1976) 2873 Fic. 6. Bright-field views of monolayer cultures of chick embryo retina cells incubated with !25I-labeled aBT and subjected to auto- radiography. Labeled and unlabeled cells and cell processes can be seen. A, Retina cells (9 x 10° cells from 8-day-old embryonic retina inoculated per 35-mm petri dish), cultured for 10 days, incubated with 125]-Jabeled aBT (300 Ci/mmol of toxin), and subjected to autoradi- ography for 36 days. The neurons are attached to a confluent mono- layer of cells resembling fibroblasts which are not labeled appreciably and thus are not seen clearly. B, Retina cells (7 X 10° cells inoculated per 35-mm petri dish) cultured for 7 days, incubated with }*5]-labeled aBT (410 Ci/mmol of toxin) and subjected to autoradiography for 30 days. The arrow indicates an unlabeled cell process. The bar repre- sents 50 um. Three types of synapses were identified in the neurite regions. Conventional synapses (Fig. 5A) which resemble amacrine- amacrine synapses of the intact retina and which are similar to those reported by Stefanelli et al. (2) were observed most frequently. At these synapses, synaptic vesicles were abundant on one side of the cleft, but were scattered or absent on the other side; electron dense material was present under pre- and post- synaptic membranes; and, in some cases, one or more tufts of electron dense material projected intracellularly from the presynaptic density. Single neurites frequently formed two or more conventional synapses; serial synapses also were observed. A second type of synapse (Fig. 5B) resembled the synapse between a bipolar neuron and two postsynaptic processes of amacrine and/or ganglion neurons. The presynaptic ending contained a short synaptic ribbon surrounded by a cluster of vesicles close to a convex electron-dense cap at the tip of the ending. The postsynaptic endings had electron-dense material under the plasma membrane in the vicinity of the cap. In some cases, one or both postsynaptic endings contained synaptic vesicles as with amacrine neurons in the intact retina. A third type of synapse (Fig. 5C), resembled the synapse of a photoreceptor cell with neurites of horizontal and/or bipolar neurons. The large presynaptic processes contained synaptic ribbons, many electron-lucid synaptic vesicles, and a few dense core vesicles. Stefanelli et al. (2) described processes of this type in retina cell aggregates and Sheffield and Moscona (1) iden- tified photoreceptor cell processes by the presence of paraboloid and ellipsoid bodies in the cell body. We also identified pho- toreceptor cell processes in this manner. Neurite endings with submembrane electron dense material were found in the in- vaginations of photoreceptor processes, sometimes near synaptic ribbons. Photoreceptor ribbon synapses with one or two post- synaptic processes were found more frequently than triad sy- napses with three processes. Perhaps these configurations rep- resent developmental stages in the assembly of triad synaps- es. Cell aggregates were cultured for 1-21 days in the presence of 2 uM aBT and then were fixed for electron microscopy. 2374 Biochemistry: Vogel et al. «-Bungarotoxin did not inhibit the formation of the three types of synapses described above and had no obvious effect upon the abundance of synapses or their morphology. These results suggest that activation of nicotinic ACh receptors by acetyl- choline is not required for the formation of at least three kinds of synapses. Photomicrographs of stationary monolayer cultures of retina cells which were incubated with labeled aBT and subjected to autoradiography are shown in Fig. 6. Small clusters of cells which were connected to other islands of cells by long neurites often were seen on top of a monolayer of fibroblast-like cells. Some cell clusters were heavily labeled with !“1-labeled aBT; others were labeled lightly or not at all. Thus, two types of cells with neuronal morphology were found: cells which bind 125]-labeled aBT (usually on both cell bodies and processes) and cells which do not bind toxin. DISCUSSION The results show that cultured neurons dissociated from chick embryo retina synthesize nicotinic ACh receptors and extend long neurites which together sort out from cell bodies. We es- timate that cultured cells form approximately 1.5 X 10° sy- napses per mg of protein. By conservative estimate, the mini- mum number of synapses formed in vitro would appear to be >1 X 108/mg of protein. At least three types of synapses are formed in vitro which resemble those of the intact avian retina: bipolar ribbon sy- napses, photoreceptor ribbon synapses, and amacrine conven- tional synapses. Serial synapses and reciprocal synapses also were found which closely resemble those of amacrine neurons in the intact retina. Additional synapse subclasses were found but neuron identification was uncertain. For example, bipolar ribbon synapses were observed with different combinations of postsynaptic neurons, some with, and some without synaptic vesicles, tentatively identified as amacrine neurons and ganglion neurons, respectively. Synapses first appear in the intact retina on the 13th em- bryonic day (13-15). Most of the synapses in cell aggregates formed from cells that were dissociated on the 8th embryonic day, appear between the 5th and 7th day in vitro, which cor- responds to the 18th and 15th day in ovo. Autoradiography of monolayer cells which had been incu- bated with !*5]-labeled aBT revealed two populations of neu- tons; those with nicotinic ACh receptors on cell bodies and processes which comprised about 20% (the range is 15-40%) of the cell population, and neurons without these receptors. Culturing monolayers of retina cells for 5-6 days in the presence of either acetylcholine (with or without eserine), carbamyl- choline, or d-tubocurarine had little or no effect on the con- centration of nicotinic ACh receptors. Thus, as reported pre- viously for striated muscle (16, 17), ligands of the nicotinic ACh Proc. Natl. Acad. Sci. USA 73 (1976) receptor are neither required nor inhibit receptor synthesis. Cells cultured in the presence of aBT for up to 21 days were not noticeably affected with respect to the number or the kinds of synapses synthesized. Thus, activation of nicotinic ACh re- ceptors is not required for the synthesis in vitro of many sy- napses, but the results do not rule out the possibility that aBT inhibits the formation of certain synapses. a-Bungarotoxin re- portedly inhibits the normal development of the neuromuscular synapse in vivo (18). Most of the nicotinic ACh receptors were associated with neurites rather than cell bodies, both in cell aggregates and in the intact retina. The sorting out of neurites and the synthesis and apparent segregation of ACh receptors, thus are early events which precede synapse formation in vitro and, as re- ported previously, in vivo (7). The formation of cell junctions such as the macula adhaerens and zonula adhaerens also are early events compared to the formation of synaptic connections. Thus, the developmental sequence of events leading to syn- aptogenesis in vitro, the types of synapses formed, and sur- prisingly, the number of synapses formed by cultured cells agrees well with the-corresponding processes in the intact retina. 1. Sheffield, J. B. & Moscona, A. A. (1970), Dev. Biol. 23, 36-61. 2. Stefanelli, A., Zacchei, A. M., Caravita, S., Cataldi, A. & Jeradi, L. A. (1967) Experientia 23, 199-200. 3. Stell, W. K. (1972) in Handbook of Sensory Physiology, ed. Fourtes, M. G. F. (Springer-Verlag, Berlin, Heidelberg), Vol. 7, part 2, pp. 111-213. 4. Dowling J. E. (1970) Invest. Ophthalmol. 9, 655-680. 5. Gouras, P. (1976) in Cell Pharmacology of the Eye, ed. Dikstein, S. (Charles C Thomas, Springfield,), in press. 6. Vogel, Z., Daniels, M. P. & Nirenberg, M. (1974) Fed. Proc. 33, 1476. 7. Vogel, Z. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 1806-1810. 8. Dulbecco, R. & Vogt, M. (1954) J. Exp. Med. 99, 167-182. 9. Vogel, Z., Sytkowski, A. J. & Nirenberg, M. W. (1972) Proc. Natl. Acad. Sci. USA 69, 3180-3184. 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 11. Sytkowski, A. J., Vogel, Z. & Nirenberg, M. W. (1973) Proc. Natl. Acad. Sci. USA 70, 270-274. 12. Dubin, M. W. (1970) J. Comp. Neurol. 140, 479-506. 13. Meller, K. (1968) in Veroffentlichungen aus der Morpholog- ischen Pathologie, eds. Buchner, F. and Giese, W. (Gustav Fischer-Verlag, Stuttgart), Vol. 77, pp. 1-77. 14. Sheffield, J. B. & Fischman, D. A. (1970) Z. Zellforsch. Mikrosk. Anat. 104, 405-418. 15. Hughes, W. F. & La Velle, A. (1974) Anat. Rec. 179, 297-302. 16. Steinbach, J. H., Harris, A. J., Patrick, J., Schubert, D. & Heine- mann, S. (1973) J. Gen. Physiol. 62, 255-270. 17. Hartzell, H. C. & Fambrough, D. M. (1973) Dev. Biol. 30, 153-165. 18. Giacombini, G., Filogama, G., Weber, M., Boquet, P. & Chan- geux, J. P. (1973) Proc. Natl. Acad. Sci. USA 70, 1708-17132.", "Daniels, Mathew P. ; Vogel, Zvi ; Nirenberg, Marshall W.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-x3u7-4jmy-mi2m", "00000000-0000-0000-DE7A-7EB314A342ED", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Opiate-Dependent Modulation of Adenylate Cyclase", "101584910X61", null, "1977", "August 1977", "This article is one in a series completed by Nirenberg and the Biochemical Genetics team at NIH that describes the insoluble enzyme system adenyl cyclase and the hormone signaling system cyclic AMP.  This article discusses opiate receptors and the role of adenylate cyclase in response to neurotransmitters and memory.", "Articles", "Adenylyl Cyclases,Neuroblastoma,Morphine,Enzyme Activation,Pharmacology", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 74, No. 8, pp. 3365-3369, August 1977 Biochemistry Opiate-dependent modulation of adenylate cyclase (neuroblastoma X glioma hybrid cells/enzyme regulation/morphine receptors /prostaglandin E, receptors /supersensitivity) ‘SHAIL K. SHARMA*t, WERNER A. KLEE!, AND MARSHALL NIRENBERGt + Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014; and +Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, June 10, 1977 ABSTRACT Reactions mediated by the opiate receptors that inhibit adenylate cyclase (EC 4.6.1.1) are closely coupled to subsequent reactions that gradually increase adenylate cy- clase activity of neuroblastoma X glioma NG108-15 hybrid cells. Opiate-treated cells have higher basal-, prostaglandin E-, and 9-chloroadenosine-stimulated activities than do control cells. However, NaF or guanosine 5’4,7-imido}triphosphate abolishes most of the difference in adenylate cyclase activity observed with homogenates from control and opiate-treated cells. Cy- cloheximide blocked some, but not all, of the opiate-dependent increase in adenylate cyclase activity. These results suggest that the opiate-dependent increase in adenylate cyclase is due to conversion of adenylate cyclase to a form with altered activity. Protein synthesis also is required for part of the opiate effect. We propose that activity of adenylate cyclase determines the rate of conversion of the enzyme from one form to the other and that opiates, by inhibiting adenylate cyclase, alter the relative abundance of low- and high-activity forms of the enzyme.   Inhibition of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by opiates is rapidly reversed upon re- moval of opiate (1-3). However, exposure of cells to an opiate for 12 or more hours results in a compensatory increase in adenylate cyclase activity that is long-lived and not readily reversed by withdrawal of the opiate (4-6). Similar dual reg- ulations of adenylate cyclase activity are mediated by a-ad- renergic receptors (7) and excitatory muscarinic acetylcholine receptors (S. K. Sharma and M. Nirenberg, unpublished data; ref. 8). Thus, dual regulation may be a general regulatory mechanism which alters the responsiveness of adenylate cyclase to activations mediated by other species of receptors. Because the mechanism of coupling inhibition of adenylate cyclase with a subsequent increase in enzyme activity is unknown, we ex- amined the properties of adenylate cyclase from control cells and cells treated with opiates. MATERIALS AND METHODS The source of each chemical, growth conditions for culturing neuroblastoma X glioma NG108-15 hybrid cells, and prepa- ration of homogenates have been described (2). Adenylate cy- clase activity was measured by the procedure of Salomon et al. (9), modified slightly (2). Each tube contained 30 mM Tris-HCl, pH 7.5/5 mM MgCl2/160 mM sucrose/20 mM creatine phos- phate/10 units of creatine kinase (65 ug of protein)/1 mM adenosine 3/:5’-cyclic monophosphate (cAMP)/0.5 mM 4-(3- butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724)/ 0.5% ethanol/l mM {a-32PJATP (3 to 5 X 10° cpm) and 50-200 ug of homogenate protein in a final volume of 100 yl. Incuba- tions were for 5 min at 37°.   The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked “advertisement” in accordance with 18 U. S. C. $1734 solely to indicate this fact. 3365 RESULTS Effect of Activators of Adenylate Cyclase. If the increase in adenylate cyclase activity of opiate-treated NG108-15 cells were due primarily to an increase in the number of molecules of adenylate cyclase, the relatively high enzyme activity of opiate-treated cells should be maintained under other assay conditions. For this reason, we studied the effects of activators and inhibitors on adenylate cyclase of cells grown in the pres- ence or absence of morphine or etorphine for 3-5 days. Sodium fluoride is known to activate basal adenylate cyclase and inhibit activation of the enzyme by hormones, presumably by un- coupling the enzyme from the hormone-receptor complex (10). The effects of NaF upon basal and prostaglandin E, (PGE)- stimulated adenylate cyclase activity from control or etorph- ine-treated NG108-15 cells as a function of time are shown in Fig. 1. In the presence of 8 mM Naf, the basal activities of adenylate cyclase from control and opiate-treated cells were increased 4.3- and 2.6-fold, respectively, and the specific ac- tivities of the two enzyme preparations then were similar. Conversely, the activities of PGE-stimulated adenylate cyclase from control and opiate-treated cells were inhibited 44% and 60%, respectively, by NaF and the final specific activities were similar. These results show that adenylate cyclase from control cells is more responsive to activation by NaF than adenylate cyclase of opiate-treated cells and raise the possibility that the relatively high activity of adenylate cyclase of opiate-treated cells may be due to enzyme activation rather than to an increase in the number of enzyme molecules. In other systems, guanosine 5’-(8,y-imido)triphosphate [Gpp(NH)p] has been shown to activate basal adenylate cyclase and to activate or inhibit adenylate cyclase synergistically with hormones initially but ultimately to uncouple adénylate cyclase from the hormone-receptor complex (11, 12). The effects of Gpp(NH)p on basal or PGE)-stimulated adenylate cyclase from control and etorphine-treated cells as a function of time are shown in Fig. 2. Incubation with Gpp(NH)p for 16 min in- creased basal adenylate cyclase activity of control cells 460% and that of opiate-treated cells, 280%. Conversely, Gpp(NH)p inhibited PGE)-stimulated adenyiate cyclase from control and opiate-treated cells 25% and 41%, respectively. In the presence of Gpp(NH)p, the specific activities of adenylate cyclase from control and etorphine-treated cells were almost the same. These results provide additional evidence for two forms of adenylate cyclase that differ in extent of response to Gpp(NH)p or NaF and suggest that the relatively high enzyme activity of opi- ate-treated cells may be due to enzyme activation.   Abbreviations: cAMP, adenosine 3’:5’-cyclic monophosphate, Ro 20-1724, 4-(8-butoxy-4-methoxybenzy])-2-imidazolidinone, PGE), prostaglandin E,; Gpp(NH)p, guanosine 5’-(8,-imido)tri phosphate. * On leave from the Department of Biochemistry, All India Institute of Medical Science, New Delhi, India. 3366 Biochemistry: Sharma et al.                2000+ 44000 c 3 9 15001 43000 a oO E = ° £ 10001 42000 a a = ; $ sool 1000 n LL 1 1 4 L 1 0 ov 4 6 ie 16 20. 0 4 8 12 16 26 Minutes Fic. 1. Effect of NaF on basal (Left) and PGE,-stimulated (Right) adenylate cyclase activities of homogenates prepared from NG108-15 cells (subculture 15) cultured for 3 days with or without 1 uM etorphine. Each point represents a 50-u] aliquot of a 400-ul re- action mixture that contained the components described in Materials and Methods and the following additions or exceptions: 15 mM magnesium acetate; 10 1M naloxone; and either 57 ug of homogenate protein from control cells cultured without etorphine or 59 ug of ho- mogenate protein from cells cultured with etorphine. When present (Right panel), PGE, was 10 uM. Symbols: ©, no addition, control homogenate; @, no addition, homogenate from etorphine-treated cells; A,8mM Naf, control homogenate; a, 8 mM NaF, homogenate from cells treated with etorphine. The effects of different concentrations of NaF or Gpp(NH)p on the activities of adenylate cyclase from control or opiate- treated NG108-15 cells are shown in Fig. 3. At relatively low concentrations of activator (2 mM NaF or 5 uM Gpp(NH)p], basal adenylate cyclase of control cells was activated but not the enzyme of opiate-treated cells. Higher concentrations of NaF or Gpp(NH)p increased the enzyme activity from both control and opiate-treated cells to the same extent, but the specific activities of the two enzyme preparations were almost the same. Conversely, PGE)-stimulated adenylate cyclase of control cells was inhibited less by NaF or Gpp(NH)p than the enzyme from opiate-treated cells. Both the PGE -stimulated           2000 T T T T T T T T T T 4000 & @ 1500} / + 43000 ° PGE, a ‘Gpp- | E (NH)p ~ = 1000F 42000 2 ‘PGE, a + o Gpp(NH)p = sool oy t 4 1000 ° yNONE 1 1 1 1 1 L 1 1 L t Oo GO 4\"\"8 “i216 20 0 4 #8 12 16 20 Minutes Fic. 2. Effect of Gpp(NH)p on the activity of basal (Left) and PGE,-stimulated (Right) adenylate cyclase from NG108-15 cells (subculture 15) treated 3 days with or without 1 uM etorphine. The conditions were as described in the legend to Fig. 1 except that reac- tion mixtures contained, where specified: 100 1.M Gpp(NH)p; 59 ug of homogenate protein from control cells; or 55 pg of homogenate protein from cells treated with etorphine. Symbols: O, no addition, control homogenate; @, no addition, homogenate from cells treated with etorphine; 4, 100 .M Gpp(NH)p, control homogenate; a, 100 uM Gpp(NH)p, homogenate from cells treated with etorphine. (Right) Each reaction mixture contained 10 uM PGE). Proc. Natl. Acad. Sci. USA 74 (1977) 200 i            c 3 9 a a E 150 \\\\ | = \\\\ PGE a on a 1 £ 100 1 | 3 NONE z PGE, _ a oa o 50 ~ 7 Ss NONE rag oe ° og basi to be     4 681012 0 7 6 5 4 3 NaF,mM Gpp(NH)p, —log M Fic. 3. Relationship between NaF (Left) or Gpp(NH)p (Right) concentration and adenylate cyclase activity in homogenates of NG108-15 cells treated for 5 days with or without 1 4M etorphine. Each 100-yl reaction mixture contained the components described in Materials and Methods with the following exceptions: 15 mM magnesium acetate; 10 1M naloxone; 10 uM PGE, where specified; NaF or Gpp(NH)p concentrations as specified. (Left) Each reaction mixture contained either (open symbols) 86 ug of homogenate protein from cells cultured 5 days without etorphine (control homogenate); or (solid symbols) 111 yg of homogenate protein from cells cultured 5 days with 1 «M etorphine (treated). The initial portions of the curves labeled “NONE” are enlarged in the Inset to. show additional detail. (Right) Reaction mixture components were as in Left except that the reaction mixtures contained Gpp(NH)p instead of NaF, and each reaction mixture contained either 138 ,:g of homogenate protein from NG108-15 cells cultured 5 days without etorphine (control) or 170 pg of homogenate protein from NG108-15 cells treated 5 days with 1 uM etorphine (treated). Symbols: ©, no addition, control homogenate; @, no addition, homogenate from etorphine-treated cells; A, 10 «M PGE,, control homogenate; a, 10 uM PGE,, homogenate from etor- phine-treated cells. 2 and basal activities of adenylate cyclase from control cells al- most equaled those of opiate-treated cells in the presence of 2 mM NaF or 5 uM Gpp(NH)p. The concentrations of Gpp(NH)p or NaF required for half-maximal inhibition of PGE,-stimu- lated adenylate cyclase were similar to the concentrations re- quired for half-maximal reversal of the difference between control and opiate-treated adenylate cyclase activity (3 uM Gpp(NH)p or 1.5 mM NaF). Higher concentrations of Gpp(NH)p (50 uM) or NaF (4 mM) were required for half- maximal activation of basal adenylate cyclase. Thus, reversal of the difference between control and opiate-treated adenylate cyclase activities resembles the uncoupling of a receptor from adenylate cyclase. The hybrid cells were incubated with or without cyclohexi- mide in the presence or absence of etorphine to determine whether inhibition of protein synthesis affected the opiate- dependent increase in adenylate cyclase activity (Fig. 4). Cy- cloheximide blocked the etorphine-dependent increase in adenylate cyclase at 10 and 20 hr of incubation but not at 4 hr. Similar results were obtained in other experiments not shown here. Although the toxic effects of cycloheximide on cells lim- ited the duration of cell incubation and decreased the magni- tude of the etorphine-dependent increase in adenylate cyclase activity, the results show that adenylate cyclase is a relatively stable enzyme that does not turn over rapidly under the con- ditions tested and suggest that most, but not all, of the increase in adenylate cyclase activity evoked by etorphine is dependent on protein synthesis. The results should be interpreted with caution because cycloheximide probably affects many reac- tions, directly or indirectly, during the long incubation peri- The effects of varying PGE, or 2-chloroadenosine (13, 14) Biochemistry: Sharma et al.   250 225 200 cAMP, pmol/min per mg protein       1 1 1 IS 20 5 10 Hours Ls 175 Os 10 i520 Fic. 4. Effect of cycloheximide on adenylate cyclase activity of control and etorphine-treated hybrid cells. (Left) Basal activity. (Right) Activity stimulated by 10 «M PGE). Triplicate cultures of NG108-15 cells (subculture 17) were treated as follows: O, no addition; 4, 70 uM cycloheximide; @, 1 uM etorphine; a, 1 uM etorphine and 70 uM cycloheximide. In each 30-mm diameter dish, 770,000 cells were plated in 2 ml of 90% Dulbecco-Vogt modification of Eagle’s medium supplemented with 10% fetal bovine serum, 100 uM hypoxanthine, 1 uM aminopterin, and 12 »M thymidine. Cycloheximide blocked incorporation of [2H] proline into protein almost completely. After 20 hr under these conditions, cells tended to detach from the dish. Cell protein was harvested quantitatively after 4, 10, or 20 hr of incubation at 37° in 10% CO./90% air. Cells were removed and recovered by centrifugation at 400 x g for 5 min and washed three times with buffered saline adjusted to 340 mosmol/liter, pH 7.4. Each washed pellet was suspended in 1 ml of 320 mM sucrose/10 mM Tris-HCl, pH 7.5, and stored at —80° prior to assay for adenylate cyclase activity and for protein. Mean protein values per dish at 4, 10, and 20 hr were: control, 0.58, 0.66, and 0.80 mg; cycloheximide, 0.46, 0.51, and 0.52 mg; etorphine, 0.53, 0.65, and 0.75 mg; etorphine and cycloheximide, 0.46, 0.45, and 0.46 mg. Protein in the adenylate cyclase assay varied between 20 and 47 ug/100 ul of reaction mixture. Each point is the mean of triplicate values obtained with three homogenates, each from a separate dish, assayed in duplicate. Each adenylate cyclase reaction mixture contained 10 uM naloxone. concentrations of adenylate cyclase activity from control and etorphine-treated cells are shown in Fig. 5. Half-maximal ac- tivations of adenylate cyclase from control and opiate-treated cells were obtained with approximately 0.15 4M PGE, or 1.8 uM 2-chloroadenosine. Concentrations of 2-chloroadenosine >50 uM were inhibitory. The results show that the increase in adenylate cyclase activity due to treatment of cells with opiates and stimulation of adenylate cyclase by PGE, or 2-chloro- adenosine are expressed simultaneously and that treatment of cells with opiates does not alter the concentrations of PGE) or 2-chloroadenosine required for half-maximal activation of adenylate cyclase by PGE, or 2-chloroadenosine. Therefore, the supersensitivity of opiate-treated cells to PGE, or 2-chlo- roadenosine results from an increase in adenylate cyclase ac- tivity. Interaction between Receptors Coupled to the Inhibition or Activation of Adenylate Cyclase. The relationship between morphine concentration and inhibition of adenylate cyclase from control and opiate-treated cells is shown in Fig. 6 A and B, and the effects of different concentrations of naloxone in reversing the inhibition of adenylate cyclase by 10 uM mor- phine are shown in Fig, 6 C and D. Half-maximal inhibition of basal adenylate cyclase from control and morphine-treated cells was obtained with 1 «.M morphine and maximal inhibition with 5-10 uM morphine (Fig. 6B). However, in the presence Proc. Natl. Acad. Sci. USA 74 (1977) 3367              OO a rr BO 8 PGE, TREATED «| 2-CHLOROADENOSINE °o 3 300; et 60 E S TREATED c 200+ 4 40 g 5 = CONTROL oO E | 5 100 q 20 8 . = - < CT po lg 098 765 409 8 7 6 5 4 3 Activator, —log M Fic. 5. Effect of PGE, (Left) or 2-chloroadenosine (Right) concentration on activity of adenylate cyclase from NG108-15 cells with or without 1 4M etorphine. (Left) Each reaction mixture con- tained the components described under Materials and Methods and 10 »M naloxone, the PGE, concentration indicated, and (O) 71 #g of homogenate protein from control cells; or (@) 87 ug of homogenate protein from cells treated 4 days with etorphine. (Right) Each reaction mixture contained the components described under Materials and Methods, 2-chloroadenosine as indicated, and (0) 87 ug of homoge- nate protein from control cells; or (@) 94 ug of homogenate protein from etorphine cells treated 5 days with etorphine. of PGE}, a 10- to 20-fold higher concentration of morphine (100 uM or more) was required for maximal inhibition of adenylate cyclase from control or opiate-treated cells (Fig. 6A). Con- versely, a 10-fold lower concentration of naloxone (5 uM) was required for complete reversal of inhibition of adenylate cyclase by morphine in the presence of PGE; than in the absence of PGE, (50 uM naloxone). These results show that inhibitory opiate receptors and stimulatory PGE, receptors interact at the level of adenylate cyclase. Similarly, PGE increased the ef- fectiveness of naloxone in reversing the inhibition of adenylate cyclase by morphine. Perhaps both phenomena are a conse-   T ™T 240r A . PGE, c PGE, +MORPHINE a4 4 220 - a \\\\ isc4-—a® 7 4 _ 160 + 140 x L. 4 , 4+ T “Tr n NM Oo o T 4 T ——   cAMP, pmol/min per mg protei   120+ 7 100 gat t+ tt pot t+ tt B NONE | D MORPHINE 25 + 1 20 4 e > oO ol } ao) oO § 1OF 4 5 4   1 a Lob 1 a 4 ro ron oi ot-— 76 5 40 7 «+6 5 4 3 Morphine, —log M Naloxone, —log M   Fic. 6. Adenylate cyclase activity from homogenates of control NG108-15 cells (open symbols) or morphine-treated cells (solid symbols), cultured for 5 days in the absence or presence of 10 uM morphine, respectively. (A) and (B) Effect of morphine concentration on inhibition of adenylate cyclase in the presence of 10 «M PGE, or in the absence of PGE, respectively. (C) and (D) Effects of different concentrations of naloxone in reversing inhibition of adenylate cyclase due to 10 uM morphine in the presence and absence of 10 uM PGE), respectively. Each adenylate cyclase reaction mixture contained 87 ug of homogenate protein from control cells or 94 ug of homogenate protein from morphine-treated cells. 3368 Biochemistry: Sharma et al. — 4h tt}——1— T Tr mT A PGE, Ic PGE, 250r + 4   200+ + 4 wo Oo a oO oO oO T T T , ' , + T T L J. A °o   cAMP, pmol/min per mg protein ny Ww + wn o 0 0 90 T T T T +++ i 1 1 i oO T 1 t L     b 11 oa | 4. —L—_L 1 4k 0 3 2 10 3 2 i Mg?*, -log M Mn?*, —iog M Fic. 7. The effects of Mg?+ (A and B) or Mn?+ (C and D) con- centrations upon adenylate cyclase activity from homogenates of control (open symbols) or morphine-treated (solid symbols) NG108-15 hybrid cells. Homogenates are described in the legend to Fig. 6. In A and C, reaction mixtures contained 10 »M PGE); in B and D, basal adenylate cyclase activity was determined.     quence of the loss of positive cooperativity in the coupling of the morphine-opiate receptor complex with adenylate cyclase (Hill coefficient = 2). Naloxone and PGE, would be expected to act by different mechanisms because naloxone and morphine compete for a site on the opiate receptor, whereas the mor- phine-opiate receptor and the PGE)-PGE, receptor either might compete for sites coupling receptors to adenylate cyclase, or both species of receptor might be coupled simultaneously to adenylate cyclase at separate sites and interact by an allosteric mechanism. The data also show that the long-lived activation of adenylate cyclase that results from prolonged exposure of NG108-15 cells to an opiate and the transient inhibition of adenylate cyclase by morphine are expressed independently and simultaneously. Tolerance and dependence produced in animals by chronic exposure to opiates can be understood at the cellular level in terms of an increase in adenylate cyclase activity (4-6). Opi- ate-treated cells are tolerant to opiates because the relatively high adenylate cyclase activity of such cells must be inhibited to a greater extent by the opiate before levels of enzyme activity and of cAMP fall below the levels of control cells. They are dependent upon opiates because withdrawal of the opiate re- verses the inhibition of adenylate cyclase and thereby increases the cAMP level well above that of control cells. Finally, opi- ate-treated cells are supersensitive to the opiate antagonist naloxone because opiate-treated cells have more adenylate cyclase activity and more opiate-inhibited enzyme activity than control cells. Consequently, at any concentration of naloxone, more adenylate cyclase activity is rescued with homogenates from opiate-treated cells than with those from control cells when both are assayed in the presence of morphine. In experiments not shown here, the pH optimum.of ade- nylate cyclase from control or morphine-treated cells was pH 8.5 for basal activity and pH 8.0 for PGE)-stimulated activity. The apparent Michaelis constant (K,,) for ATP was 0.16 mM with adenylate cyclase from control cells and opiate-treated cells with or without PGE). Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Properties of adenylate cyclase from control and opiate- treated NG108-15 hybrid cells Control cells Opiate-treated cells     Addition Basal PGE, Basal PGE, uM ATP 160 160 160 160 Mg?+ 7000 1000 5000 1000 Mn?+ 1000 600 1000 500 PGE; _— 0.14 _ 0.16 2-Chloroadenosine 1.8 — 18 — Gpp(NH)p 50 15 50 1.2 NaF 4000 2000 . 8600 1800 Morphine 1.0 2.0 1.0 2.0 Naloxone 1.4 0.5 0.9 0.4 pH optimum 8.5 8.0 8.5 8.0   The numbers shown are #M and are K,,, values for ATP, K, values for PGE, and 2-chloroadenosine, or concentrations required for half-maximal stimulation or inhibition of adenylate cyclase by MgClo, Gpp(NH)p, NaF, morphine, or naloxone (the last in the presence of 10 »M morphine). The PGE; concentration was 10 uM except where indicated. The effects of Mg2+ and Mn?* concentrations on basal and PGE)-stimulated adenylate cyclase activities from control on morphine-treated cells are shown in Fig. 7. The concentrations of Mg?+ required for maximal activation of basal and PGE- stimulated adenylate cyclase were approximately 30 and 2 mM, respectively, with both enzyme preparations. Thus, Mg2+ and PGE, activate adenylate cyclase synergistically. Higher con- centrations of Mg2* inhibited adenylate cyclase, and the en- zyme from morphine-treated cells was more sensitive to inhi- bition than that from control cells. The concentrations of Mn2+ required for maximal stimulation of basal and PGE)-stimulated adenylate cyclase were 2 and 1 mM, respectively. Higher concentrations of Mn?* were inhibitory. In Table 1, K,, values for ATP, K, values for PGE, or 2- chloroadenosine, or concentrations of Mg2+, Mn2+, Gpp(NH)p, or NaF required for 50% of the maximal effect are summarized and are shown to be the same for adenylate cyclase from control and opiate-treated cells. Similarly, no difference was found in the concentration of morphine required for half-maximal in- hibition of adenylate cyclase or in the concentration of naloxone required for reversal of morphine inhibition of adenylate cy- clase. DISCUSSION Opiates regulate adenylate cyclase in two ways, both mediated by the opiate receptor. Opiates inhibit adenylate cyclase rapidly and reversibly, and the inhibition is coupled to a gradual, long-lived increase in adenylate cyclase activity that is expressed even in the absence of the opiate. In this report, the properties of adenylate cyclase from neuroblastoma X glioma NG108-15 hybrid cells cultured with or without opiates for several days are compared. Adenylate cyclase preparations from control and opiate-treated cells do not differ appreciably with respect to the concentrations of PGE, 2-chloroadenosine, morphine, naloxone, ATP, Mg?t, or Mn+ required for half-maximal ef- fects. However, Gpp(NH)p or NaF abolishes most, but not all, of the opiate-dependent increase in adenylate cyclase activity. Basal adenylate cyclase of opiate-treated cells is activated less by Gpp(NH)p or NaF, and the PGE)-stimulated enzyme is inhibited more by these compounds, than the enzyme from control cells. Because the mechanisms of activation and inhibition of adenylate cyclase by Gpp(NH)p or NaF are not understood and Biochemistry: Sharma et al. the extent of activation of adenylate cyclase by Gpp(NH)p or NaF is low compared to that found with PGE, the effects of Gpp(NH)p or NaF on adenylate cyclase should be interpreted with caution. However, the results show that the opiate-de- pendent increase in adenylate cyclase is reversed by agents that are thought to uncouple adenylate cyclase from receptors and suggest that NG108-15 cells contain two or more forms of adenylate cyclase that differ in basal activity and in respon- siveness to activators. Inhibition of protein synthesis with cy- cloheximide blocked most, but not all, of the opiate-dependent increase in adenylate cyclase. These results show that adenylate cyclase is a relatively stable enzyme that does not turn over rapidly and suggest that protein synthesis is required for the full expression of the opiate-evoked increase in adenylate cyclase activity. Further work is needed to identify the protein that is required for opiate-modulation of adenylate cyclase activity and the function of the protein. Changes in cell membrane lipids have persistent effects on adenylate cyclase activity that resemble the opiate-dependent increase in adenylate cyclase activity. Increases were reported in basal activity and in hormone-, Gpp(NH)p-, or NaF-stimu- lated activity; however, NaF-stimulated activity was least af- fected (15-17). Cholera toxin also activates basal- and hor- mone-stimulated adenylate cyclase but not the NaF-activated enzyme (18). A simple hypothesis that provides a mechanism for coupling inhibition of adenylate cyclase by opiates with a subsequent long-lived increase in enzyme activity is that activation of adenylate cyclase desensitizes the enzyme (19), perhaps via a cAMP-dependent reaction. Alternatively, inhibition of ade- nylate cyclase by opiates results in the conversion of the enzyme to an activated form. In either case, inhibition of adenylate cyclase by opiates would lead to an increase in enzyme activity, and stimulation would decrease enzyme activity by altering the relative proportions of low- and high-activity forms of the en- zyme. Other species of receptor coupled to the inhibition of ade- nylate cyclase have also been shown to regulate the amount of adenylate cyclase activity. Exposure of NG108-15 cells to car- bamylcholine, which activates muscarinic acetylcholine re- ceptors of the cells, or to norepinephrine, which activates a- receptors, also inhibits adenylate cyclase, and slowly increases adenylate cyclase activity (refs. 7 and 8; S. K. Sharma and M. Proc. Natl. Acad. Sci. USA 74 (1977) 8869 Nirenberg, unpublished data). Such reactions that modulate the amount of adenylate cyclase activity may provide mecha- nisms for relatively long-lived (hours) regulation of the sensi- tivity and extent of neural responses to various neuro- transmitters and thus may play a role in memory. We thank Doyle Mullinax, Richard Streaty, Deborah Carper, and Linda Lee for their excellent assistance. _ Collier H. O. J. & Roy, A. C. (1974) Nature 248, 24-27. 2. Sharma, S. K., Nirenberg, M. & Klee, W. A. (1975) Prac. Nail. Acad. Sct. USA 72, 590-594. 3. Traber, J., Fisher, K., Latzin, S. & Hamprecht, B. (1975) Nature 253, 120-122. 4. Sharma, S. K., Klee, W. A. & Nirenberg, M. (1975) Proc. Natl. Acad. Sct. USA 72, 3092-3096. 5. Collier, H. O. J., Francis, D. L., McDonald-Gibson, W. J., Roy, A. C. & Saeed, S. A. (1975) Life Sci. 17, 85-90. 6. Traber, J., Gullis, R. & Hamprecht, B. (1975) Life Sci. 16, 1863-1868. 7. Sabol, S. L. & Nirenberg, M. (1977) Fed. Proc. 36, 736. 8. Klein, W. L., Nathenson, M. N. & Nirenberg, M. (1976) Fed. Proc. 35, 1576. 9. Salomon, Y., Londos, C. & Rodbell, M. (1974) Anal. Biochem. 58, 541-548. 10. Manganiello, V. C. & Vaughn, M. (1976) J. Biol. Chem. 251, 6205-6209. 11. Londos, C., Salomon, Y., Lin, M. C., Harwood, J. P., Schramm, M., Wolff, J. & Rodbell, M. (1974) Proc. Natl. Acad. Sct. USA 71, 3087-3090. 12. Lefkowitz, R. J. & Caron, M. G. (1975) J. Biol. Chem. 205, 4418-4422. 13. Blume, A. J. & Foster, C. J. (1976) J. Biol. Chem, 251, 3399- 3404. 14, Sturgill, T. W., Schrier, B. K. & Gilman, A. G. (1975) J. Cyclic Nucleotide Res. 1, 21-30. 15. Orly, J. & Schramm, M. (1975) Proc. Natl. Acad. Sct. USA 72, 3433-3437. 16. Engelhard, V. H., Esko, J. D., Storm, D. R. & Glaser, M. (1976) Proc. Natl. Acad. Sci. USA 73, 4482-4486. 17. Tallman, J. F., Smith, C. C. & Henneberry, R. C. (1977) Proc. Natl, Acad. Sci. USA 74, 873-877. 18. Sahyoun, N. & Cuatrecasas, P. (1975) Proc. Natl. Acad. Sci. USA 72, 3438-3442. 19. Constantopoulos, A. & Najjar, V. A. (1973) Biochem. Biophys. Res. Commun. 53, 794-799.", "Nirenberg, Marshall W. ; Sharma, S. K. (Shail K.) ; Klee, Werner A.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xqn7-4i7y_czav", "00000000-0000-0000-B060-2607F7D2DFBC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Muscarinic Acetylcholine Receptors of the Developing Retina", "101584910X62", null, "1977", "December 1977", "As part of the work in neurobiology by Nirenberg and the Biochemical Genetics lab at NIH, this article describes the properties of acetylcholine neurotransmitter receptors and their distribution within chick retina during embryonic development.", "Articles", "Receptors, Cholinergic ; Receptors, Muscarinic ; Binding, Competitive", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad, Sci. USA Vol. 74, No. 12, pp. 5524-5528, December 1977 Biochemistry Muscarinic acetylcholine receptors of the developing retina (synapse formation/receptor localization /3-quinuclidinyl benzilate) HIROYUKI SUGIYAMA, MATHEW P. DANIELS, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda. Maryland 20014 Contributed by Marshall Nirenberg, October 7, 1977 ABSTRACT Six- and 13-day chicken embryo retinas contain 10 and 320 fmol per mg of protein of specific binding sites for 343H|quinuclidinyl benzilate, a ligand of muscarinic acetyl- choline receptors. Most of the receptors of 13-day embryo retina were found, by autoradiography, to be localized in two sharp bands within the inner synaptic layer of the retina. In the adult, the receptors were found almost exclusively in three bands in the inner synaptic layer of the retina. A possible mechanism for generating sets of stratified or columnar neurons and relating one set to another is proposed.   The vertebrate retina provides a model system for synapse formation because synaptic circuits may be assembled with relatively few types of cells and because cultured neurons dis- sociated from retina form synapses in profusion in vitro (1, 2). Biochemical (3-7), histological (8, 9}, and electrophysiological (10-13) evidence strongly suggests that acetylcholine (ACh) functions as a neurotransmitter in the retina. Developmental] and histological studies of chicken retina acetylcholinesterase (EC 3.1.1.7 AChE) (8), ACh (14), choline acetyltransferase (EC 2.3.1.6) (2), and nicotinic ACh receptors (15, 16) have been reported. In this report, the properties of muscarinic ACh receptors of chicken retina, the number of receptors, and their distribution within the retina during embryonic development are de- scribed. MATERIALS AND METHODS Homogenate Preparations. Neural retinas of White Leg- horn chicken embryos were homogenized in 50 mM sodium phosphate buffer, pH 7.4 (buffer A). In some experiments, homogenates were diluted several times with buffer A and centrifuged at 17,300 X g for 20 min at 3°. The pellet was suspended in buffer A (membrane fraction). All experiments were performed with freshly prepared homogenates or mem- branes. Binding Assay. (3-+)-Quinuclidiny] benzilate (QNB), a gift from Hoffman-La Roche, Inc., was labeled by catalytic 9H exchange and purified as described by Yamamura and Snyder (17); the specific activity was 8.4 Ci/mmol. 3(+)-[3-3H}] QNB used in some experiments was obtained from Amersham /Searle (13 Ci/mmol). [3H|QNB binding was measured by a modification of the method of Yamamura and Snyder (17). Homogenates were combined with [7HJQNB in buffer A and incubated for various periods. Each 100- to 150-ul portion of the reaction mixture (usually containing 100-200 yg of protein) then was diluted into 5 ml of ice-cold buffer A, immediately filtered, and washed three times, each with 5 ml of buffer A. Binding kinetics were   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U. S. C. §1734 solely to indicate this fact. . 5924 measured at 25° by using Whatman glass fiber GF/C filters. The concentration of (+)-[(3HJQNB in the reaction mixture was 0.5-1.0 nM. When the effects of competing ligands were tested, homogenates were incubated with desired concentrations of ligands for 5-10 min at 25° and then mixed with (SH]QNB solution containing the same concentrations of the ligands. Equilibrium studies were performed at 4° with Millipore HAWF filters or, in some cases, GF/C filters (results were es- sentially the same). For the determination of nonspecific binding, homogenates were incubated with 0.4-10 uM atropine sulfate for 10 min in ice and then mixed with [HJQNB solution containing the same concentration of atropine. The number of [SH|QNB binding sites was determined by Scatchard analysis in some experiments but more often was determined at one saturating concentration of (+)-[7H]QNB (6-10 nM). [7HJQNB Autoradiography. Neural retinas were dissected in cold Dulbecco’s phosphate-buffered saline with Ca?+ and Mg?+ (PBS). Pieces of retina from 13-day embryos or adult chickens were incubated for 90 min in 5 ml of PBS containing 4 or 2 nM (+)-[SH|QNB (13 Ci/mmol), respectively, and then washed eight times, each with 5 ml of PBS. In control experi- ments, pieces of retina were preincubated in 5 ml of PBS con- taining 0.4 4M atropine sulfate for 10 min, followed by incu- bation in 5 ml of [SHJQNB solution in PBS containing 0.4 uM atropine sulfate for 90 min. The tissue then was washed twice with 5 ml of PBS containing 0.4 uM atropine sulfate and six times with 5 ml of PBS. Samples were kept in an ice bath at each step. Both experimental and control retinas were washed for 25 min (all washes). Retinas then were sandwiched between two pieces of mouse liver and frozen quickly in liquid Freon cooled in liquid nitrogen. Frozen pieces were sectioned (12 wm thick) and thaw-mounted onto glass slides coated with Kodak NTR-2 nuclear emulsion. To minimize diffusion of [2H|QNB, mounted sections were immediately dried under a stream of nitrogen gas. Slides were stored in the dark at 4° with a desiccant. Autora- diographs were developed, fixed, and then immersed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, for | hr at room temperature. Some slides were stained with 0.02% tolu- idine blue for 5 min at room temperature. Retina Cell Cultures. Cells were prepared from 8-day em- bryos and cultured in rotating petri dishes as described (1) with minor modifications: 1.5 X 107 cells in 3 ml of medium (95% Eagle’s basal medium with Earle’s salts and 5% fetal bovine serum) were cultured in a bacterial petri dish (35 mm; Falcon no. 1008) placed on a rotary shaker with an excursion of 2.6 cm (75-80 rpm) in a 37° incubator in a humidified atmosphere of 5% CO./95% air. Half of the medium was replaced each day.   Abbreviations: ACh, acetylcholine, AChE, acetylcholinesterase: QNB. 3-quinuclidiny] benzilate; PBS, phosphate-buffered saline with Ca? and Mg?*. Biochemistry: Sugiyama et al.   ool DRATES OF GNB BINDING JBQNS CONCENTRATION, 2 [AND RELEASE TOTAL |                9 100 a b @ 80 mam Zz Co Tt mou vy g Oa a aT OPE SA SEO MINUTES nV BH (+) ONB Fic. 1. QNB binding to receptors in homogenates of 13-day chicken embryo retina. (A) Kinetics of QNB binding to and release from sites on membrane preparations at 25°. The ordinate represents specific binding of [SH]QNB per 0.273 mg of protein (0.15 and 3.0 ml reaction mixtures for on and off reactions, respectively). The initial concentration of (+)-{3H]QNB was 2.0 nM. For the release kinetics experiment, the reaction mixture was incubated for 3 min and then diluted with 19 volumes of buffer A containing 0.4 uM atropine. Fifty-seven min after the first dilution, the reaction mixture was again diluted 20-fold with buffer A with 0.4 uM atropine. No release of QNB was observed during further incubation (not shown). (B) QNB con- centration curve and Scatchard plot (Inset). Specific binding (@) is the difference between total binding (0) and nonspecific binding (O) in the presence of 0.4 4M atropine. Each point represents the mean of triplicate values. B and F correspond to concentrations (nM) of specifically bound QNB and free (—)-QNB, respectively. The line without points represents the concentration of active isomer (~)-QNB added, The concentration of protein in the reaction mixture was 2.86 mg/m. Tubes were incubated at 4° for 100 min. RESULTS Receptor Properties. The rates of (7H|QNB binding to and release from receptors in homogenates of 13-day chicken em- bryo retina are shown in Fig. 1A. [H|QNB bound rapidly to retina membranes; in the presence of 2 nM (+)-(3HJQNB, half maximal binding was achieved in 2 min and maximal binding, in approximately 15 min. Some, but not all, of the reactions are reversible. The addition of 0.88 uM atropine and dilution of reaction mixtures 20-fold resulted in the dissociation of ap- proximately 50% of the (3H|QNB-receptor complex. QNB association and dissociation reactions both exhibited biphiasic kinetics with fast and slow association reactions and dissociation reactions. The kinetics will be discussed elsewhere; however, the rate constants (k) for fast and slow QNB association reactions were estimated, by assuming bimolecular irreversible reactions asa first approximation, to be 2.7 X 108 M7! min7! and 1.4 X 108 M~! min7}, respectively. Both fast and slow QNB-receptor dissociation reactions were first-order reactions with rate con- stants of 1.2 min7! and 0.041 min7!, respectively. The relationship between [3H|QNB concentration and binding to receptors in retina is shown in Fig. 1B. The binding of the pharmacologically active isomer, (—)-[7H|QNB, to retina receptors is a saturable process. In the presence of 0.4 4M at- ropine, relatively little nonspecific (3H|QNB binding was found with homogenates prepared from 13-day chicken embryo retina; however, nonspecific (3H |QNB binding was markedly increased when homogenates are prepared from >15-day chicken embryo retina or posthatched retina. The dissociation constant (Kp) determined by Scatchard analysis (Fig. 1B and inset) was 0.12 nM (—)-[(3H|QNB. However, we consistently observed higher apparent dissociation constants (~0.4 nM) with homogenates from retina of chickens 2 weeks after hatching and of adult chickens. The calculated concentration of specific ONB binding sites in 13-day chicken embryo retina is 325 Proc. Natl. Acad. Sci. USA 74 (1977) 5525           —T tT ia MUSCARINE OF INTIAL RATE OF QNB BINDING on °   —O     4 L S LOG [B/Bpg-B)] % ° v T NX \\\\ t \\\\ . . ‘N =, 1 J 4   1 4 o oo OO @ 6 [LIGAND] (M) Fic. 2. (A) Inhibition of [[H]QNB binding by various com- pounds. (B) Hill plot. Thirteen-day embryo retinas were used. Whole homogenates and membrane fractions were used and no significant difference was noted. The final concentration of (+)-[PHJQNB was 0.5 nM (1.0 nM in some experiments). Protein concentrations in re- action mixtures were 0.5-2.6 mg/ml. Receptor concentrations were 0.1-1.0 nM. Initial rates of binding (usually 0 to 3-4 min) were fitted to a model of bimolecular irreversible reaction mechanism, and the bimolecular association rate constant was calculated. The apparent rate constant in the presence of protecting drugs was expressed as the percentage of the control value in A. Hill plots were obtained by as- suming that the percentage decrease of [*H]QNB binding rate rep- resents the percentage of the receptor sites occupied by unlabeled ligands which corresponds to B/Bmax- When ACh was tested the ho- mogenate was preincubated with 3 uM eserine for 30 min at 25° before addition of ACh. Symbols: 0, scopolamine; ©, atropine sulfate; ¢, oxotremorine; @, AChCI; a, carbamylcholine chloride; +, pilocarpine; * muscarine. fmol/mg of protein and each retina contained 818 fmol per retina (4.9 X 10!! sites per retina) of specific QNB binding sites. The apparent Hill coefficient is 1.0 (plot not shown), which suggests that QNB binds to independent, noninteracting re- ceptors. Although only one population of QNB binding sites was detected by Scatchard analysis, kinetics of the QNB binding to and release from receptors show that (7H|QNB-receptor complexes are heterogeneous. The effects of different concentrations of unlabeled ligands known to activate or inhibit muscarinic ACh receptors on the initial rate of (3H}QNB binding to receptors in homogenates prepared from 13 day chick embryo retina are shown in Fig. 2A. [SHIQNB binding was markedly decreased in the presence of antagonists of muscarinic ACh receptors such as scopolamine or atropine or receptor activators such as oxotremorine, ACh, carbamylcholine, pilocarpine, or muscarine at expected physiological concentrations. (7H|]QNB binding was not af- fected by prior incubation of homogenates with 10 nM a- bungarotoxin for 3 hr at 25° (not shown). Thus, the specificity of QNB binding sites for ligands closely resembles that of muscarinic ACh receptors. As shown in Fig. 2B, the apparent Hill coefficients of acti- vators of the muscarinic ACh receptor such as oxytremorine, ACh, and carbamylcholine were 0.6 to 0.8, whereas those of receptor antagonists were approximately 1. These results agree well with those of Birdsall et al. (18). The apparent Hill coef- ficients of pilocarpine and muscarine were approximately 1. Pilocarpine has been shown to be both an activator and an an- 5526 Biochemistry: Sugiyama et al. Table 1. Apparent dissociation constants and Hill coefficients of ligands for muscarinic acetylcholine receptors of 13 dav chick embryo retina   App Kp,* Ligands nM h Antagonists QNB 4° 0.12 1.0 25° 0.44 1.0 0.29\" — Atropine 0.69 1.0 Scopolamine 0.17 1.1 Activators Oxotremorine 130 0.7 Acetylcholine 1,100 0.8 Carhamylcholine 1,700 0.6 Muscarine 8,700 Ll Pilocarpine 1,100 1.0 Local anesthetics Tibucaine 30,000 1.0 Tetracaine 21,000 1.4   * Values at 25° except otherwise specified. The Kp values for QNB were obtained by determining the binding of |\"H}QNB at equilib- rium. The Kapp values for the other antagonists and activators represent the concentrations that result in 50% inhibition of the initial rate of [\"H]QNB binding; values were not corrected for h< 1. The K app values for local anesthetics are estimated from experi- ments where the retina homogenate with or without different con- centrations of a local anesthetic were incubated for 60 min at 25° in the absence of [*H|QNB, then 0.50 nM (+)-[7H]QNB was added and the reaction mixtures were incubated for an additional 5 min. + 0.29 and 4.4 nM (—)-QNB are the dissociation constant values de- termined from rate constants for slow and fast association and dissociation reactions, respectively. tagonist of the muscarinic ACh receptor; although muscarine is an activator of the muscarinic ACh receptor in other organ- isms, the apparent Hill coefficient with chicken embryo retina receptors resembles that of a receptor antagonist. The apparent Hill coefficients of <1 observed with oxytremorine, ACh, and carbamylcholine can be interpreted in various ways such as negative cooperativity, heterogeneity of binding sites, or de- sensitization of the ACh receptor. The demonstration by W. Klein in this laboratory that heterogeneity of muscarinic ACh receptors can be detected by kinetic studies was confirmed. Muscarinic ACh Receptors during Embryonic Develop- ment. The concentration and number of muscarinic ACh re- ceptors in chicken embryo retina are shown in Fig. 3 A and B, respectively, as a function of developmental age. Values re- ported for nicotinic ACh receptors (16) also are shown for comparison. Muscarinic ACh receptors were detected in 5.5- day embryo retina, but the concentration was relatively low (10 fmol/mg of protein). Between the 6th and 14th days of em- bryonic development, the concentration of specific QNB binding sites increased 30-fold. In the 5.5- to 9-day chicken embryo retina, specific QNB binding sites accumulated expo- nentially with a doubling time of approximately 26 hr; in 9- to 14-day embryo retina, the doubling time was approximately 60 hr. The maximal concentration of QNB binding sites (320 fmol/mg of protein) was attained in the retina of the 13- to 14-day embryo. No further change was detected during later embryonic development; however, receptor concentrations were lower in retina 2 weeks after hatching and in adult retina. Although the concentration of receptors decreased after hatching, the number of receptors per retina increased slightly after hatching (Fig. 3B). The adult chick retina contained 1200 Proc. Natl. Acad. Sci. USA 74 (1900)   RET RORLNE RECEPTOR ROETYIOHOUNE RECEPTORS, yey 2O000F CONCENTRATION PER RETINA ee 2000 ® ® o e 1ooob + F487 1000 sooF NN NO “pauses 4 500        CULTURE x L ey, t t ‘ ‘ 100 NICOTINIC 4 100 go oO tT eu oO fMOL QNB OR a-BT BOUND/RETINA HATCH fMOLS QNB OR a-BT BOUND /mg PROTEIN °   14 sail       Shitty yt tye) 5 05 10 15 20 4ADLTS 10 18 20) ADULT DAYS Fic. 3. The accumulation of QNB binding sites in chicken em- brvo retina as a function of development age. The number of specific sites per mg protein (A) or per retina (B) is shown. For comparison, «-bungarotoxin (a-BT) binding to nicotinic ACh receptors (16) is shown (broken lines). Circles. intact retina in vivo; triangles, cultured cells dissociated from 8-day chicken embryo retina. Filled circles are values obtained by Scatchard analysis. Open symbols represent the specific binding obtained at 6-10 nM (+)-[SH]QNB. Tubes were in- cubated at 4° for 60 min. In some cases, the retina dissection was not complete; the amount of protein per retina then was estimated from the published values (19). Each point represents the mean of at least three determinations. fmol of specific QNB binding sites per retina(7.2 X 10!! sites per retina), These results show that genes for muscarinic ACh receptors are expressed early in the development of the retina and suggest that some neurons synthesize muscarinic ACh receptors but not neuroblasts as reported for nicotinic ACh receptors (16). The number of muscarinic and nicotinic ACh receptors increase more than 30-fold and the receptors accumulate at similar rates between the sixth and ninth days in embryos. The maximal concentration of muscarinic ACh receptors is attained in the retina of the 13-day embryo, whereas nicotinic ACh receptors continue to increase until hatching. The concentration of spe- cific QNB binding sites in retina of the 6- to 13-day embryo is 2- to 3-fold higher than the concentration of a-bungarotoxin binding sites; however, this ratio is reversed in the adult retina. Thus, the ratio of muscarinic to nicotinic ACh receptor changes markedly during retina development. Cells dissociated from 8-day chicken embryo retina were cultured for various times in rotating petri dishes. At various times, homogenates were assayed for specific binding of (3HJQNB (Fig. 3A). The concentration of QNB binding sites increased from 50 fmol of specific QNB binding sites per mE of protein in retina of the 8-day embryo to 225 fmol/mg ol protein after 4 days of culture. Thus, the accumulation 0! muscarinic ACh receptors in cultured retina cells resembled that in the intact retina. Receptor Distribution in Retina. The distribution of [SH|QNB binding sites in intact 13-day chicken embryo retina and in adult retina is shown in Fig. 4. In 13-day embryo retina. most of the silver grains were localized in two narrow bands within the inner synaptic layer of the retina (also termed “inner plexiform layer”) (Fig. 4 A and B). In adult retina (Fig. 4 C and Biochemistry: Sugiyama et al. SS ee ve Fic. 4. Autoradiography of sections of chicken retina incubate with [\"H|QNB in the absence of atropine. (A) Phase-contrast view and (B) dark-field view of stained section of 13-day embryo retina exposed for 390 days. (C) Phase-contrast view and (D) dark-field view of stained section of adult chicken retina exposed for 173 days. (Bars represent 100 um.) Lines at the left of each photograph represent the boundaries of layers: R, photoreceptor layer; O, outer synaptic layer; IN, inner nuclear layer; IS, inner synaptic layer: G, ganglion cell layer: A, ganglion axon layer. D), two or three bands of silver grains could be seen within the inner synaptic layer of the retina. Histograms relating the density of silver grains on autora- diographs that had been exposed for shorter times with grain location over the retina are shown in Fig. 5. Two sharply de- fined bands of silver grains of equal density can be seen within the inner synaptic layer of 13-day embryo retina. Fewer silver grains were associated with the lower portion of the inner nu- clear layer (cell bodies of amacrine and bipolar neurons and Muller cells) and with ganglion neuron soma and axons but were not associated with other regions of the retina. The average number of silver grains over the entire retina incubated in the absence or presence of 0.4 4M atropine (nonspecific (RH|QNB binding) was 3.83 and 0.87 grain per 100 um?, respectively.                                                25 & ~ 1.5 2 eS 2 S.0Fr Oo 2 a =. 2 a 2.97 0.5 S$ a AQ ~ uy | UONSPECIFIC _° : | Loa iC a IT 4 qe | ~~ eL LOX a = io 2 io on Ceo Tr I 0.5 \\\\ I pepe Oo jeg Joey tieins po 0 RO N S Ga RON S Fig. 5, Histograms showing the grain distribution in ]H|QNB autoradiographs of sections of 13-day chicken (A) or adult chicken (B) retina. Sections of retina of both ages, treated for both total binding and nonspecific binding, were subjected to autoradiography for 65 days. Grains were counted at X600 magnification by using a camera lucida. Specific binding was obtained by subtracting non- specific from total binding. Number of grains counted were: 543 and 220 for total and nonspecific binding, respectively, for 13-day embryo retina: 1592 and 847 for total and nonspecific binding, respectively, tor adult retina. Abbreviations are as in Fig. 4.       Proc. Natl. Acad. Sci. USA 74 (1977) 5527 EMBRYO ADULT O DAY \\\\ Ar ~ ob) acre acre ud \\\\ oT 4 s O 20 oe tT > <t <I 240 az & 2 5 , i sy ee < od { zo. 3 2 = 3 5 go 4° 32 Ica = 5   Fic. 6. Schematic representation of the relative distributions and concentrations of muscarinic (M) and nicotinic (N) ACh receptors (16) and ACHE activity (8) in the inner synaptic layer of chick retina. M and N are from 13-day embryo or adult retinas; AChE is from 12-day embryo and newly hatched chicken retinas. Open, stippled, hatched, and filled areas represent relative ACh receptor concen- trations or AChE activity in increasing order. Numbers refer to peak positions. The top and the bottom of the figure (0 and 100%) corre- spond to the inner nuclear and ganglion neuron boundaries of inner synaptic layer, respectively. Cajal’s layers for chicken retina are from plates 4 and 5 of ref. 20. Thus, specific QNB binding accounted for 77% of total QNB binding. In the adult retina (Fig. 5B), three bands of specific QNB binding sites were localized in the inner synaptic layer of the retina. Few, if any, specific binding sites for QNB were detected elsewhere in the retina; thus, muscarinic ACh recep- tors are localized to a greater extent in the adult retina than in the 13-day embryo retina. In other sections, the first band of specific QNB binding sites near the inner nuclear layer over- lapped the first and second fractions of the inner synaptic layer and the demarcation between the second and third bands was less distinct than that shown. The average number of silver grains over the entire retina in the absence or presence of 0.4 uM atropine was 1.39 and 0.96 grain per 100 um”, respectively. Specific (3H|QNB binding was 31% of total QNB binding, in accord with ligand binding results. Adult retina was incubated with 2 rather than 4 nM (3H|QNB to decrease nonspecific (3H |QNB binding; however, the grain density was somewhat lower than expected. These results show that muscarinic ACh receptors are lo- calized in the inner synaptic layer of the retina and suggest that the receptors are present in some amacrine and ganglion neu- rons but not in other cell types in the retina. DISCUSSION The distribution of muscarinic ACh receptors within the inner synaptic layer of chicken embryo and adult retina is compared with previously reported distributions of nicotinic ACh re- ceptors (16) and AChE activity (8) in Fig. 6. Muscarinic and nicotinic ACh receptors and AChE activity are localized in bands within the inner plexiform layer of chick retina. In the embryo, two bands, each with high concentrations of muscar- inic ACh receptors and high AChE activity, can be seen; nico- tinic ACh receptors are distributed diffusely in two broad bands throughout most of the inner synaptic layer. After hatching, the inner synaptic layer of the retina contains three muscarinic ACh receptor bands, four nicotinic ACh receptor bands (16), and four bands with high AChE activity (8). Nicotinic ACh receptors are present in the outer synaptic layer (16), but not muscarinic receptors. Most, but not all, of the ACh receptor bands are associated with AChE bands. The bands appear in 5528 Biochemistry: Sugiyama et al. an ordered sequence during development, with respect to temporal and positional relationships. The maximal concen- trations of muscarinic and nicotinic ACh receptors are attained on the 18th and 21st days of embryonic development. Thus, most of the synapses mediated by muscarinic ACh receptors probably are formed at an earlier developmental stage in retina than those mediated by nicotinic ACh receptors. Vogel et al. (21) have shown that nicotinic ACh receptors are localized at sites of synapses in chicken retina. The localized bands of muscarinic ACh receptors within the inner synaptic layer and the apparent absence of the receptors from cell bodies and axons of ganglion neurons in adult retina raise the possibility that muscarinic receptors also may be localized at certain synapses. Further work is needed to resolve this question. Bipolar neurons and photoreceptors of retina form double or triple synapses (ribbon synapses) wherein one cell transmits information across one synapse simultaneously to two or three neurons. ACh probably is the transmitter at some double sy- napses of bipolar neurons because localized nicotinic ACh re- ceptors have been found on the processes of one or both post- synaptic cells (21). Because three species of ACh receptor— muscarinic excitatory, muscarinic inhibitory, and nicotinic—are widely distributed in the nervous system, ACh released at one synapse may excite and/or inhibit the recipient neurons, de- pending on the species of ACh receptor that are present. The inner synaptic layer is composed predominantly of neurites of amacrine, bipolar and ganglion neurons, and syn- aptic connections with processes of ganglion, amacrine, or bi- polar neurons. Five layers can be distinguished by phase-con- trast microscopy but not by transmission electron microscopy within the inner synaptic layer. However, Dubin (22) has shown that three classes of synapses that can be identified by ultra- structural features are stratified in different ways in the inner synaptic layer of pigeon retina; stratification was not detected in the retina of other organisms examined. Eleven layers can be distinguished within the inner synaptic layer of chick retina on the basis of ACh receptor concentrations and AChE activity. Three additional layers rich in catechol- amines have been identified in the inner synaptic layer of chicken retina (5), and four or five glutamic acid decarboxylase bands have been detected in rat retina (23). These results show that neurites of one type sort out from those of other types. A neuron that forms synaptic connections with two or more neurons is, in effect, a polyvalent crosslinking agent. Thus, neighboring neurons that form synapses with two or more cells of the same type, at the same stage of development, become linked to one another and sort out from other sets of neurons. Since a single neuron may both send and receive information Prac. Natl. Acad. Sci. USA 7-4 (1977) across synapses and may form multiple kinds of synapses, such neurons may link sets of neurons that form different types of synapses. The extent of sorting out and the relationship of one class of neurons to another may be determined by the number and kinds of synapses formed by each class of neurons (both pre- and postsynaptic connections), the sequence of synapse for- mation, and the initial spatial relationships of the neurons. We thank Linda Lee for excellent assistance. 1. Vogel, Z., Daniels, M. P. & Nirenberg, M. (1976) Proc. Natl Acad. Sci. USA 73, 2370-2374. 2. Puro, D. G. DeMello, F. G. & Nirenberg, M. (1977) Proc. Nail. Acad, Sci. USA 74, 4977-4981. 3. Graham, L. T., Jr. (1974) in The Eye, eds. Davson, H. & Graham, L. T., Jr. (Academic Press, New York), Vol. 6, pp. 283-342. 4. Neal, M. J. (1976) in Transmitters in the Visual Processes, ed. Bonting, S. L. (Pergamon Press, New York), pp. 127-143. 5. Ehinger, B. (1976) in Transmitters in the Visual Processes, ed. Bonting, S. L. (Pergamon Press, New York), pp. 145-168. 6. Ross, C. D. & McDougal, D. B., Jr. (1976) J. Neurochem. 26, 521-526. 7. Lam, D. M. K. (1976) Cold Spring Harbor Symp. Quant. Biol. 40, 571-579. 8. Shen, S. C., Greenfield, P. & Boell, E. J. (1956) J. Comp. Neurol. 106, 433-461. 9. Spira, A. W. (1974) J. Histochem. Cytochem. 22, 868-880. 10. Masland, R. H. & Ames, A., JI (1976) J. Neurophysiol. 39, 1220-1235. 11. Straschill, M. & Perwein, J. (1973) Pfliigers Arch. 339, 289- 298. 12. Vivas, I. A. & Drujan, B. D. (1977) 6th Meeting International Soc. Neurochem., p. 149 (Abstract). 13. Noell, W. K. & Lasansky, A. (1959) Fed. Proc. 18, 115. 14. Lindeman, V. F. (1947) Am. J. Physiol. 148, 40-44. 15. Wang, G. K. & Schmidt, J. (1976) Brain Res. 114, 524-529. 16. Vogel, Z. & Nirenberg, M. (1976) Proc. Natl Acad. Sci. USA 73, 1806-1810. 17. Yamamura, H. I. & Snyder, S. H. (1974) Proc. Natl. Acad. Sci. USA 71, 1725-1729. 18. Birdsall, N. J. M., Burgen, A. S. V., Hiley, C. R. & Hulme, E. C. (1976) J. Supramol. Struct. 4, 367-371. 19. DeMello, F. G., Bachrach, U. & Nirenberg, M. (1976) J. Neuro- chem. 27, 847-85]. 20. Ramon y Cajal, S. (1972) The Structure of the Retina, translated by Thorpe, S. A. & Glickstein, M. (Charles C Thomas, Springfield, IL). 21. Vogel, Z., Maloney, G. J., Ling, A. & Daniels, M. P. (1977) Proc. Natl. Acad. Sci. USA 74, 3268-3272. 22. Dubin, M. W. (1970) J. Comp. Neurol. 140, 479-505. 23. Barber, R. & Saito, K. (1976) in GABA in Nervous System Function, eds. Roberts, E., Chase, T. N. & Tower, D. B. (Raven Press, New York), pp. 113-132.", "Daniels, Mathew P.,Nirenberg, Marshall W. ; Sugiyama, Hiroyuki", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4x8d~hmac-2he8", "00000000-0000-0000-DF25-3839B1B1B9D3", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Regulation of Acetylcholine Release from Neuroblastoma x Glioma Hybrid Cells", "101584910X63", null, "1978", "March 1978", "In this article the authors describe the process of synaptic communication in hybrid cells and show that the ability of cells to release neurotransmitters in response to stimuli can be regulated.", "Articles", "Acetylcholine,Neurons,Neuroblastoma,Synaptic Membranes,Metabolism", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 13814-1318, March 1978 Biochemistry Regulation of acetylcholine release from neuroblastoma X glioma hybrid cells (synapse formation/dibutyryl cyclic AMP/neuron development) RICHARD MCGEE®®, PAUL SIMPSON®°, CLIFFORD CHRISTIAN’, MARINA MATA‘, PHILLIP NELSON?, AND MARSHALL NIRENBERG? * Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, and 4Behavioral Biology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, January 10, 1978 ABSTRACT Neuroblastoma X glioma NG108-15 hybrid cells exposed to N®,O0?'-dibutyryladenosine 3':5'-cyclic mono- phosphate for several days release [SH]acetylcholine in response to serotonin, prostaglandin F2,,, KCI, or veratridine. NG108-15 cells grown in the absence of dibutyryl cyclic AMP do not re- spond to an excitatory stimulus by releasing [2H]acetylcholine but can be shifted to a responsive state by treatment with di- butyryl cyclic AMP. Thus, the reactions that are required for acetylcholine release can be regulated in NG108-15 cells, thereby regulating the ability of cells to form synapses and the efficiency of synaptic communication.   Neuroblastoma X glioma NG108-15 hybrid cells form synapses with cultured striated muscle cells (1-4). Synaptogenesis by NG108-15 cells is greatly increased when hybrid cells are treated for a period of days with N®,O?-dibutyryladenosine 3’:5’-cyclic monophosphate (BtpcAMP). Culture of NG108-15 cells with BtgcAMP also results in increases in cell body diam- eter, neurite extension, abundance of clear vesicles 600 A in diameter (5), membrane excitability, and specific activities of choline acetyltransferase (acetyl-CoA:choline O-acetyltrans- ferase, EC 2.3.1.6) and acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7).¢ The cells generate both Nat and Ca?* action potentials and have endorphin receptors, excitatory muscarinic acetylcholine {ACh) receptors, a-adrenergic receptors, prostaglandin E) (PGE) receptors, PGF2, receptors, and adenosine receptors that are coupled to shifts in cyclic AMP and/or cyclic GMP levels of the cells (6-14).¢£8 Serotonin (13), ACh (13), PGF, (13),8 or dopamine (14) can depolarize the cells and initiate action potentials. In this report, we describe the properties of stimulus-de- pendent release of [7H]ACh from NG108-15 cells, a response required for synaptic communication, and show that the ability of the cells to release [[H]ACh in response to stimulation can be regulated by BtgcAMP. A preliminary report of this work has appeared (15). MATERIALS AND METHODS Cell Culture. The culturing of NG108-15 hybrid cells has been described (1). To switch cells to a more differentiated state, growth medium [90% Dulbecco's modified Eagle’s minimal essential medium (DMEM) (GIBCO H-21)/10% fetal bovine serum/100 »#M hypoxanthine/] uM aminopterine/16 uM thymidine] was supplemented with 1 mM BtecAMP, purified   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S. C. §1734 solely to indicate this fact. 1314 as described (1), and the fetal bovine serum concentration was decreased from 10% to 5%. Cells to be used for experiments were dissociated and transferred to 200-ul disposable glass capillary pipettes (total volume, 300 ul) bent in the form of a “U” and connected in series (1.5-2.0 X 10° cells per capillary) and incubated for 3 hr at 37° to promote cell attachment to the glass. The tubes then were perfused with 2 ml of medium per hr for 2 days unless indicated otherwise.\" Each U tube then contained 200-400 yg of cell protein. Measurement of [3H|Choline Uptake and [7HJACh Re- lease. Cells in capillaries were washed for 3 min by perfusion (0.4 ml/min) with medium A [DMEM without choline, NaHCOs, phenol red, and fetal bovine serum but with 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) and 125 mM NaCl, adjusted to pH 7.4 and 340 mOsm/liter]. Cells then were incubated without perfusion in medium A supplemented with [methyl-°H]choline (10.1 Ci/mmol, Am- ersham/Searle) as indicated in figure legends. For cells grown in the presence of BtgpcAMP, 1 mM BtgcAMP also was added to the [2H]choline uptake medium but not to the wash medium. After incubation, most of the [2H ]choline in the medium was removed by perfusion with medium B (medium A with 20 uM eserine sulfate), When only intracellular [SHjcholine, (8H]ACh, and other 3H-labeled metabolites of choline were to be deter- mined, the tubes were perfused for 1.5 min (0.7 ml/min) with medium A, a small air bubble was introduced into the line, and the capillary was filled with acetone/1 M formic acid, 85:15 (vol/vol), to precipitate protein and extract SH-labeled com- pounds from cells (16). The acetone/formic acid extract was collected and each tube was washed twice with 100 yl of the acetone /formic acid solution; the extracts and washes then were combined. Greater than 98% of the intracellular 9H-labeled compounds were recovered. Cell protein in each capillary then was dissolved in 0.4 M NaOH and assayed by a modification of the method of Lowry et al. (17) with crystalline bovine serum albumin as the standard. The 2H-labeled compounds extracted from cells were frac- tionated by high-voltage paper electrophoresis with 1.5 M acetic   Abbreviations: BtgcAMP, N®,O9-dibutyryladenosine 3':5’-cyclic monophosphate; ACh, acetylcholine; PG, prostaglandin; DMEM. Dulbecco's modification of Eagle’s minimum essential medium; 5-HT. 5-hydroxytrypamine (serotonin). > Present address: Department of Pharmacology, Georgetown Uni- versity, Washington, DC 20007. © Present address: Cardiology 111C, Veterans Administration Hospital. San Francisco, CA 9412). ¢ B. Hamprecht, T. Amano, and M. Nirenberg, unpublished data. f S, Sharma and M. Nirenberg, unpublished data. & H. Matsuzawa and M. Nirenberg, unpublished data. h Details of the capillary culture tube system will appear elsewhere. Biochemistry: McGee et al.   T T T T T t                 4 (HI ACh [°H} Choline a) \" 3 50 3 L 450 & 2 £5 s £ % (—) Enzyme x B40} & . 1 2 x a9 5 10 ( | = ® O18 fmt X 10° | 00's gE 2 q |* x 207 4 any a (+) or (—) enzymef 450 3 oo IOF 1 £ E (+) Enzyme 1 1 4 L 1 1 i Oo 16 20736 40 560 50 io 150° fmot [9H] ACh or [3H] choline Fic. 1. [2H]ACh was separated from [?H]choline by conversion of [3H] choline to [7H}phosphorylcholine with choline kinase and ex- traction of [SHJACh into 3-heptanone/30 mM tetraphenylboron. Indicated amounts of [7H]ACh (Left) or [?H]choline (Right) (both 10.1 Ci/mmol) were incubated in the absence or presence of 2.5 munits of choline kinase. [7H]ACh or unreacted {*H]choline then were ex- tracted into the organic phase and radioactivity was determined. Each point represents the mean of four values; SEM <4%. acid/0.75 M formic acid (18). ACh, choline, phospholipids, and a fraction containing phosphorylcholine and CDP-choline were identified by coelectrophoresis with authentic standards. The paper was treated with Ip vapor to visualize spots, 9H-labeled compounds in each fraction were eluted from the paper with 1 ml of water, the eluate was mixed with 10 ml of Hydromix (Yorktown Research), and radioactivity was determined by using a liquid scintillation spectrometer. When [8H]ACh release from cells was measured, cells that had been incubated with [°H]choline were washed by perfusion with medium B at 0.4 ml/min for 8-14 min as indicated in the figure legends and fractions then were collected at 2-min in- tervals unless indicated otherwise. Cells were exposed to dif- ferent compounds by stopping the peristaltic pump for a few seconds to switch from one perfusion medium to another. To separate (7H]ACh and [°H]choline in the perfusate, a modification of the method of Goldberg and McCaman (19) was used in which (8H|choline was converted to (7H |phospho- rylcholine during incubation with choline kinase, and then (SHJACh and remaining traces of [3H]choline, but not (3H]- phosphorylcholine, were extracted with 3-heptanone/30 mM tetraphenylboron (Sigma). Choline was converted to phos- Phorylcholine in a reaction mixture containing, in a final vol- ume of 0.275 ml, the following: 0.25 ml of perfusate, 10 mM NaATP, 10 mM MgCl, and 2.5 munits of choline kinase (ap- proximately 10 ug of protein, Sigma). Reaction mixtures were incubated for 15 min at 36° and then extracted with 2 ml of 3-heptanone/30 mM tetraphenylboron. The organic phase was removed and part (1.7 ml) was evaporated at 60° under a stream of air, the residue was dissolved in Hydromix, and ra- dioactivity was determined with a liquid scintillation spec- trometer. The amount of [3H]JACh or [*H]choline released by cells was calculated with the assumption that the specific ra- dioactivity of [SH]choline was not diluted. Because the (°H]- choline probably is diluted by unlabeled choline of cells, the values reported are lower than true values. As shown in Fig. 1, >97% of authentic [3H}]ACh or [2H |cho- line is extracted into the organic phase over the wide range of concentrations tested. However, after incubation with choline kinase, 97% of the [SH]ACh, but only 3-5% of the [3H |choline (or other 3H-labeled compounds present), is extracted into the organic phase. Purification of the [methyl-2H|choline by paper electrophoresis prior to use decreased the amount of 3H-labeled Proc. Natl. Acad. Sci. USA 75 (1978) 1315 material extracted after incubation with choline kinase to <1% of the (8H]choline present initially. The amount of 3H-labeled material extracted into the organic phase after incubation of [H]choline with choline kinase was determined for each ex- periment and the amount, <25% of the total 3H-labeled com- pounds extracted was subtracted from the total to calculate the amount of [SHJACh. The sensitivity of the assay is limited by the specific activity of the [SH]ACh and the quantity of other 3H-labeled compounds which are extracted with [SH]ACh. The maximum sensitivity in our experiments was 2 fmol of (SH|ACh per sample. The assay also is rapid, and as many as 300 samples were analyzed in a single day by one person. Characterization of Released [*H]JACh. Perfusate fractions from three tubes were pooled and eserine was removed by ex- traction with equal volumes of CHCls. The aqueous phase then was warmed to 36° to remove residual CHCl, and the solution was incubated with choline kinase as described above in the presence or absence of 0.5 unit of acetylcholinesterase (0.5 yg of protein, electric eel, Sigma) in a 0.25-ml! reaction mixture. Greater than 95% of authentic [SH]ACh was hydrolyzed under these conditions. RESULTS Release of [2HJACh from Cells Grown with or without BtgcAMP. NG108-15 cells incubated with (3H choline release both ([SHJACh and [H]choline into the medium. The release of [7H]ACh and [°H]choline into the medium from NG108-15 cells grown with or without BtgcAMP is shown in Fig. 2. NG108-15 cells grown without BtgcAMP released (SH|ACh into the medium but 80 mM K* had little or no effect on the rate of release. In contrast, the rates of [SH]ACh release from cells grown in the presence of 1 mM BtgcAMP increased in response to KCI. The basal unstimulated rates of [SH]ACh release from cells treated with BtseAMP were twice those of control cells, Both basal and KC]-stimulated rates of (3HJACh release in- creased throughout the 5 days of treatment with BtgcAMP. These results show that populations of NG108-15 cells can be shifted from an unresponsive to a responsive state with respect to KCl-dependent ACh release by exposure of cells to BtocAMP. KC] also stimulated the release of [8H]choline from both control and BtgcAMP-treated cells to approximately the same extent. The rates of basal and KCl-stimulated [2H]choline re- lease decreased between the 1st and 5th days of culture. These results show that BtgcAMP has little or no effect on basal or KCl-stimulated release of [SH choline. The (SHJACh released from cells was characterized further by determining the sensitivity of the 3H-labeled material re- leased by hydrolysis catalyzed by acetylcholinesterase (Table 1). After incubation in the presence of acetylcholinesterase, the 3H-labeled material recovered in the organic phase was <5% of the amount obtained in the absence of acetylcholinesterase. In experiments not shown here, >97% of the $H-labeled com- pounds released from cells exhibited the electrophoretic mobilities of ACh or choline. Veratridine, which activates action potential Na* ionophores, also stimulated (7H]ACh release from NG108-15 cells grown in the presence of BtgcAMP but had little or no effect on cells grown without BtgcAMP (Fig. 3A). The response to veratridine decreased with time. Veratridine-stimulated ACh release was abolished in the presence of 1 uM tetrodotoxin, a specific in- hibitor of action potential Na+ ionophore activation, but te- trodotoxin had no effect on the basal, unstimulated rate of [SHJACh release. Veratridine increased the rate of [SH ]choline release, after a delay, from cells grown with or without 1316 Biochemistry: McGee et al.                eo T r rr r : 7 50k A Control cells I B Bt,cAMP-treated cells Acetylcholine c g 2 4 (OOF aD E ~ , e KCI 5 days £ 3 50r 4 o 3 days s 3 days K 3 c 1 day g Ow + + ; oy + +t + t 3 900F c yD | £ = Choline a 3 KL = 600f / 1 day, 5 / <f 4 day Wood xi a Na LS Qa 3 days 0200 . | E> NOR - bt—H 3 days 1 ‘ KCI KCl 5 days © 10 \\\\6 22 28 10 6 22 28 Minutes Fic. 2. Effect of culturing NG108-15 cells without or with BtscAMP on KCl-stimulated (solid symbols) release of (SHJACh (A and B) and of [8H] choline release (C and D). A cell suspension (ap- proximately 200 ug of cell protein) was added to each capillary and the tubes then were perfused with DMEM supplemented with 5% fetal bovine serum for 24 hr. Some tubes then were perfused with the above medium supplemented with 1 mM BtocAMP. One (0), 3 (0), or 5 (A) days later, the release of (7H]ACh and {?H] choline from cells was determined. Cells were incubated with 10 uM [*H]choline for 45 min and washed for 10 min with medium without choline but with 20 uM eserine sulfate (wash discarded), and then fractions were collected at 2-min intervals (0.8 ml per fraction). Stimulation was with 80 mM KCI for 6 min as indicated. Each point represents the mean of four values obtained with separate cultures. Protein ranged from 200 yg per culture on the first day to 550 wg on the fifth day with or without BtocAMP. BtpcAMP but tetrodotoxin did not inhibit veratridine-stimu- lated [2H]choline release. This suggests that the increase in choline release due to veratridine is not mediated by the acti- vation of action potential Nat ionophores. Choline Metabolism. The effect of growing cells in the presence of BtgcAMP for 0, 9, or 16 days on [°H]choline me- tabolism is shown in Table 2. The intracellular concentrations of (SHJACh and [H]choline increased and those of phospho- Table 1. Effect of acetylcholinesterase on released [>H]ACh   [SHJACh released, Stimulation fmol/mg protein of cells ~AChE +AChE None (before stimulation) 126 6 80 mM KCl] 211 3 None (after stimulation) 136 0   NG108-15 cells grown for 9 days with 1 mM BtgcAMP were incu- bated with 10 uM [H]choline for 45 min and washed by perfusion for 10 min. Six-minute fractions were collected before, during, and after stimulation with 80 mM KCl and perfusates from three cultures were then combined. Eserine was then extracted with CHCl, and the [2H]ACh content of the perfusates was determined after incubation in the presence or absence of acetylcholinesterase (AChE). Each value is the mean of duplicate determinations. Proc. Natl. Acad. Sci. USA 75 (1978)                       3 — oO | Acetylcholine A |. Choline B | o c 2100) > fA 500 2 \\\\. Treated 3 E 3 re ; ® € 490 o£ £* Jee Treated|“” £3 o 3 z° 3 =a © 0 TTX Control | o> 3 SO CE C = £ as g Control UE — 8 H—H 150 6 = Veratridine £ = Veratridine 2 Olu 1 1. 1. lye po 1 0 E 6 0 4 2&8 2 66 M0 4 BW RW 3 Minutes Fic. 3. Effect of culturing NG108-15 cells with or without BtgcAMP on veratridine-stimulated release of [PHJACh (A) or [3H] choline (B). Cells cultured with or without 1 mM BtgcAMP for 7 days were dissociated and transferred to U tubes (about 300 ug of protein per tube) and then perfused with the same growth medium with or without BtecAMP for 2 additional days. Cells were incubated with 10 uM [H]choline for 45 min and washed for 16 min as described in the legend to Fig. 2. Cells were exposed to 0.2 mM veratridine for 6 min as indicated. @,0, Cells treated with BtecAMP for 9 days; 4,4, cells cultured without BtscAMP for 9 days. 0,4, 1 uM tetrodotoxin (TTX) added at the start of the 16-min wash. Each point is the mean of four values obtained with separate cultures. rylated 3H-labeled compounds derived from [2H]choline de- creased as the time of exposure of cells to BtpeAMP was in- creased. After 16 days of exposure to BtgecAMP, NG108-15 cells had 2- and 5-fold higher levels of (SH|ACh and [°H]choline, respectively, than cells not exposed to BtgeAMP. The results shown in Table 2 were obtained with NG108-15 cells that had been incubated with [8H ]choline and then washed in the absence of extracellular choline for 36 min. The results shown in Fig. 4 were obtained with cells that had been incu- bated with [2H]choline and washed only 1.5 min prior to the extraction of 3H-labeled compounds from cells. The intracel- lular level of [2H]choline increased rapidly during the first 10 min of incubation and then plateaued, whereas the amount of (SHIACh in cells increased throughout the 60-min incubation period. (8HJPhosphorylcholine accumulated at a rapid linear rate for 60 min. After a short lag, [SH ]phospholipids also ac- cumulated rapidly, which suggests a precursor—product rela- tionship between [*H|phosphorylcholine and [8H]phospho- lipids. The relationship between extracellular choline concentration and uptake and metabolism of [*H]choline by NG108-15 cells is shown in Fig. 4 right. The accumulation of 3H-labeled phosphorylated compounds derived from [3H|choline and of Table 2. [H]Choline metabolism by NG108-15 cells grown with       or without BtgcAMP Intracellular levels, pmol/mg protein Days Phosphorylated Total with {H]- [H]- [3H] choline (SH] BtacAMP ACh Choline metabolites compounds 0 19.3 3.3 685 708 9 33.8 11.3 704 750 16 40.0 16.2 519 575   NG108-15 cells grown with BtgcAMP for 0, 7, or 14 days were transferred to U tubes and perfused with medium with or without BtocAMP for 2 days. Cells then were incubated with 10 2M [3H]cho- line for 45 min and washed for 36 min with medium B. 3H-Labeled metabolites in the cells were extracted and subjected to paper elec- trophoresis, Each value represents the mean of three values obtained with separate cultures.   Biochemistry: McGee et al.   900; + PL, Pch L Choline;      300 Choline + 4 pmol [*H] choline incorporated/mg protein ACh   L i L i 10 2 30 40 50 60051 5 10 50 100 500 Choline, uM Fic. 4. Choline uptake and metabolism by BtecAMP-treated NG108-15 cells. (Left) Cells that had been grown with 1 mM BtecAMP for 16 days were incubated with 10 4M [8H] choline for the times indicated and then washed for 1.5 min at a perfusion rate of 0.7 ml/min. The cells then were extracted with acetone/1 M formic acid, 85:15 (vol/vol), the 3H-labeled compounds extracted were separated by high-voltage paper-electrophoresis, and the protein content of each culture was determined. Each point represents the mean of three values from separate cultures. PCh, (?H]phosphorylcholine and [SH]CDP-choline; PL, [H] phospholipids; choline, [2H] choline; ACh, (83H}ACh. (Right) Cells that had been treated with BtzcAMP for 11 days were incubated with [*H]choline at the concentrations indicated for 15 min and then washed; then, [H]choline and metabolites were extracted as for Left. PCh + PL, phosphorylcholine, CDP-choline, and phospholipid; choline, [3H]choline; ACh, [SHJACh. (Inset) Total uptake of ['H]choline by cells.   Minutes total 3H-labeled compounds increased as the extracellular choline concentration was increased and plateaued at 250 uM (2H |choline. However, [2H]choline and [SH]ACh levels in cells increased over the entire range of extracellular [SH|choline concentrations tested (0.5-500 uM). Data analysis by the method of Lineweaver and Burk suggests that NG108-15 cells have both high- and low-affinity choline uptake mechanisms (not shown); however, further work is needed to define more accurately the properties of the choline uptake systems. The results also show that, as the extracellular choline con- centration is increased, the levels of intracellular (H|choline and (3H]ACh increase at approximately the same rate. The distribution of 3H-labeled compounds accumulated by the cell remained constant between 0.5 and 250 uM extracellular (3H choline. These results suggest that the synthesis of (7HJACh is not preferentially coupled to the high- or low-affinity choline uptake systems. Properties of [7HJACh and {3H]Choline Release. KCl repetitively stimulated [7H]ACh release from NG108-15 cells perfused with control medium containing 1.8 mM Ca?* and 0.8 mM Mg?*; but the second response to KC] was smaller than the first response (Fig. 5). Omission of Ca?* and increasing Mg?+ to 4 mM abolished the KC]-dependent (9H]ACh release and also decreased the unstimulated rate of [7H]JACh release. Omission of Ca?+ and increasing Mg?* partially decreased the KCl-stimulated release of [2H |choline from cells. The relationship between KCI concentration and [SH]ACh release from cells is shown in Fig. 6 left. KCI at 30 mM stimu- lated the release of (2HJACh from cells but not as effectively as 40 or 80 mM KCI. A second addition of KCl resulted in the release of approximately half the amount of [H]ACh compared to the first response to KC] at each concentration of KC] tested. These results suggest that the decreased (8H|ACh release evoked by the second application of KCl may not be due to depletion of an intracellular pool of releasable [SHJACh. As shown in Fig. 6 right, 10 uM 5-hydroxytryptamine (5-HT) Proc. Natl. Acad. Sci. USA 75 (1978) 1317             £ aan 7 7 ph r + ; r . 120+ + = A Acetylcholine 8 Choline aad a ~ & 100} Contrat 4+ 4750 3 & Control 2 a & 3s — 80 4600 £3 ~ ® 5 3 ex oe aa ® 60 4450 2a 3 SE 2 No Ca” - re 6 4mM Mg 490 CLE s KH 4 8 = 2 KCl KCI 4 KCI KCI digo E 6 &€ oY 1 i 1 1 4 4 4 i 1 1 1 0 - 4 #18 2 26 20 4 4 8 2B MW HU Minutes Fic. 5. Effects of omission of Ca2+ and increasing Mg?* on KCl-stimulated release of [7H]ACh (A) and [8H]choline (B) from NG108-15 cells. Cells grown with 1 mM BtecAMP for 35 days were incubated with 13 »M [H]choline for 45 min and washed, as described in the legend to Fig. 2. Cells then were perfused with control medium (0,@) (1.8 mM Ca2*+, 0.8 mM Mg?*) or medium without Ca2+ and adjusted to 4 mM MgCl (4,4). Cells were stimulated with 80 mM KC] for 4 min as indicated (solid symbols). Perfusate fractions were assayed for [7H]ACh and [*H]choline. Each point represents the mean of three values from separate cultures. or PGF, stimulated (SHJACh release from NG108-15 cells; whereas, PGE, stimulated [SH]ACh release only slightly. Per- fusate fractions were collected at 1-min intervals since cell re- sponses to 5-HT at PGF, rapidly desensitized. The addition of 5-HT, PGF», or PGE; had little or no effect on the rate of [3H]choline release from cells (not shown). DISCUSSION The results show that the ability of NG108-15 cells to respond to excitatory stimuli by releasing ACh into the medium can be f 8 | | | | yw 8. 200    fmol [7H] ACh released/min/mg protein | 3a? | fmol {°7H] ACh released/min/mg protein wage! ow. 100 Ma : KCI KCI gl s00l-- es Se 3 10 16 #22 28 3M 40 14 «#16 18 20 22 Minutes Minutes Fic. 6. (Left) Effect of KC] concentration on the amount of (PHJACh released from NG108-15 cells. Cells cultured with BtecAMP for 11 days were incubated with 20 uM (#H]choline for 45 min. The cells then were washed and perfusate fractions were collected and assayed for [3H]ACh as in the legend to Fig. 2. Each point represents the mean of three values from separate cultures. Solid symbols cor- respond to fractions collected during stimulation with KCl, open symbols correspond to fractions collected from cells before and after the stimulus. A, 80 mM KCI; 0, 40 mM KCi, 5, 30 mM KCl. (Right) Effects of serotonin (5-HT), PGF 2q, or PGE, on the release of (°H]- ACh from NG108-15 cells. Cells grown in BtpcAMP for 9 days were incubated with 35 »M [°H|choline for 45 min and then washed for 14 min as in the legend to Fig. 2; fractions then were collected at 1-min intervals. Cells were stimulated for 3 min (solid symbols) with 10 «M serotonin creatinine sulfate (A), 10 uM PGF, (@), or 10 .M PGE; plus 0.1% ethanol (0). Perfusate fractions were assayed for [H|ACh. Each point represents the mean of three values from separate cul- tures. 1818 Biochemistry: McGee et al. regulated. Cells grown without BtgcAMP do not respond to excitatory stimuli by releasing [2H]ACh but they can be shifted toa responsive state by treatment with BtgcAMP. The response to stimulation slowly increases over a period of at least 5 days while cells are cultured with BtgcAMP. Because ACh release in response to stimulation is required for synaptic communi- cation, the ability of the cells to form synapses and the efficiency of synaptic communication can be regulated by factors that control stimulus-dependent ACh release. Exposure of NG108-15 cells to BtpeeAMP also results in an increase in cell body diameter, neurite length, number of clear vesicles, membrane excitability, and the specific activities of acetyl- cholinesterase and choline acetyltransferase (5-14). Thus, many reactions required for synapse formation are regulated, directly or indirectly, by BtgcAMP. The properties of evoked ACh release from NG108-15 cells treated with BtgcAMP are similar to those of neurons. The amount of ACh release due to exposure of cells to KCI is a function of KCI concentration, 40-80 mM KCl evokes maximal ACh release. The response to KCI is abolished by removal of extracellular Ca?+ and elevation of Mg?* from 0.8 to 4 mM. Veratridine-stimulated ACh release is completely inhibited by 1 uM tetrodotoxin, a specific inhibitor of action potential Nat ionophore activation. Serotonin or PGF2,-stimulated ACh re- lease ceases rapidly, possibly due to receptor desensitization, whereas veratridine- or KCl-dependent ACh release can be maintained for longer periods (4-8 min). In each case, however, only 1% or Jess of the intracellular [7H]ACh is released before cell responsiveness to the stimulating agent decreases or dis- appears. If [SH]ACh is released into the medium from vesicles that contain approximately 10,000 molecules of ACh per vesicle, then one can estimate that ACh may be released from 10 ves- icles per min per cell without stimulation and 15 vesicles per min per cell when stimulated by serotonin. Both basal and serotonin-stimulated rates of ACh release are compatible with muscle responses observed at synapses between NG108-15 cells and striated muscle cells (1-4, 13), but response rates at different synapses vary greatly. Such data suggest that the NG108-15 cell population is heterogeneous with respect to the amount of ACh released and the responsiveness of cells to stimulation. Bio- chemical measurements of [7H]ACh represent the mean values for the population of cells. The results suggest that ACh is released from NG108-15 by two mechanisms because, Ca?* is required for KC]-stimulated release of ACh but not for unstimulated release of ACh. KC] stimulates the release of both [SH]ACh and (*H]choline; how- ever, these compounds seem to be released by different mechanisms. About 15% of the [H]choline taken up by NG108-15 cells is converted to [SH]ACh at each choline concentration tested between 0.5 and 250 uM. Thus, the synthesis of [7H]ACh is not preferentially coupled to high- or low-affinity choline uptake systems. In contrast, Yavin (20) has observed that an increased percentage of accumulated [H]choline is converted to [SHJACh by cultured rat embryo brain cells as the external concentration of (8H]choline is increased. The reasons for the differences between these two systems is unknown at present but both differ Proc. Natl. Acad. Sci. USA 75 (1978) substantially from what has been observed with synaptosomes, in which 50-80% of the {[*H]choline accumulated is converted to [SHJACh (21). One major difference between choline me- tabolism in cultured cells and synaptosomes is that much of the choline probably is taken up by cell bodies rather than nerve terminals and is thus exposed to both acetylation and phos- phorylation pathways; little if any phosphorylation of choline is observed with synaptosomes (21). The results show that stimulus-dependent [SH]ACh release from NG108-15 cells is acquired slowly by cells over a period of at least 5 days while cells are exposed to BtpcAMP. The slow increase in cell responsiveness to stimuli suggests that one or more components required for ACh release may be formed during this time. Whether regulation of stimulus-dependent ACh release by BtecAMP is mediated by cyclic AMP is not known; however, if the process is regulated by cyclic AMP, neurotransmitters, hormones, or other molecules coupled to the activation or inhibition of adenylate cyclase might then regulate both the ability of a neuron to form functional synapses and the efficiency of communication across the synapse. 1. Nelson, P., Christian, C. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 123-127. 2. Puro, D. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 3544-3548. 3. Christian, C., Nelson, P., Peacock, J. & Nirenberg, M. (1977) Science 196, 995-998. 4. Nelson, P. G., Christian, C. N., Daniels, M. P., Henkart, M., Bullock, P., Mullinax, D. & Nirenberg, M. (1978) Brain Res., in press. 5. Daniels, M. P. & Hamprecht, B. (1974) J. Cell Biol. 63, 691- 699. 6. Traber, J., Fischer, K., Latzin, S. & Hamprecht, B. (1975) Nature 253, 120-122. 7. Sharma, S. K., Klee, W. & Nirenberg, M. (1975) Proc. Natl. Acad. Sci. USA 73, 3092-3096. 8. Traber, J., Fischer, K., Buchen, C. & Hamprecht, B. (1975) Na- ture 255, 558-560. 9. Klein, W. L., Nathanson, N. M. & Nirenberg, M. (1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1576 (Abstr.). 10. Traber, J., Reiser, G., Fischer, K. & Hamprecht, B. (1975) FEBS Lett. 52, 327-332. . ll. Sabol, S. L. & Nirenberg, M. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 736 (Abstr. ). 12. Traber, J., Fischer, K., Latzin, S. & Hamprecht, B. (1974) FEBS Lett. 49, 260-263. 13. Christian, C. N., Nelson, P. G., Bullock, P., Mullinax, D. & Nirenberg, M. (1978) Brain Res., in press.\\", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dgy7_pt8g~yd6b", "00000000-0000-0000-C806-04E06B36864C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Neurotransmitter Synthesis by Neuroblastoma Clones", "101584910X55", null, "1972", "January 1972", "This article demonstrates the connection Nirenberg sees between biochemical genetic research and neurobiology.  The authors suggest that because genes determine neurotransmitter species, and gene expression is inherited and perpetuated for hundreds of generations, the study of neuronal tumors allows the researcher to explore the relationship between disease, heredity, and environmental influences.", "Articles", "Neuroblastoma,Clone Cells,Synaptic Transmission", "Neuroblastoma Research, 1967-1976", "6", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Nat. Acad. Sci. USA Vol. 69, No. 1, pp. 258-263, January 1972 Neurotransmitter Synthesis by Neuroblastoma Clones (neuroblast differentiation/cell culture/choline acetyltransferase/acetylcholinesterase/ tyrosine hydroxylase/axons-dendrites) TAKEHIKO AMANO, ELLIOTT RICHELSON, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, November 15, 1971 ABSTRACT Neuroblastoma clones were examined for choline acetyltransferase (EC 2.3.1.6), tyrosine hydroxyl- ase (EC 1.14.3.a), acetylcholinesterase (EC 3.1.1.7), and also for neurite formation. One clone does not form axons or dendrites. Three types of clones were found with respect to neurotransmitter synthesis: cholinergic, adrenergic, and clones that do not synthesize acetylcholine or cate- chols, All clones contain acetylcholinesterase. These re- sults show that genes determining neurotransmitter species can be expressed in dividing cells, that the parental programs of gene expression are inherited, and that dividing cells can be programmed with respect to their ability to communicate with other cells.   Elegant biological studies have vielded much information pertaining to the problem of how neural circuits form as the nervous system is assembled. However, virtually nothing is known about the molecular mechanisms for synapse forma- tion. The problem ultimately must be defined in terms of the genetic program for generating different cell types and the steps that determine the specificity of neurons in forming functional synapses. The neuroblastoma system established by Augusti-Tocco and Sato (1) provides an unusual opportunity to explore steps in neuron differentiation and function. The cells multiply rapidly in vitre, yet exhibit many properties characteristic of differentiated neurons (2-7). In this report, the properties of additional clones derived from the mouse neuroblastoma are described. Three cell types, cholinergic cells, adrenergic cells, and cells that do not synthesize acetylcholine or catechol- amines, were detected. METHODS AND MATERIALS Cells. Mouse neuroblastoma C-1300 cells were grown as described (7). Some clones were obtained in two stages: first, a well-isolated colony of cells in agar was picked and then cloned by isolation of a single cell with a stainless-steel cylinder. In other cases, cells were added to petri dishes con- taining broken coverslips; each glass shard with a single cell was then transferred to a separate dish. Chromosomes were analyzed by incubation of cells in logarithmic growth for 6-12 hr with 15-300 uM colcemide (N-desacetyl-N-methyl-colchicine) obtained from Ciba; chromosomes were spread by the method of Merchant, Kahn, and Murphy (8}. Choline Acetyltransferase (EC 2.3.1.6) Assay. Cell mono- layers were washed 3 times with an isotonic salt solution; then cells and protein were harvested by scraping and washing with 10 mM potassium phosphate buffer (pH 6.8)— 258 1mM EDTA (potassium salt). The recovered suspension was sonicated for 5 min at 3°C, divided into small portions, and stored in a vapor-phase liquid-nitrogen freezer. Choline acetyltransferase activity was assaved by a method modified (manuscript in preparation) from that of Schrier and Shuster (9). Each reaction contained the following components in a final volume of 0.05 ml, except where noted: 50 mM _ potassium phosphate buffer (pH 6.8), 200 mI NaCl, 1 mM EDTA (potassium salt), 2.6 mM _ choline iodide, 0.5% Triton X-100 (Packard), 2.2 mM [#C]lacetyl CoA (10 Ci/mol), 0.1 mM neostigmine methylsulfate, and 0-0.5 mg of homogenate protein. Each reaction was incubated at 37°C for 10 min; then 0.5 ml of H.O at 3°C was added and the diluted reaction and 2 subsequent 1.0-ml washes were passed through a 0.6 * 5 em column of Bio-Rad AG 1-X8 resin (CI~ form, 100-200 mesh). Each eluate was collected in a scintillation vial; 10 ml of scintillation solution [1000 ¢ Triton X-100-2 liters of toluene-165 ml Liquifluor (New England Nuclear Co.)] was added and radioactivity was determined. The counting efficieney for “4C was 80-90%. Duplicate or triplicate homogenates were prepared and each was assayed for choline acetvltransferase activity at 4 con- centrations of protein. The rate of reaction was proportional to enzyme concentration within the range 5-350 pmol of [!C acetylcholine formed per 10 min. Assay reproducibility with replicate homogenates was +15%. Each value reported is the average of values obtained with 2-3 homogenates. M4C-Labeled reaction products in column eluates were characterized by paper chromatography or electrophoresis. Reactions were modified so that the specific activity of the [4C]acety] CoA was 40-50 Ci/mol, and choline chloride rather than choline iodide was used. Solutions containing M4C-labeled products (25-30 yl of a column eluate); 0.2 nmol of unlabeled acetylcholine, and 0.2 wmol of unlabeled acetylearnitine were subjected to ascending paper chroma- tography for 16-24 hr with 1-propanol-0.1 N acetic acid 3:1. Chromatograms were dried and sprayed with the Dragendorf reagent (18) to visualize acetylcholine or acetylcarnitine. The chromatogram was cut into 1.0 x 0.5 cm segments, and the radioactivity of each was determined with a scintillation counter. Acetylcholinesterase (EC 8.1.1.7) Assay. The enzyme was assaved as described by Blume et al. (7). Tyrosine Hydroxylase (EC 1.14.8.a) Assay. Cell monolayers were washed, harvested, and sonicated as described above, Proc. Nat. Acad. Sct. USA 69 (1972) except that cells and protein were harvested in 0.1 M potas- sium phosphate buffer, pH 6.2. Tyrosine hydroxylase activity was assayed by a modifica- tion of the methods described by Nagatsu, Levitt, and Udenfriend (10), and by Shiman, Akino, and Kaufman (11). Each reaction contained the following components in a final volume of 0.05 ml: 0.1 M potassium phosphate buffer (pH 6.2), 0.6 mM L-[3,5-di *H]tyrosine (12 Ci/mol, from Amersham-Searle), 0.3 mM 6,7-dimethyl-2-amino-4-hydroxy- 5,6,7,8-tetrahydropteridine (Calbiochem), 0.25 mM NADPH (sodium salt), about 27 ug of sheep-liver dihydropteridine reductase protein (purified through the second ammonium sulfate-precipitation step of Kaufman (12)], and 0-0.5 mg of homogenate protein. Reactions were incubated at 34°C for 10 min, and were stopped by the addition of 0.5 ml of 0.17 N acetic acid at 3°C and assayed as described by Nagatsu et al. (10). 93% of the *H* released from t-[3,5\"H |tyrosine was recovered in the column eluate; appropriate corrections were applied to reported values. An internal standard of *H OH then was added to each sample and radioactivity again was determined. The counting efficiency for 3H was about 30%. The rate of reaction was proportional to the concentration of homogenate protein for values reported. Duplicate homoge- nates were prepared and each was assayed for tyrosine hydroxylase at four protein concentrations; reproducibility was £25%,. Average values are reported. Protein was assayed by a modification of the method of Lowry (13). Characterization of the *#H-labeled product of the Tyrosine Hydroxylase Reaction. The tyrosine hydroxylase reaction contained 0.1 mM p-bromo-m-hydroxybenzyloxyamine, an inhibitor of aromatic L-amino acid decarboxylase, in addition to the components described above. 3H-Labeled products formed during incubation were adsorbed to alumina and separated from {*H tyrosine as described by Nagatsu et al. (14), except that ’H-labeled products were eluted with 0.2 N HCl. Recovery of 3,4-dihydroxypheny alanine was 78%. An appropriate correction was applied to values reported. The [Hleatecholamines then were characterized by paper and thin-layer chromatography with the following solvents: 1-butanol-glacial acetic acid-H.,O 12:38:58; methylethy]- ketone-formie acid-H,O 24:1:6; ethylacetate-glacial acetic acid-HLO 15:15:10; and 1-butanol-L N acetic acid-ethanol 35:10:10. Thin-layer chromatography was performed with Eastman Chromogram Sheet 6065 (20 X 20 cm). Spots were located after development by spraying with ethylenediamine ferricyanide solution (15) to locate vatechols, or with nin- hydrin to locate tyrosine. RESULTS Cell types The specific activities of tyrosine hydvosylase, choline acetyitransferase, and acetylcholinesterase found with homog- enates of the neuroblastoma tumor grown in vivo and different clonal cell lines derived from this tumor are shown in Table 1. Values obtained with mouse L-cells, a fibroblastic cell line, and mouse brain are also given for comparative purposes. The specific activity of choline acetyltransferase from tumor was about 2% that of mouse brain. Acetylcholine synthesis was detected with most cell extracts, ineluding L-cells, and other estublished cell lines not shown here: however, the rate of acetylcholine synthesis was <2% that of mouse brain Neurotransmitter Synthesis by Neuroblastoma 259 (1-10 pmol of acetylcholine formed per min per mg of pro- tein). The neuroblastoma-tumor and mouse-brain specific activities of tyrosine hydroxylase were <3% that of adrenal medulla. Of the 21 clones derived from neuroblastoma C-1300 that were assayed for tyrosine hydroxylase and choline acetyl transferase, 12 clones were inactive with respect to both enzymes; 6 clones were cholinergic with high choline acetyl- transferase activity; and 1 clone was adrenergic, with tyrosine hydroxylase specific activity about 200-fold higher than that of brain. Two cell lines were found with low activities of both tyro- sine hydroxylase and choline acetyltransferase, but further evi- dence is needed to distinguish between a cholinergic-adren- ergic cell type and a mixture of adrenergic cells and cholin- ergic cells. No cells were found with high activities of both tyrosine hydroxylase and choline acetyltransferase. These re- sults show that three, possibly four, classes of neuroblastoma cells with respect to neurohormone synthesis can be derived from neuroblastoma C-1800. The specific activity of acetylcholinesterase was high with all neuroblastoma clones tested. In addition, electrically excitable cells were found with each cholinergic, adrenergic, and inactive neuroblastoma clone tested. Homogenates were prepared from stationary-phase cells rather than from logarithmically growing cells because neuroblastoma C-1300 tyrosine hydroxylase, choline acetyl- transferase and acetylcholinesterase activities respond to regulatory mechanisms, and are 30-, 4-, and 25-fold higher, respectively, in nondividing than in logarithmically multi- plying cells (7, 16, and unpublished data). Genetic heterogeneity was examined by determination of the modal number of chromosomes per cell. The mouse neuroblastoma arose spontaneously in 1940; a modal value of 66-70 chromosomes was found by Levan in 1956 (17). Modal values of 59 and 118 were found with cholinergic clones, 104 and 192 with adrenergic clones, and 100-120 with in- active clones. It is clear that neuroblastoma cells contain more chromosomes than does a diploid mouse cell, and that clones differ from one another in their chromosome content. Neuroblastoma clones without tyrosine hydroxylase or choline acetyltransferase may synthesize other transmitter- like compounds. Histamine was detected in uncloned neuro- blastoma cells that had been subcultured frequently at a concentration of 17 pmol/mg protein (unpublished data), a value similar to that of rat brain. However, glutamic acid decarboxylase was not detected with neuroblastoma extracts (unpublished data). Subclones A cholinergic neuroblastoma clone and two inactive clones were recloned, and the properties of the sublines then were studied (Table 2). 23 of the 26 subclones derived from cholinergic clone NS-20 contained active choline acetyl- transferase, and closely resembled the parent-cell type; however, 3 clones were found with relatively low activities of choline acetyltransferase. Three sublines derived from inactive clone N-4, and 5 sublines derived from inactive clone N-18, were similar to the parent cells. However, 2 subclones of N-18 contained higher activities of choline acetyltransferase than the parent clone. These results show that most, but not all, subclones resemble the parental type. Clone N-1, with both tyrosine hydroxylase and choline 260 Cell Biology: Amano eé al. Proc. Nat. Acad. Sct. CSA 69 (1972)     Taste l. Types of neuroblastoma C-1300 clones Choline Acetyl- Modal number Tyrosine acetyl- cholin- of Source of homogenate hydroxylase transferase esterase chromosomes —————-pmol product formed per min per mg of protein ————~ Mouse L cells, clone A9 2 440 Mouse brain 5 590 69, 000 Neuroblastoma tumor, 71 vivo 0 10* 130,000 66-70T Cholinergic clones NS-20Y 920 47,000 NS-20 0 490 48,000 59 NS-26 1 437 47,000 116 NS-25 1 125 55,000 59 NS-18 1 76 80, 000 59 NS-21 0 A2 36,000 61 NS-16 0 43 67,000 60 Adrenergic clones N1-106 60 a* 179, 000 104 Nik-115 980 0.1 256, 000 192 Inactive clones N-1A-103 0 2 19,000 101 N-3 9 4 174,000 N-4 4 0.1 46,000 105 N-7 6 0 99 , 000 N-8 2 2 42,000 N-9 11 1 53, 000 N-10 1 3 55,000 N-11 0 5* 404, 000 N-12 0 4 346, 000 N-13 0 6 130,000 N-18 2 2 105, 000 110 13 other clones 5* Uneertain (clonal homogeneity not established) N-1 70 22 23,000 106 N-5 25 13 151,000 109   * Estimate based on determination of C-labeled products in column eluates. t Data of Levan, cited by Hauschka (17). acetyltransferase, was recloned, and the enzyme activities of the sublines were determined (Table 3). Unlike the other neuro- blastoma clones, N-1 cells do not extend axons or dendrites. The cell population was homogeneous initially, consisting of cells that were attached well to the surface of a petri dish an TaBLE 2. Subclones of cholinergic and inactive clones   Choline acetyl- Tyrosine Cell line transferase hydroxylase   pmol of product formed per min per mg of protein Cholinergic clone NS-20 100-750* 23 Subelones 100-930* 3 Subclones 25-55* Inactive clone N-4 0.1 5 3 Subclones 1 0 Inactive clone N-18 2 2 5 Subclones 5 2 2 Subclones 16 1   * Estimate based on determination of 4C- labeled products in column eluates. usually formed short spikes (<30 um), but were entirely devoid of neurites 100-2000 ym in length. However, after the 10-20th subculture, some relatively large cells were observed with long, branched neurites. N-1 then was recloned and 19 subclones and 10 colonie= were examined. Two N-1 subclones were found that were without neurites, tyrosine hydroxylase, or choline acety!- transferase; however, acetylcholinesterase was present and cells with electrically active membranes were found when one clone was examined. Fifteen adrenergic clones with neurites were found. Each clone studied contained tyrosine hydroxy- lase and acetylcholinesterase activities, but cells were almo-t devoid of choline acetyltransferase activity. One clone (N1-106) with neurites contained 104 chromosomes per cell; other cell lines contained about 200 chromosomes per cell, Several nonadrenergic clones with neurites were found: clone NIE-113 contained 205 chromosomes per cell. These results show that the N-1 cell population is heterogeneous and contains adrenergic and nonadrenergic cells. No cholin- ergic or adrenergic- cholinergic cell type was found. Clonal morphology Four types of neuroblastoma clones, incubated in the absence of serum to stimulate neurite extension, are shown in Fig. 1: proc. Nat. Acad. Sci. USA 69 (1972) Neurotransmitter Synthesis by Neuroblastoma 261 Tape 3. Azon-minus and adrenergic subclones of neuroblastoma clone N-1       Choline Acetyl- Modal number Tyrosine acetyl- cholin- of Subclones hydroxylase transferase esterase chromosomes -—————-pmol of product formed per min per mg of protein Parent clone N-1f 70 22 23, 000 106 Axon-dendrite minus subclones N1A-103t 0 2 19,000 101 N1A-1047 1 5 Adrenergic subclones N 1-106 60 5* 179,000 104 NLE-115 980 O.L 236, 000 192 NLE-124 350 0.9 175, 000 207 NLE-125 330 1.3 98, 000 N1E-fi6 225 0.5 109, 000 202 N Le-122 215 0.4 40, 000 N 1-110 150 N 1LE-126 122 0.3 73,000 202 NLE-114 93 0.1 74, 000 Nile-112 63 N 114-128 60 NL}E-127 60 NLE-123 40 NIE-111 32 NILE-118 17 Minus tyrosine hydroxylase subclones NIE-113 6 205 N1iE-117 1   * Estimate based on determination of C-labeled products in column eluates. ; All subelones were positive for neurites, except those marked f.   Fig. 1. Axon-dendrite formation by (A) cholinergic clone NS-20; (B) adrenergic clone NIE; (C) inactive clone N-18; and (D) inactive clone N1A-103, which does not form axons or dendrites. Cells were incubated in growth medium without serum for five days to stimulate neurite formation. The scale shown in A applies to all panels, and corresponds to 10 um. 262 Cell Biology: Amano et al. Proc. Nat. Acad. Sct. USA 69 (1972) Tass 4. Neuroblastoma C-1300 clones         Acetylcholin- esterase and Choline Modal number excitable acetyl- Tyrosine of Cell type membranes Neurites transferase hydroxylase chromosomes Axon-minus + _ _ _ 101 Inactive + + - - 110 Cholinergic + + + - 59, 116 Adrenergic + + - + 104, 200   cholinergic, adrenergic, inactive with neurites, and the clone lacking neurites. Cells from each clone adhered well to the surface of the petri dish, but only the axon-minus line (N1A-103) was devoid of long neurites. Axon-minus cells and N-18 cells that form long neurites were mixed and cultivated in the same flasks for more than a week ; however, no influence of one cell type upon the other was detected. Product identification Neuroblastoma N-1E tyrosine hydroxylase activity was dependent upon a pteridine cofactor (6,7-dimethy]-2-amino- 4-hydroxy-5,6,7,8-tetrahydropteridine). Tritium release from L-[3,5-*H Jtyrosine agreed well (within 5%) with PH]di- hydroxyphenylalanine formation. The labeled product of the tyrosine hydroxylase reaction formed in the presence of an inhibitor of aromatic amino-acid decarboxylase was character- ized by paper and thin-layer chromatography with four solvents. Between 86 and 99% of the applied tritiated product was identical in chromatographic mobility with authentic dihydroxyphenylalanine. Clone N-1 cells, in the absence of an inhibitor of aromatic amino-acid decarboxylase, synthesize 3,4-dihydroxy phenylalanine, 3,4-dihydroxyphenylethylamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, —3,4- dihydroxyphenylethylglycol, 3-methoxytyrosine, 3-methoxy- tyramine, and 3-methoxy-4-hydroxyphenylacetic acid (un- published data). Catechols formed by clone C-1300 have been characterized also by Schubert, Humphreys, Baroni, and Cohn (2), and by Anagnoste, Goldstein, and Broome (4). The '4C-labeled products of the choline acetyltransferase reaction were eluted from the column and routinely character- ized by paper chromatography. 84-99% of the labeled product formed with homogenates of cholinergic cells was identical in chromatographic mobility to authentic acetyl- choline. However, only 2-25% of the labeled material formed with adrenergic or inactive homogenates was acetylcholine. The major contaminant was identified as [*C acetyl carnitine (unpublished data). DISCUSSION Clones of neuroblastoma C-1300 were examined for two enzymes required for neurotransmitter synthesis, choline acetyltransferase and tyrosine hydroxylase, catalyzing acetyl- choline formation and the first step in norepinephrine syn- thesis, respectively. A summary of data is shown in Table 4. Three types of clones were found: (a) clones that form little or no acetylcholine or catechols; (6) cholinergic clones; and (c) adrenergic clones. One additional clone was found with relatively low cholinergic and adrenergic activities, but we do not know whether this is a fourth cell type or a mixture of adrenergic and cholinergic cells. However, no cells were found that actively synthesize both acetylcholine and cate- chols. Tumors of cholinergic neurons have not been reported before. They probably afflict man, but may not have been recognized for lack of a diagnostic test. Since few mammalian cells other than neurons have high activities of choline acetyltransferase, the enzyme and acetylcholine can be used as specific diagnostie markers for cholinergic neuroblastomas. Since acetylcholinesterase was found with all neuroblastoma cell types, the enzyme apparently is not a specific marker of cholinergic neurons. At least six types of neurons or corresponding tumors that arise from the neural crest can be distinguished on the basis of transmitter synthesis: those synthesizing acetylcholine: 3,4-dihydroxy phenylalanine, 3,4-dihydroxyphenylethylamine, norepinephrine, epinephrine, and sensory neurons that do not synthesize these compounds. The three types of neura- blastoma thus exhibit properties expected of neural-crest neurons. Although neuroblastoma stem cells capable of giving rise to cholinergic, adrenergic, and inactive cells were not found, it seems likely that such cells normally exist. The available information suggests that there are relatively few kinds of universal neurotransmitters in the nervou- system of both vertebrates and invertebrates. Most neurons probably are greatly restricted with respect to the number of kinds of neurotransmitters that can be synthesized. We find cholinergic, adrenergic, and inactive neuroblastoma cell types, but have not detected cells capable of synthesizing both acetylcholine and catechols at rapid rates. It seems likely that the expression of a gene required for the sythesi= of one neurotransmitter may restrict the expression of genex for alternate neurotransmitters. For example, a product derived from the choline acetyltransferase gene directly or indirectly might restrict the expression of genes for tyrosine hydroxylase, and vice versa. Simultaneous expression of acetyltransferase and tyrosine hydroxylase genes might inactivate both genes, or might result in a balanced state of mutual inhibition; i.e., a cell with low cholinergic and adren- ergic activities. Whether a product of one neuron affects the expression of genes for transmitter svnthesis of neighboring neurons is a problem for future study. Analysis of sublines derived from clonal cells demot- strates that most, but not all, resemble the parent type. The mechanisms underlying altered gene expression are nol known. Gene expression may be reversible or mutations may affect structural or regulatory genes. In any event, the loss of a step that commits a cell to one developmental pattern may enable the cell or its descendants to differentiate along an alternate pathway. Thousands of cell generations have elapsed since tumor C-1300 originated, and extensive genetic heterogeneity in the proc. Nat. Acad, Sei. USA 69 (1972) vell population is expected. By a relatively simple experi- mental approach, it may be possible to obtain lines that express genes that are characteristic of many kinds of neurons jrom a single neural tumor cell; i.e, with two sequential lective procedures: first, selection for dedifferentiated cells might yield cell populations enriched in stem cells; then lection for differentiated cells might yield cells that follow alternate programs of differentiation. We conclude that genes determining the species of neuro- transmitter synthesized can be expressed in dividing cells, aud that the parental programs of gene expression are ittherited and perpetuated for hundreds of cell generations. Similarly, normal neuroblasts may be destined with respect to trans- mitter synthesis and may generate different types of clonal cell populations that are programmed with regard to their ability to establish functional communication with other cell types before synapses are formed. We thank Miss Eve Cutler for excellent technical assistance. 1, Augusti-Tocco, G. & Sato, G. (1969) Proc. Nat. Acad. Sct. USA, 64, 311-315. 2. Schubert, D., Humphreys, 8., Baroni, C. & Cohn, M. (1969) Proc. Nat. Acad. Sci. USA, 64, 316-323. 3. Olmsted, J. B., Carlson, K., Klebe, R., Ruddle, F. & Rosen- baum, J. (1970) Proc. Nat. Acad. Set. USA, 65, 129-136. 4. Anagnoste, B. F., Goldstein, M. & Broome, J. (1970) Pharmacologist, 12, 269. we 11. 12. 13. 14. 16. 17. 18. Neurotransmitter Synthesis by Neuroblastoma 263 Nelson, P., Ruffner, W. & Nirenberg, M. (1969) Proc. Nat. Acad. Sci. USA, 64, 1004-1010. Seeds, N. W., Gilman, A. G., Amano, T. & Nirenberg, M. W. (1970) Proc. Nat. Acad. Sci. USA, 66, 160-167. Blume, A., Gilbert, F., Wilson, 8., Farber, J., Rosenberg, R. & Nirenberg, M. (1970) Proc. Nat. Acad. Sei. USA, 67, 786-792. Merchant, D. J., Kahn, R. H. & Murphy, W. H., (1960) Handbook of Cell and Organ Culture (Burgess Publishing Co., Minneapolis, Minn.), pp. 198-200. Schrier, B. K. and Shuster, L. (1967) J. Neurochem. 14, 977- 985. Nagatsu, T., Levitt, M. & Udenfriend, S. (1964) Anal, Biochem. 9, 122-126. Shiman, R., Akino, M. & Kaufman, 8. (1971) J. Biol. Chem., 246, 1330-1340. Kaufman, S. (1962) in Methods in Enzymology, Vol. VY, ed. Colowick, 8. P. & Kaplan, N. O. (Academic Press, New York) p. 802. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem., 193, 265-275. Nagatsu, T., Levitt, M. & Udenfriend, 8. (1964) J. Biol. Chem., 239, 2910-2917. Schneider, F. H. & Gillis, C. N. (1965) Biochem. Pharm. 14, 623-626. Amano, T., Richelson, E. & Nirenberg, M. (1971) Fed. Proc. 30, 1085. Hauschka, T. S., Kvedar, B. J., Grinnell, S. T. & Amos, D. B. (1956) Ann. N.Y. Acad. Sci. 63, 683-705. Bregoff, H. M., Roberts, E. & Delwiche, C. C. (1953) J. Biol. Chem. 205, 565-574.", "Nirenberg, Marshall W. ; Richelson, Elliott ; Amano, Takehiko", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-59i4~jm95_wdwb", "00000000-0000-0000-27E2-24A64F1D59F8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Regulation of Adenylate Cyclase Activity Mediated by Muscarinic Acetylcholine Receptors", "101584910X64", null, "1978", "April 1978", "This follow-up to earlier reports on the enzyme system adenylate cyclase finds that activators of excitatory acetylcholine in hybrid cells have \"both transient and long-lived effects\" that may be involved in transmission across certain synapses.  The experimental evidence points to an increase in enzyme release as regulated by neuroreceptors.", "Articles", "Receptors, Cholinergic ; Receptors, Muscarinic ; Adenylyl Cyclases,Neurons,Carbachol", "From Neuroblastoma to Homeobox Genes, 1976-1992", "4", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 75, No. 4, pp. 1788-1791, April 1978 Biochemistry Regulation of adenylate cyclase activity mediated by muscarinic acetylcholine receptors (synapse plasticity /cyclic AMP/neuroblastoma X glioma hybrid cells) NEIL M. NATHANSON*, WILLIAM L. KLEIN', AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, February 10, 1978 ABSTRACT Carbachol, an activator of muscarinic acetyl- choline receptors of NG108-15 hybrid cells, inhibits adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] rapidly and reversibly and slowly evokes a 200-300% increase in ade- nylate cyclase activity over a period of 24-30 hr. Both the inhi- bition of adenylate cyclase and the gradual increase in enzyme activity are dependent on muscarinic acetylcholine receptors and the receptor activator. Withdrawal of carbachol results in a gradual return of adenylate cyclase activity to control levels over a period of 6 hr; the half-life for decay of enzyme activity is 1.6 hr. These results show that muscarinic acetylcholine re- ceptors mediate both transient and long-lived effects on ade- nylate cyclase activity that resemble those of opiates. The possibility that cyclic AMP (cAMP) or cyclic GMP (cGMP) functions as an intermediate or a modifier of transynaptic communication at certain synapses has been considered fre- quently. Evidence has been reported that suggests that cAMP plays a role at synapses terminating on Purkinje neurons of the cerebellum (1, 2) and evidence for (3-6) and against (7) roles for cAMP and cGMP at synapses in sympathetic ganglia has been described. Mouse neuroblastoma X rat glioma NG108-15 hybrid cells resemble neurons in many ways. The cells synthesize acetyl- choline, have electrically excitable membranes, generate Na* and Ca?* action potentials,* have clear vesicles and large dense-core vesicles (8), and form synapses with cultured striated muscle cells (9). The cells possess receptors that are coupled to the activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], such as adenosine and prostaglandin E, (PGE) receptors, and have other receptors coupled to the inhibition of adenylate cyclase, such as opiate receptors, mus- carinic acetylcholine receptors, and a-adrenergic receptors (10-13). When NG108-15 cells are cultured in the presence of morphine (14, 15) or norepinephrine (13) for 10-24 hr, the specific activity of adenylate cyclase slowly increases and thus compensates for the inhibition of enzyme activity (14-16). Growth of NG108-15 cells in the presence of carbachol, a rel- atively stable analog of acetylcholine, was reported by Traber et al. (15) to result in an increase in cAMP accumulation by the cells. In a preliminary communication (16), we reported that growth of NG108-15 cells in the presence of carbachol gradu- ally results in an increase in the specific activity of adenylate cyclase and a decrease in the number of muscarinic acetyl- choline receptors of cells. In this paper we examine the increase in adenylate cyclase mediated by muscarinic acetylcholine receptors.     The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1788 MATERIALS AND METHODS Materials. The sources of some of the chemicals have been described (10). In addition, [a-3*P]ATP was obtained from New England Nuclear; atropine sulfate, arecoline hydrogen bromide, acetylcholine chloride, carbamylcholine chloride, and eserine sulfate, from Sigma; N-2-hydroxyethylpiperazine-N’-2-eth- anesulfonic acid (Hepes), from Calbiochem; and oxotremorine, from Aldrich. Cell Culture. Mouse neuroblastoma X rat glioma NG108-15 hybrid cells? derived by fusion of mouse C1300 neuroblastoma clone N18TG2 (17) and rat glioma C6BU-1 (18) were grown as described (19). cAMP Assay. Confluent NG108-15 cells (8-5 mg of protein per 60-mm dish, 20 cm? surface area) were washed three times, each time with 5 ml of the Dulbecco-Vogt modification of Eagle’s minimal essential medium (DMEM, Gibco, cat. no. H-21) supplemented with 10 mM Hepes (pH 7.4) instead of NaHCOsz and adjusted to 340 mOsmol/liter with NaCl. To examine the cAMP-synthesizing capacity of the cells, 3.5 ml of DMEM (as above except 25 mM Hepes) containing 0.5 mM 4-(3-butoxy-4-methoxybenzy])-2-imidazolidinone (Ro20-1724) and 1 pM atropine was added and cells were incubated for 10 min at 37° (atropine was added to inhibit the possible effects of traces of carbachol, present during growth, that may not have been removed by washing). Cells were incubated for an addi- tional 5 min to test cAMP accumulation. cAMP levels were determined as described (10) by the method of Gilman (20). Values reported are corrected to 100% recovery of cAMP; each is the mean of values obtained from two or three dishes. Three or four concentrations of the extract from each dish were as- sayed for cAMP. The standard errors of the mean were +£10%. Adenylate Cyclase Assays. Confluent cells were washed three times, harvested (10-13 mg of protein per 100-mm dish), suspended in 290 mM sucrose/25 mM Tris-HCl, pH 7.4, at 4-6 mg of protein per ml, frozen rapidly in small aliquots, and stored in a liquid No freezer. Before use, extracts were thawed and homogenized in a ground-glass homogenizer with 10 strokes of a motor-driven pestle for 20 sec at 1200 rpm. Ade- nylate cyclase activity in cell-free homogenates was determined by a procedure (14) based on method C of Salomon et al. (21).   Abbreviations: cAMP, adenosine 3’,5’-cyclic monophosphate, cGMP. guanosine 3’,5’-cyclic monophosphate; Hepes, N-2-hydroxyethylpi- perazine-N’-2-ethanesulfonic acid; Ro20-1724, 4-(3-butoxy-4- methoxybenzy])-2-imidazolidinone; PGE), prostaglandin E. * Present address: Department of Physiology, University of California, San Francisco, CA 94143. t Present address: Department of Biological Sciences, Northwestern University, Evanston, IL 60201. + B. Hamprecht, T. Amano, and M. Nirenberg, unpublished data. Biochemistry: Nathanson et al. Table 1. cAMP accumulation by NG108-15 cells grown with or without carbachol     Growth conditions Addition 10 uM 10 uM for None carbachol carbachol test 43 hr 19 hr 43 hr pmol cAMP in cells and medium/mg protein Control 467 1258 1046 PGE, 10 uM 4530 6905 6280   Cells were grown with or without carbachol for the time indicated and then washed and incubated for 10 min at 37° with 0.5 mM Ro-20 1724 and 1 uM atropine sulfate. Cells were incubated for an additional 5 min with or without PGE, to test cAMP accumulation in cells and medium. All cells were tested and deproteinized at the same time. Values reported are mean values obtained from two or three homogenates, prepared from separate dishes, each assayed at four protein concentrations (40-200 ug of protein per reaction mixture) with standard errors of the mean < +10%. Where indicated, 0.5-1 4M atropine was added to reaction mixtures to inhibit possible effects of trace amounts of carbachol not removed by washing the cells. RESULTS As shown in Table 1, levels of cAMP in NG108-15 cells are el- evated after growth of cells in the presence of carbachol, a relatively stable analog of acetylcholine. Cells were grown with or without 10 uM carbachol for 19 or 43 hr, washed, and incu- bated for 10 min with a phosphodiesterase inhibitor and then for an additional 5 min with or without PGE, to test cAMP accumulation in cells and medium. Growth of cells in the presence of carbachol for 19 hr resulted in a 300% increase in basal cAMP and a 50% increase in PGE)-stimulated cAMP accumulation. In Fig. 1, the relationship between the carbachol-dependent increase in specific activity of adenylate cyclase and the car- bachol concentration during cell growth is shown. Growth of cells for 2 days in the presence of 0.1-100 uM carbachol resulted in increases in basal and PGE)-stimulated adenylate cyclase activities. When ceils were treated with 100 4M carbachol, basal adenylate cyclase activity was 225% higher and PGE-stimu- lated activity was 63% higher than control activities. The muscarinic acetylcholine receptor antagonist atropine   th T T —- T T T AG ¥ am Basal + B PGE, ase 4200 Is +150 pmo! cAMP/min/mg protein + q- pmol cAMP/min/mg protein     j_1 . 1 1. 2p. i 1 1 Or Ss 5 4° 0° 7 6 5 4 —Log carbachol, M   FIG. 1. Change in adenylate cyclase specific activity after 48 hr of growth of NG108-15 in the presence of various concentrations of carbachol. (A) Basal specific activity; (B) with 10 uM PGE). Reaction mixtures contained 0.5 uM atropine sulfate. Proc. Natl. Acad. Sci. USA 75 (1978) 1789 Table 2. Effects of growing NG108-15 cells with carbachol and atropine on adenylate cyclase activity   Cell growth conditions Adenylate cyclase activity for 2 days Basal 10 uM PGE, pmol cAMP/min/mg protein Control 5 42 Carbachol, 100 nM 16 137 Atropine, 1 uM 7 61 Carbachol, 100 uM + atropine, 1 uM 5 43   Each reaction mixture contained 0.5 uM atropine sulfate. blocked the carbachol-dependent increase in adenylate cyclase activity (Table 2). Growth of cells for 2 days in the presence of 100 uM carbachol resulted in 3-fold increases in basal and PGE)-stimulated adenylated cyclase activities. However, the presence of | 4M atropine had little effect on adenylate cyclase activity, which shows that occupancy of the receptor by a re- ceptor antagonist does not result in an increase in the specific activity of adenylate cyclase. However, 1 uM atropine blocked the carbachol-dependent increase in adenylate cyclase activity, which suggests that the increase in enzyme activity is mediated by muscarinic acetylcholine receptors. Exposure of cells to acetylcholine for 24 hr also increased basal and PGE)-stimulated activities of adenylate cyclase 65% and 32%, respectively (Table 3). Serum was omitted in this experiment to eliminate possible effects of serum cholinesterase (EC 8.1.1.7). The specific activities of adenylate cyclase in homogenates of cells grown for different times with or without 100 4M car- bamylcholine are shown in Fig, 2. In this experiment, the spe- cific activities of adenylate cyclase from both control and car- bachol-treated cells decreased with time; however, the decrease in specific activity found with homogenates from carbachol- treated cells was less than that found with homogenates from control cells. In most experiments, however, the specific activity of adenylate cyclase increased when NG108-15 cells were treated with carbachol. The maximum difference in adenylate cyclase specific activity of cells grown with or without carbachol usually was attained in 20 to 30 hr. NG108-15 cells were grown in the presence of carbachol for 24 hr and then for 8 hr in the absence of carbachol. As shown in Fig. 3. the increased specific activity of adenylate cyclase returned to control values in 5-8 hr. The half-life of the car- Table 3. Adenylate cyclase activity of NG108-15 cells grown with or without acetylcholine   Additions Adenylate cyclase assay during growth Basal 10 uM PGE, pmol (3?P]cAMP/min/mg protein Control 15 204 Eserine sulfate, 2.5 uM 16 210 Acetylcholine chloride, 1 mM + eserine sulfate, 2.5 uM 27 272   Cultures of NG108-15 cells, approximately 85% confluent, were washed twice with medium without serum and then cultured for an additional 24 hr (with two changes of media) with the indicated compounds in the absence of serum to eliminate possible effects of serum cholinesterase. Adenylate cyclase was assayed as described in Materials and Methods, in the presence of 0.5 uM atropine sul- fate. 1790 Biochemistry: Nathanson et al. ap no a         1247 A c e\"\\\\ 2 \\\\ Carbachol 2 iot \\\\ 4 5 E Br 1 aa a = Cf Control! 7 $ Be a ab 4 9 IO 20 30 40 50 60 Hours Fic. 2. NG108-15 cells were grown with or without carbachol and, at the times shown, homogenates were prepared and assayed for adenylate cyclase activity. @, 0.9% NaCl; a, 100 »M carbachol chloride dissolved in 0.9% NaCl. Adenylate cyclase reaction mixtures contained 0.5 uM atropine sulfate. bachol-dependent enzyme activity in the absence of carbachol was 1.6 hr; enzyme activity decreased exponentially with an estimated first-order rate constant (k) for loss of enzyme activity of 0.007 min7! (Fig. 3 inset). Thus, the carbachol-dependent increase in adenylate cyclase activity is acquired slowly and is relatively long-lived compared to hormone- or neuro- transmitter-dependent activation or inhibition of the en- zyme.                          504 1 at Carbachol 40 £ £ 5 4 £30 £ £ Remove E— carbachol $ q 20 2 Control ° € a 100 lO F 50 4 fo 10 OL¢y r , 1 . O 2 4 6 8 8 24 Hours Fic. 3. Decrease in adenylate cyclase activity after carbachol withdrawal. NG108-15 cells were grown for 24 hr without carbachol (Q) (control cells), or with 100 uM carbachol (GQ). At 24 hr (zero time for withdrawal of carbachol), cells were washed twice and the medium was replaced with fresh medium without carbachol (0, control cells; A, carbachol-treated cells), or with 100 «M carbachol (QO, carba- chol-treated cells). (Inset) Rate of decay of the carbachol-dependent increase in adenylate cyclase activity due to withdrawal of carba- chol. Proc. Natl. Acad. Sci. USA 75 (1978) Table 4. Decreased sensitivity of adenylate cyclase to carbachol in NG108-15 cells grown in the presence of carbachol Adenylate cyclase assay   Cell growth 0.5 uM 100 uM % conditions atropine carbachol inhibition pmol cAMP/min/mg protein Control 44 32 27 Carbachol, 100 uM, 0.25 hr 43 31 28 Carbachol, 100 uM, 24 hr 81 74 9   Adenylate cyclase assays were performed as described in Materials and Methods, except that the pH of reaction mixtures was 8.0, which increases both the activity of adenylate cyclase and the sensitivity of the enzyme to inhibition by carbachol. Adenylate cyclase from NG108-15 cells exposed to carbachol for 24 hr was less sensitive to inhibition by carbachol than was the enzyme from control cells (Table 4). In contrast, treatment of cells with carbachol for 15 min did not reduce the sensitivity of adenylate cyclase to carbachol. This phenomenon, first ob- served by S. Sharma,$ probably results from a gradual loss, over 5-10 hr, in the number of muscarinic acetylcholine receptors when NG108-15 cells are grown in the presence of carbachol (16). DISCUSSION The results show that carbachol, an activator of muscarinic acetylcholine receptors of NG108-15 hybrid cells, inhibits adenylate cyclase rapidly and reversibly and also slowly evokes an increase in adenylate cyclase activity over a period of 24-30 hr. Both the inhibition of adenylate cyclase and the gradual increase in enzyme activity are dependent on muscarinic ace- tylcholine receptors and the receptor activator, and both effects are blocked by 1 «M atropine. However, the carbachol-de- pendent increase in adenylate cyclase activity, once acquired, can be expressed independently in the absence of carbachol. NG108-15 cells also possess opiate receptors and a-adrenergic receptors that similarly are coupled to an inhibition of adenylate cyclase and a gradual increase in enzyme activity. The cells seem to acquire tolerance to the inhibitor but instead become dependent on the inhibitor to maintain normal cAMP levels because withdrawal of the inhibitor can result in a long-lived increase in adenylate cyclase activity. Withdrawal of carbachol from NG108-15 cells that had been grown for 24 hr in the presence of carbachol resulted in an increase in adenylate cy- clase activity for 4-5 hr. Carbachol-dependent adenylate cy- clase activity decayed with a half-life of 1.6 hr. These and previous results show that acetylcholine and norepinephrine (13) have biological activities that resemble those of opiates (22, 23). The results also suggest that other species of receptors mediating an inhibition of adenylate cyclase also may function to increase the specific activity of the enzyme. Thus far, NG108-15 cells have been shown to respond to activators of muscarinic acetylcholine receptors in five ways: cells depolarize,? intracellular cGMP levels increase slightly,\" adenylate cyclase is inhibited and thus cAMP levels fall (10, 12), a receptor-mediated increase in adenylate cyclase activity is slowly evoked, and the number of muscarinic acetylcholine receptors slowly decreases over a period of hours in the presence of carbachol (16). Carbachol-dependent depolarization of cells and the increase in cGMP levels can desensitize completely in   §S. Sharma and M. Nirenberg, unpublished data. ‘| H. Matsuzawa and M. Nirenberg, unpublished data.   Biochemistry: Nathanson et al. 1 min. In contrast, carbachol-dependent inhibition of adenylate cyclase is not desensitized under these conditions. It seems likely that the desensitized form of the muscarinic acetylcholine re- ceptor, with respect to depolarization and cGMP accumulation, may be the active form with respect to inhibition of adenylate cyclase. Adenylate cyclase in homogenates from NG108-15 cells grown in the presence of carbachol for 24 hr is inhibited less by carbachol than is adenylate cyclase from control cells. The decreased sensitivity of adenylate cyclase to carbachol probably is due to a carbachol-dependent decrease in muscarinic ace- tylcholine receptors (16). The recovery of receptors requires protein synthesis and a 24- to 48-hr incubation. Thus, an acti- vator of the muscarinic acetylcholine receptors of NG108-15 cells can decrease cell responsiveness to that compound and, in addition, can regulate cell responsiveness to ligands that activate other species of receptors coupled to adenylate cyclase. For example, the acquisition of supersensitivity to PGE) was shown to be mediated by muscarinic acetylcholine recep- tors. In summary, we find that activators of excitatory acetyl- choline receptors of NG108-15 cells have both transient and long-lived effects on macromolecules that may be involved in transmission across certain synapses. N.M.N. is a postdoctoral fellow of the Muscular Dystrophy Associ- ation. 1. Siggins, G. R., Hoffer, B. J. & Bloom, F. E. (1971) Brain Res. 25, 535-553. 2. Bloom, F. E., Siggins, G. R., Hoffer, B. J., Segal, M. & Oliver, A. P. (1975) in Advances in Cyclic Nucleotide Research, eds. Drummond, G. L., Greengard, P. & Robinson, G. A. (Raven Press, New York), Vol. 5, pp. 603-618. 3. Kebabian, J. W. & Greengard, P. (1971) Science 174, 1346- 1349. 10. 11. 12. 18. 14. 15. 16. 17. 18. 19. 20. 21. 23. Proc. Natl. Acad. Sci. USA 75 (1978) 1791 McAfee, D. A. & Greengard, P. (1972) Sctence 178, 310-312. Weight, F. F., Petzold, G. & Greengard, P. (1974) Science 186, 942-944. Kebabian, J. W., Bloom, F. E., Steiner, A. L. & Greengard, P. (1975) Science 190, 157-159. Busis, N. A., Schulman, J. A., Smith, P. A. & Weight, F. F. (1978) Br. J. Pharmacol., in press. Daniels, M. P. & Hamprecht, B. (1974) J. Cell Biol. 63, 691- 699. Nelson, P., Christian, C. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 123-127. Sharma, S. K., Nirenberg, M. & Klee, W. A. (1975) Prac. Nail. Acad. Sci. USA 72, 590-594. Traber, J., Fischer, K., Buchen, C. & Hamprecht, B. (1975) Na- ture 255, 558-560. Traber, J., Reiser, G., Fischer, K. & Hamprecht, B. (1975) FEBS Lett. 52, 327-332. Sabol, S. L. & Nirenberg, M. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 736. Sharma, S. K., Klee, W. A. & Nirenberg, M. (1975) Proc. Nail. Acad. Sci. USA 72, 3092-3096. Traber, J., Gullis, R. & Hamprecht, B. (1975) Life Sci. 16, 1863-1868. Klein, W., Nathanson, N. M. & Nirenberg, M. (1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1576. Minna, J., Glazer, D. & Nirenberg, M. (1972) Nature New Biol. 235, 225-231. Amano, T., Hamprecht, B. & Kemper, W. (1974) Exp. Cell Res. 85, 399-408. Klee, W. A. & Nirenberg, M. (1974) Proc. Natl. Acad. Sci. USA 71, 3474-3477. Gilman, A. G. (1970) Proc. Natl. Acad. Sci. USA 67, 305-312. Salomon, Y., Londos, C. & Rodbell, M. (1974) Anal. Biochem. 58, 541-548. Lampert, A., Nirenberg, M. & Klee, W. (1976) Proc. Natl. Acad. Sct. USA 73, 3165-3167. Sharma S. K., Klee, W. A. & Nirenberg, M. (1977) Proc. Natl. Acad. Sci. USA 74, 3365-3369.", "Nirenberg, Marshall W. ; Klein, William L. ; Nathanson, Neil M.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rqfp-4ee9~93ws", "00000000-0000-0000-C727-6A689237368C", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Synapse Turnover: A Mechanism for Acquiring Synaptic Specificity", "101584910X65", null, "1978", "May 1978", "In previous studies on synapse formation, \"chick embryo retina neurons were shown to form synapses rapidly with striated muscle cells\" but were transient and eventually disappeared.  This report uses striated muscle cells to define the number of neurons that are able to form synapses at different stages of development and suggests that both correct and incorrect synapses are formed during normal embryonic development, with correctly matched synapses being retained.", "Articles", "Neuromuscular Junction,Synapses", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Prac. Natl. Acad. Sci. USA Vol. 75, No. 5, pp. 2281-2285, May 1978 Biochemistry Synapse turnover: A mechanism for acquiring synaptic specificity (retina /muscle cell /neurons/development/cell culture) ROBERT R. RUFFOLO, JR., GEORGE S. EISENBARTH, JEFFREY M. THOMPSON, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Nirenberg, March 10, 1978 ABSTRACT Neurons are generated in chick retina that are able to form synapses with striated muscle cells for only a brief period during embryonic development. The ability to form syn- apses is lost with a half-life of 21 hr. Retina neuron-myotube synapses form rapidly but soon are terminated. Chick embryo spinal cord neurons also form synapses with muscle cells for only a limited time during development, but these synapses are long lived. These results show that different classes of synapses turn over at different rates and suggest that part of the specificity of synaptic circuits may be acquired during development by a process of selection based on synapse termination rates.   Cultured cells dissociated from chick embryo retina have been shown to form approximately 1 X 109 synapses per mg of pro- tein (1), and ultrastructural studies reveal different types of synapses, which closely resemble those formed in ovo (1-3). Because relatively few classes of neurons have been identified in the retina and retina synaptic circuits have been studied extensively (4), the developing retina would appear to be a fa- vorable model system for studies on the mechanism of assembly of synaptic circuits. Acetylcholine probably is a neurotransmitter at some syn- apses in chick retina. High choline acetyltransferase (EC 2.3.1.6) (5) and acetylcholinesterase (EC 3.1. 1.7) (6) activities, acetyl- choline (6), a high-affinity choline uptake mechanism (7), and nicotinic (8, 9) and muscarinic (10) acetylcholine receptors are present in chick retina. In previous studies on the mechanism of synapse formation, chick embryo retina neurons were shown to form synapses rapidly with striated muscle cells; such synapses were transient and eventually disappeared (5). In this report we have used striated muscle cells as synaptic targets to define further the process of synapse formation and to determine the number of neurons that are able to form synapses at different stages of development. We now show that retina neurons are able to form synapses for only a short time during development, that synapses of different types are terminated at different rates, and that populations of synapses can be selected on the basis of differences in synapse termination rates. METHODS AND MATERIALS Primary muscle cultures were prepared by fusion of myoblasts obtained from fetal or newborn Fisher rats as described pre- viously (5, 11); however, to select for nondividing cells, 10 uM 5-fluorodeoxyuridine and 100 uM uridine were added on the 3rd or 4th day of culture and removed on the 6th to 9th day. Except where indicated, chick neural retina and spinal cord cells were dissociated with trypsin crystallized three times (Worthington Biochemical Corp.) and cultured with myotubes previously cultured for 8-20 days. Neuron-myotube synapses were detected by recording   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked paeerinerent in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2281 spontaneous depolarizing synaptic muscle responses, usually 0.5 mV in amplitude and 10 msec in duration, with intracellular microelectrodes filled with 3 M KCI solution as described by Nelson et al. (12). Only those myotubes with stable membrane potentials of at least —40 mV were used. A myotube was con- sidered to be innervated if at least three spontaneous synaptic responses of the muscle cell, judged free of artifacts, were identified during a 2-min recording period. RESULTS Neurons dissociated from chick embryo retina at different stages of development were cocultured with rat striated muscle cells. The percent of muscle cells tested that were innervated by retina neurons after 2 or 24 hr of coculture is shown in Fig. 1A, and the average frequency of spontaneous synaptic re- sponses of innervated muscle cells, in Fig. 1B. Neurons that are able to form synapses with muscle cells appear in chick embryo retina on the 6th or 7th day, are most abundant on the 8th day, and disappear by the 16th day of embryo development. Almost as many synapses were found at 2 hr of coculture as at 24 hr. The frequency of spontaneous muscle responses of innervated myotubes follows a similar developmental sequence (F ig. 1B); however, the frequency of muscle responses was maximum after 24 hr of coculture with neurons from 8-day chick embryo retina, whereas, with more mature neurons, the maximum frequency of muscle responses was observed after 2 hr of co- culture. Glutamate (5 mM) applied locally by diffusion from a micropipette increased the frequency of muscle responses at synapses between neurons from 8-day embryo retina and muscle cells, but failed to evoke responses from muscle cells cocultured with neurons from 16-day embryo retina (not shown). The frequency of muscle responses after 24 hr of co- culture decreased exponentially as a function of retina devel- opment with a half-time of 31 hr and a first-order rate constant of 0.022 hr~! (Fig. 1B inset). To rule out the possibility that cell damage resulting from dissociation with trypsin was responsible for the inability of older neurons to synapse with muscle, 8- and 16-day embryo retinas were dissociated with [ethylenebis(oxyethyleneni- trilo) tetraacetic acid (EGTA) (0.02%), and the resulting cells were cocultured with myotubes. Under these conditions, functional synapses were detected with retina neurons from 8-day but not 16-day embryos. Neurons in retina explants (nondissociated tissue fragments) from 8-day embryos formed synapses with striated muscle after 24 hr of coculture, but not neurons in explants from 16- or 18-day embryos (data not shown). These results indicate that the loss of ability of neurons from 16-day embryo or older retinas to form synapses with muscle cells is not due to cell dissociation. The relation between the percent of muscle cells tested with synapses and the number of retina cells per dish, dissociated   Abbreviations: EGTA, {ethylenebis(oxyethylenenitrilo) |tetraacetic acid; BME, basal medium (Eagle's). 9282 Biochemistry: Ruffolo et al. yiTt Titty tT Ty TTT 7 B, RESPONSES/MIN 1120                       ta Y | P24 HR 69 mm oy < | 100 24 HR iE $1 ' T Z sol 71002 no | B i \\\\ 25 197 31 HR - = z= ' ra \\\\ \\\\ a - { ! 2 = 80) Bro: Wye pee a \\\\ i & 10: : g ao he 3 Lge nS weo-) \\\\\\\\ ¢ [LR sr 760 & rs} rt WV ght Sond! \\\\ t | w O 8 12 6 DUA \\\\ fo wee uy pavsovo 1/7 | \\\\% 42 46 201 tg oO ; ~ 40 4 2 a0} DAYS IN OVO = ) 8 Oo Ss Hi a s | 3 e 5 20 = Ww Oo ira Ww a \\\\m — 20 HATCH | {| 0 6 8 1012141618201 6 8 10121416 1820 1 AGE OF RETINA NEURONS (DAYS /N OVO) FIG. 1. Synapse formation between neurons dissociated from chick embryo retina at various stages of development and rat striated muscle cells. Thirty million cells dissociated from chick embryo retina with trypsin were cocultured for 2 or 24 hr with myotube monolayers in collagen-coated petri dishes 35 mm in diameter (9.6-cm? surface area). The percent of muscle cells tested with synapses is shown in A and the mean frequency of spontaneous synaptic responses of in- nervated muscle cells is shown in B. A, Retina cells and muscle cells cocultured for 2 hr; the assay medium was 90% basal medium (Eagle’s) (BME) and 10% fetal bovine serum; O, retina cells and muscle cells cocultured for 24 hr; 1 hr before testing for synapses the medium was changed to BME with the CaClz concentration increased from 1.8 to 3.8 mM and the choline chloride concentration adjusted from 6 to 106 uM. The data shown in the Insets are from cells cocultured for 24 hr and are plotted with a logarithmic ordinate. Kach point represents an average of 30 muscle cells (range 10-70) tested for synapses. T'1/2 represents the half-time for decrease in muscle response frequen- cy. oS from embryos 6-19 days after fertilization, is shown in Fig. 2. As retina cells aged in ovo, the number of neurons that formed synapses with myotubes increased between the 6th and 8th day of embryonic development and decreased thereafter. With 20% of the myotubes innervated, most muscle cells that had synapses were innervated by only a single neuron. Because each dish contained approximately 20,000 myotubes, the percent of retina cells synapsing with muscle (Fig. 2 inset) is easily calculated (5). At least 1.6% of the retina cells from 8-day chick embryos formed synapses with muscle cells, and this fraction declined logarithmically with embryo age with a half-life of 21 hr. This suggests that the loss of ability of retina neurons to synapse with muscle is an apparent first-order process with a rate constant of 0.035 hr-!. Because the retina cell concentration—percent myotube innervation curves are parallel, concentration ratios (concentration of 8-day embryo retina cells needed to form synapses with 50% of the muscle cells, divided by the concen- tration of retina cells from 8-day or older embryos needed to innervate 50% of the muscle cells) may be calculated in order to determine the ability of retina neurons at different embryo ages to form synapses relative to retina cells from 8-day em- bryos. The decline in concentration ratio with embryo age also is exponential and is described by the same regression line as the percent of retina cells synapsing with muscle cells. The developmental age of the retina cells and the duration of coculture of neurons with muscle were varied and the con- centration of retina cells was kept constant (3 X 107 retina cells per dish). After 1 day of coculture, 100% of the myotubes tested were innervated by neurons dissociated from 8-day embryo retina (Fig. 3A); however, the maximum percent of muscle cells innervated by neurons from older retina declined progressively Proc, Natl. Acad. Sci. USA 75 (1978) TTT 8 DAY - Y   TTY TTT TTiny TTT try TOTTI]         8 16 2 z 10DAY - $ eg i < 7, OF 016 £ 12 DAY xr n | E 4 aad = oe o 7 3 : — 40 # BT ees Naoay 2 Ss DAYS IN OVO 5 20 wi oO ao o / 15 0 pak op peti pt) 49 104 105 106 107 108 RETINA CELLS/DISH Fic. 2. Loss of neuronal ability to synapse. The percent of muscle cells tested with synapses is shown as a function of the number of dissociated retina cells per dish and of embryo age. Cells were disso- ciated with trypsin from chick embryo retina of the following ages: v, 6 days; O, 8 days; @, 10 days; >, 12 days; #, 14 days; V, 15 days; d, 16 days; and A, 19 days. One hour before assay for synapses the me- dium was changed to BME adjusted to 3.8 mM calcium chloride and 106 uM choline chloride. Each point represents an average of 40 muscle cells (range 10-70). One-hundred percent corresponds to 20,000 myotubes innervated per dish. (Inset) Retina cells and my- otubes were cocultured for 24 hr prior to assay for synapses; 4, the percent of retina cells that form synapses with muscle cells is shown on the right ordinate; and ©, on the left ordinate the concentration ratio (the number of 8-day embryo retina cells needed to innervate 50% of the muscle cells divided by the concentration of retina cells from 8-day or older embryos needed to innervate 50% of the muscle cells). T1/2 corresponds to the half-life for loss of ability of retina neurons to form synapses with striated muscle cells as a function of neuron maturation. as a function of embryo age. No synapses were detected be- tween neurons from 16-day embryo retina and muscle cells cocultured for 20 min to 48 hr. Increasing the concentration of CaCly from 1.8 to 3.8 mM and the choline concentration from 6 to 106 uM had little or no effect on the percent of myotubes innervated by retina neurons from 8- to 16-day embryos co- cultured 24 hr. The percent of innervated muscle cells was maximum at 24 hr with retina neurons at all developmental ages tested and decreased thereafter. The period of culture required for loss of all synapses depended upon the develop- mental age of the retina neurons cultured. Retina neurons from 10-day chick embryos required 5 days in culture to terminate all synapses with muscle cells, whereas only 2 days of culture were required to terminate all synapses between neurons from 14-day embryo retina and muscle cells. The time required for termination of synapses appears to be related to the total age of the retina neurons (days in ovo plus days of coculture) as shown in Fig. 3A inset. Retina neurons terminated all synapses with muscle cells 15-16 days after fertilization. Because synapses are terminated at approximately the same rate that neurons lose the ability to form synapses between the 11th and 16th embryo day, the results suggest that the half-life of synapses between retina neurons and muscle cells is relatively short (less than 21 hr). To eliminate the possibility that synapse termination is 4 consequence of cell dissociation, fragments of tissue from 8-day chick embryo retina were explanted and cultured with muscle cells for 5 days. Neurons in explants from 8-day chick embryo Biochemistry: Ruffolo et al.   Ss & 8 8B ~ MUSCLE RESPONSES/MINUTE          PERCENT MUSCLE CELLS WITH SYNAPSES L oa 012 3 46 650 12 3 4 § DAYS OF COCULTURE Fic. 3. Percent of muscle cells tested with synapses (A) and mean num ber of spontaneous synaptic responses of innervated muscle cells (B). The developmental age of retina cells and duration of coculture of retina cells with muscle cells were varied and the concentration of retina cells was kept constant (3 X 10’ retina cells per dish). Cells were dissociated with trypsin from chick embryo retina at the following ages: O, 8 days; A, 10 days; >, 12 days; v, 14 days; O, 16 days. The medium was changed 1 hr before assay; open symbols represent an assay medium of 90% BME and 10% fetal bovine serum. Filled-in symbols correspond to an assay medium of BME adjusted from 1.8 to 3.8 mM calcium chloride and from 6 to 106 uM choline chloride. &, Nondissociated explants of retina tissue from 8-day chick embryo retina. Only those myotubes in contact with retina explants were as- sayed for synapses. Each point represents an average of approximately 20 muscle cells (range 10-70) tested. (Insets) Total days shown on the abscissa represents days in ovo plus days of coculture and thus cor- responds to the age of the retina cells since fertilization. Symbols are as above. retina formed fewer synapses than dissociated retina cells after coculture with muscle cells for 2 hr, but 100% of the muscle cells were innervated at 24 hr and the rate of termination of synapses by neurons in explants thereafter was identical to that found with dissociated cells from 8-day embryo retina. The average frequency of spontaneous muscle responses in innervated myotubes is shown in Fig. 3B as a function of age of retina neurons and duration of culture. In general, the de- velopmental pattern was similar to that shown in Fig. 8A except that the maximum muscle response frequencies found with 10- and 14-day embryo retina neurons were obtained after 2 rather than 24 hr of culture, and the muscle response frequency ob- served with explants from 8-day embryo retina declined ap- proximately 1 day later than that found with cells dissociated from 8-day embryo retina. Increasing the concentrations of calcium ions and choline had little effect on the frequency of muscle responses in innervated myotubes cocultured for I day. In general, the frequency of spontaneous muscle responses decreased faster than the loss of myotubes with synapses (Fig. 3 insets). These results show that synapses are transient, and that neurons lose the ability to form synapses at approximately the same rates in vitro and in ovo. Cells from 8-day chick embryo retina were dissociated and cocultured with myotubes for 1-6 days and then again disso- ciated with trypsin and added to fresh myotube monolayers and cultured for an additional 24 hr to determine the effects of in vitro aging and denervation on the ability of neurons to form synapses (Fig. 4). The results show that neurons progressively lose the ability to form synapses with muscle cells as the neurons mature in vitro. Neurons dissociated from 8-day chick embryo retina, cocultured with muscle cells for 6 days, formed few synapses with myotubes. The number of muscle cells with synapses and the frequency of spontaneous muscle responses were slightly higher than expected for cultures with 1 X 108 Proc. Natl. Acad. Sci. USA 75 (1978) 2283 i oy 805 ~ —50 a i : > 40.- ee) w 40, sywapses = E = = \\\\ \\\\ iH a 30; \\\\ -30 0” = 5 ud \\\\ a. oO v2 W a ~20 wy g..lCUN 3 = 10- \\\\ OK 108 5 RESPONSES ae Ww x Qo * 4 m1 2 3 4 5 6 DAYS OF CULTURE Fic. 4. Effects of in vitro aging and denervation on the ability of neurons to form synapses. Cells from 8-day chick embryo retina were dissociated with trypsin and cocultured (1 X 10’ retina cells per dish) with myotubes for 1-6 days as shown on the abscissa. Retina cells were again dissociated with 0.05% trypsin and retina cells were added to fresh muscle monolayers (1 X 108 retina cells per dish) and cultured for an additional 1 day. 0, Percent of muscle cells tested with syn- apses; A, mean number of spontaneous synaptic responses of inner- vated myotubes per min. Muscle cells were tested for synapses in growth medium (90% BME and 10% fetal bovine serum). Each point represents 10-21 muscle cells tested for synapses. retina cells per dish compared to retina neurons that matured in ovo (see Fig. 2). Elsewhere we will show that neurons from chick embryo spinal cord gain the ability to form synapses with striated muscle cells at approximately 2 days of embryonic development and lose the ability by the 16th or 17th day of embryogenesis:* this is similar to the pattern of synaptogenesis found with retina neurons. Thus, the question was asked, “Are synapses between spinal cord motor neurons and striated muscle cells, which normally form during development, longer lived than mis- matched synapses between retina neurons and muscle cells?” Dissociated cells from 8-day chick embryo retina and spinal cord both formed synapses with striated muscle cells rapidly, but synapse termination was only observed with mismatched retina~muscle cell synapses (Fig. 5). No loss of synapses between spinal cord neurons and muscle cells was detected after 14 days of culture. These results show that retina neurons form transient synapses with striated muscle cells, whereas spinal cord neurons form stable, long-lived synapses; the results strongly suggest that at least part of the specificity of synaptic connections is acquired after synapses form by selection for synapses with slow termi- nation rates. DISCUSSION The results show that neurons are generated in chick embryo retina that are able to form synapses with striated muscle cells but then lose the ability to form synapses with a half-life of 21 hr. These neurons appear in retina on the 6th and 7th day of chick embryo development, are most abundant on the 8th day, and lose the ability to form synapses by the 16th day. Synapses between retina neurons and muscle cells form rapidly, but are transient and turn over with a half-life of 21 hr or less. Chick embryo spinal cord neurons also appear by the 2nd day of embryo development and disappear by the 16th or 17th day* but form stable, long-lived synapses with striated muscle cells.   * J. Thompson, G. Eisenbarth, R. Ruffolo, and M. Nirenberg, unpub- lished observations. 9984 Biochemistry: Ruffolo et al. a sof 1 > le | Za | = 60 ~ ho. ao . nl Vers ye 5x 108 = SPINAL CORD NEURONS | B40) \\\\ aN a 4 : x Ay ae 1x 106 3 20 | e 4x10 2 5x 108 5 » a Ae RETINA NEURONS co 2 4” 6 g 10 12 14 o DAYS OF COCULTURE Fic. 5. Synapse turnover by cells dissociated from 8-day chick embryo retina or spinal cord cocultured with muscle for various times. Filled-in symbols correspond to retina cells; open symbols, spinal cord cells. Circles represent 5 X 106 and triangles 1 X 10° retina or spinal cord cells per dish cocultured with muscle cells. The growth medium and assay medium consisted of Eagle’s minimum essentia] medium supplemented with 10% fetal bovine serum, and 10 pM 5-fluo- rodeoxyuridine and 100 pM uridine (added 16 hr after retina or spinal cord cells were added to myotube monolayers to prevent rapidly di- viding cells from overgrowing cultures). Each point represents 10 or 20 muscle cells assayed for synapses. These results strongly suggest that at least part of the specificity of synaptic connections can be acquired after synapses form by a process of selection based on the rate of synapse termina- tion. The mechanism of conversion of retina neurons from a syn- apse-competent to an incompetent state is unknown. The spe- cific activity of choline acetyltransferase is high in ovo and in vitro and does not decrease while neurons lose the ability to form synapses (5). Thus, the loss of ability of retina neurons to form synapses is not due toa decrease in the number of neurons that are able to synthesize acetylcholine. Nicotinic acetylcholine receptors are present in 6-day chick embryo retina in low concentration and increase in concen- tration throughout embryo development (8, 9). Thus, synapses mediated by nicotinic acetylcholine receptors probably form in chick retina between the 6th and 16th day of embryonic development. Synapses recognized by ultrastructural special- izations first appear in chick embryo retina on the 13th day of development (13-15). Synapses are formed in abundance be- tween cultured retina neurons (1 X 10° synapses per mg of protein) also starting approximately 13 days after fertilization (1), during the same period that retina neuron-myotube syn- apses decrease in number. Many ultrastructural specializations that are used to identify synapses were not found with retina neuron-muscle cultures at a time when many synapses were detected by electrophysiological methods.t Thus, early stages of synaptogenesis may be detected by electrophysiologic methods although they are not detected by electron micros- copy. Dissociated neurons and neurons in explants of 8-day chick embryo retina extended long neurites in vitro, whereas few neurites were extended by neurons from 16- to 19-day embryo retina or chick retina after hatching. Retina neurons have been + M. Daniels, D. Puro, and M. Nirenberg, unpublished observations. Proc. Natl. Acad. Sci. USA 75 (1978) shown to become less adhesive as they mature, thus, the di- ameter of cultured retina cell aggregates decreases during the course of embryonic development (16). However, in stationary cultures, cells dissociated from 16- to 19-day embryo retina formed large, loose aggregates of retina cells when cocultured with muscle. Further work is needed to determine whether the loss of ability of neurons from older chick embryo retina to form synapses with muscle cells is related to the change in adhesive properties of the neurons and/or to the decrease in ability to extend neurites. A summary of the steps in synapse formation that have been detected is shown in Fig. 6. Neurons that synthesize acetyl- choline and are able to form synapses (N+) are generated in chick retina on the 6th to 7th day of embryonic development (reaction 1). The ability to form synapses is lost (N7) via an apparent first-order process with a half-life of 21 hr (reaction 9). The apparent rates of synapse termination that were re- ported previously (5) thus reflect both the rate of loss of ability of neurons to form synapses and the rate of synapse termination. Ninety-four percent of the synapses formed by these neurons therefore are made within 84 hr (4 half-lives) of the time the neurons acquire the ability to form synapses. Thus, the se- quence of genesis of neurons, their relative positions, and the duration of the synapse-competent state may restrict synaptic targets to cells of approximately the same or older age in neighboring areas. As shown in reaction sequence B, neurons dissociated from 8-day chick embryo retina adhere to muscle cells (M) and neurons (N) (reaction 3) with a half-time of 10 min (5) and form synapses with muscle cells and probably also with neurons (reaction 5) with a half-time of 25 or 60 min for retina neurons dissociated with EGTA or trypsin, respectively (5). Neurons lose the ability to form synapses (reaction 2), but not the ability to transmit information across synapses formed previously. Sy- napses may be terminated by reversal of synapse formation (reaction 6) or by cell dissociation as indicated by reaction 7. In summary, we find that synapses formed by retina neurons and spinal cord neurons with muscle turn over at different rates and that synapses between spinal cord neurons, which pre- sumably include developmentally correct synapses, are retained while synapses between retina neurons and muscle cells are terminated, perhaps due to differences in cell adhesiveness. (a) NEUROELAST ——l—>NT 2 sN7 WILL, FORM SYNAPSES WILL NOT FORY. SYNAPSES VM a we) NTEMSANENT SBRNAN SSNAN +N +M “Ny WN ADHESION SYNAPSE SYNAPSE FORMATION SELECTION Fic. 6. ‘Summary of proposed steps in synapse formation. In reaction sequence A, neurons with choline acetyltransferase activity (N+) are generated which are able to form synapses (reaction 1). During embryonic development the ability of the neurons to form synapses is lost (N-) (reaction 2). In reaction sequence B, dissociated retina neurons that are able to form synapses (N*) attach to striated muscle cells (M) and other neurons (N) via reaction 3; © represents cell adhesion. Synapses form via reaction 5; —® represents synapse formation. Synapses may be terminated by reversal of synapse for- mation (reaction 6) or by dissociation of cells (reaction 7). The loss of the ability of neurons to form synapses (reaction 2) does not affect communication across synapses formed previously. Incorrect, mis- matched synapses are terminated at markedly faster rates than those formed by correctly paired synaptic partners. Thus, populations © synapses may be selected on the basis of rate of synapse termina- tion. Biochemistry: Ruffolo et al. Similarly, synapses between cultured retina neurons are re- tained after nearly all synapses between retina neurons and muscle cells have been terminated.t These results strongly suggest that both correct and incorrect synapses aré formed during embryonic development and that correctly matched synapses are selectively retained, as suggested by Changeux and Danchin (17). The demonstration that neurons acquire the ability to form synapses for a brief period during embryonic development and that some neurons form stable, long-lived synapses whereas other neurons form transient synapses suggests that selection for stable synapses or synapses with slow rates of termination may be a general mechanism for the assembly of some synaptic circuits during development. R.R.R. isa Pharmacology Research Associate of the National Institute of General Medical Sciences. 1. Vogel, Z., Daniels, M. P. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 2370-2374. 2. Stefanelli, A., Zacchei, A. M., Caravita, S., Cataldi, A. & Ieradi, L. A. (1967) Experientia 23, 199-200. 3. Sheffield, J. B. & Moscona, A. A. (1970) Dev. Biol. 23, 36-61. 4. Stell, W. K. (1972) in Handbook of Sensory Physiology, ed. ll. 12. 13. 14. 15. 16. 17. Proc. Natl. Acad. Sci. USA 75 (1978) 2285 Fourtes, M. G. F. (Springer-Verlag, Berlin, Heidelberg, W. Germany), Vol. 7, part 2, pp. 111-213. Puro, D. G., DeMello, F. G. & Nirenberg, M. (1977) Proc. Natl. Acad. Sci. USA 74, 4977-4981. Lindeman, V. F. (1947) Am. J. Physiol. 148, 40-44. Baughman, R. W. & Bader, C. R. (1977) Brain Res. 138, 469- 485. Vogel, Z. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 1806-1810. Wang, G.-K. & Schmidt, J. (1976) Brain Res. 114, 524-529. Sugiyama, H., Daniels, M. P. & Nirenberg, M. (1977) Proc. Nail. Acad, Sci. USA 74, 5524-5528. Puro, D. G. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 3544-3548. Nelson, P. G., Peacock, J. H., Amano, T. & Minna, J. (1971) J. Cell. Physiol. 77, 337-352. Meller, K. (1968) in Veroffentlichungen aus der morpholog- ischen Pathologie, eds. Buchner, F. & Giese, W. (Gustav Fischer- Verlag, Stuttgart, W. Germany), Vol. 77, pp. 1-77. Sheffield, J. B. & Fischman, D. A. (1970) Z. Zellforsch. Mikrosk. Anat. 104, 405-418. Hughes, W. F. & LaVelle, A. (1974) Anat. Rec. 179, 297-302. Moscona, A. A. (1962) J. Cell. Comp. Physiol. 60, 65-80. Changeux, J.-P. & Danchin, A. (1976) Nature 264, 705-712.", "Thompson, Jeffrey M. ; Eisenbarth, George S. ; Ruffolo, Robert R. ; Nirenberg, Marshall W.", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xthx~8spb~efzk", "00000000-0000-0000-715A-11EFA14290F8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Adenylate Cyclase and Acetylcholine Release Regulated by Separate Serotonin Receptors of Somatic Cell Hybrids", "101584910X66", null, "1979", "March 1979", "In the 1970s, Nirenberg and the Biochemical Genetics team at NIH extended their work in neurobiology to the study of neurotransmitter function in clonal hybrid cells.  This article presents evidence from the study of serotonin receptors and their relationship to acetylcholine release.", "Articles", "Receptors, Serotonin ; Serotonin,Acetylcholine,Adenylyl Cyclases,Neuroblastoma,Binding, Competitive,Enzyme Activation", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "proc. Natl. Acad. Sci. USA vol. 76, No. 3, pp. 1135-1139, March 1979 Biochemistry Adenylate cyclase and acetylcholine release regulated by separate serotonin receptors of somatic cell hybrids (lysergic acid diethylamide/neurotransmitters /synapse/neuroblastoma) JOHN MACDERMOT*, HARUHIRO HIGASHIDAt, STEVEN P. WILSON, HIROSHI MATsuUzAwaf, JOHN MINNAS, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014 Contributed by Marshall Warren Nirenberg, December 13, 1978 ABSTRACT Serotonin activates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of NCB-20 neuro- blastoma-brain hybrid cells with an activation constant of 530 nM, but has little or no effect on cellular cyclic AMP or cyclic GMP content of N1E-115 neuroblastoma or NG108-15 hybrid cells. In homogenates of NCB-20 hybrid cells, lysergic acid di- ethylamide stimulates adenylate cyclase activity (Kace = 12 nM) and partially inhibits (K; = 10 nM) the stimulation of adenylate cyclase activity by serotonin. No desensitization was detected of serotonin receptors coupled to adenylate cyclase. Serotonin also depolarizes NCB-20, NG108-15, and NIE-115 cells and in- creases acetylcholine release. Serotonin receptors mediating depolarizing responses desensitize rapidly and reversibly, and the depolarizing effects of serotonin are neither mimicked nor inhibited by lysergic acid diethylamide. These results indicate that (i) NCB-20 cells possess at least two species of serotonin receptors, which independently regulate cellular functions, (ii) activation of adenylate cyclase does not directly affect mem- brane potential or acetylcholine release, and (iii) serotonin- dependent cell depolarization does not affect cyclic AMP or cyclic GMP synthesis in the cell lines tested.   Serotonin activates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in mammalian brain (1-3) and increases or decreases the frequency of spontaneous action potentials in cerebral neurons (4, 5). Serotonin also induces contractions in ileal smooth muscle (6). However, central neuronal responses to serotonin in mammals are predominantly inhibitory and are mediated through either pre- or postsynaptic receptors, which can be distinguished by the differences in their responses to D-lysergic acid diethylamide (LSD) (5, 7). In molluscan neu- rons, as many as six kinds of response to serotonin have been detected (8). Serotonin-dependent activation of adenylate cyclase is pre- served after electrolytic lesion of the raphe nuclei, which greatly reduces the number of serotonergic neurons innervating the colliculus. This suggests that collicular serotonin receptors that mediate activation of adenylate cyclase are postsynaptic re- ceptors (2). In this report, effects mediated by two svecies of serotonin receptor in NCB-20 neuroblastoma—brain hybrid cells are de- scribed. One receptor is coupled to the activation of adenylate cyclase, and the other mediates cell depolarization and ace- tylcholine release. The receptors are distinguished on the basis of functional response, specificity, and desensitization.   the publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “ad- fe ement in accordance with 18 U.S. C. §1734 solely to indicate this fact. 1135 MATERIALS AND METHODS Cells. The N1E-115 adrenergic neuroblastoma cell line was cloned from C-1300 mouse neuroblastoma (9). The NG108-15 neuroblastoma-glioma hybrid cell line (unpublished results) was derived by Sendai virus-induced fusion of C-1300 mouse neuroblastoma clone N18TG2 resistant to 6-thioguanine (10) with rat glioma clone C6BU-1, resistant to 5-bromodeoxyuri- dine (11). The NCB-20 neuroblastoma-fetal Chinese hamster brain hybrid cell line (12) was obtained by Sendai virus-induced fusion of NI18TG2 with fetal Chinese hamster brain cells dis- sociated from 18-day embryos. The NBr-10A and NBr-20A neuroblastoma-rat liver hybrid cell lines (unpublished results) were obtained by Sendai virus-induced fusion of NI8STG2 with the BRL30E Buffalo rat liver cell line, resistant to 5-bromo- deoxyuridine (H. G. Coon, personal communication). Adenylate Cyclase Assay. Cells were cultured as described (13). Confluent cells were detached from flasks by gentle tapping and were washed three times, each with 10 ml of Dulbecco's phosphate-buffered saline (without Ca** or Mg?+ ions) adjusted to 340 mOsm with NaCl. Cells were recovered by centrifugation (150 X g for 5 min) after each wash, and after the third wash were suspended in 25 mM Tris-HCl, pH 7.4/290 mM sucrose (2 ml/75-cm? flask) and homogenized at 4°C with 50 strokes of a Dounce homogenizer. Homogenates were frozen and stored in a liquid No freezer. Each 100-pl reaction mixture contained 50 mM Tris-HCl (pH 7.4); 87 mM sucrose; 20 mM creatine phosphate, disodium salt (Sigma); 10 International Units of creatine kinase, 150 units/mg of protein (ATP:creatine N-phosphotransferase, EC 2.7.3.2) from Sigma; 1 mM cyclic AMP (cAMP), sodium salt (Sigma); 0.25 mM Ro20-1724 (a gift from Hoffmann-La Roche); 0.25% ethanol; 1 mM {@-32P|ATP (3 wCi, New England Nuclear; 1 Ci = 3.7 X 10!° Bq); and 100-200 ug of homogenate protein. Homogenates were thawed and maintained at 4°C in an ice bath for no longer than 10 min prior to incubation. Reaction mixtures were incubated for 8 min at 37°C. Adenylate cyclase activity was determined by a modification (14) of method C   Abbreviations: LSD, D-lysergic acid diethylamide; PGE), prostaglandin E); cAMP, cyclic AMP; BtzcAMP, N®, O2-dibutyryl cyclic AMP; cGMP, cyclic GMP; Gpp(NH)p, guanylyl-5’-imidodiphosphate. * Present address: Dept. of Clinical Pharmacology, Royal Postgraduate Medical School, London W12 OHS, England. + Present address: Dept. of Aerospace Physiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464, Japan. + Present address: Dept. of Agricultural Chemistry, University of Tokyo, Tokyo, Japan. 8 Present address: Human Tumor Biology Laboratory, VA Hospital. Washington, DC 20422.   1136 —_ Biochemistry: MacDermot et al. of Salomon et al. (15). The production of [8?P]cAMP was pro- portional to protein concentration within the range 50-250 ug of homogenate protein per reaction mixture; similarly [8?P]- cAMP synthesis increased linearly for 30 min. Values are the means of two to four determinations, except where noted; replicates usually varied by <8%. cAMP and Cyclic GMP (cGMP) Assays. Methods for cell culture and incubation, and for cAMP and cGMP separation and assay have been described by Matsuzawa and Nirenberg (16). Protein was determined by a modification of the method of Lowry et al. (17), with bovine serum albumin, crystallized three times, as standard. Electrophysiological Measurements. Hybrid cells were usually cultured for 5-15 days in the presence of 1 mM N 6 O2-dibutyryl-cAMP (BtscAMP). Myoblasts were dissociated from newborn Fisher rat hind limb and cultured for 3 days. Nondividing myotubes were selected for by incubating cells with 10 uM 5-fluorodeoxyuridine and 100 uM uridine for 48 hr; then hybrid cells that had been treated with 1 mM BtocAMP for 7-10 days were added and cells were cocultured for an additional 1-3 days in the presence of 1 mM BtgcAMP (18). The techniques for intracellular recording of membrane potentials of cultured cells have been described (18, 19). Serotonin and LSD were applied to cells iontophoretically or by addition of a solution from a micropipette. The following generous gifts were received: LSD from Judith R. Walters: bufotenine-creatinine sulfate (Upjohn) and N,N’- dimethyl-5-methoxytryptamine (free base) (Aldrich) from J. C. Gillin; mianserin-hydrochloride (Organon Products, West Orange, NJ) from S. H. Snyder; methysergide-hydrogen mal- eate from C. R. Creveling; prostaglandin E, (PGE,) from Upjohn; metergoline from Farmitalia (Milan, Italy); cypro- heptadine-hydrochloride from Merck, Sharp and Dohme; fluphenazine-2 HCl from Squibb; phentolamine-hydrochloride from Ciba Pharmaceuticals (Summit, NJ); and pimozide (free base) from McNeil Laboratories Inc. (Fort Washington, PA). Serotonin-creatinine sulfate, 5-methoxytryptamine-hydro- chloride, and propranolol-hydrochloride were obtained from Sigma; tryptamine-hydrochloride from Regis Chemical Co.; and guanylyl-5’-imidodiphosphate [GPP(NH)p] from P-L Biochemicals. RESULTS Serotonin stimulated adenylate cyclase activity 75-115% in homogenates of NCB-20 hybrid cells (Fig. 1). The serotonin concentration required for half-maximal activation (Kact) was consistently within the range 400-800 nM and was not affected by 50 uM ascorbate or 100 uM pargyline. Homogenates were prepared and frozen for subsequent use since freezing and thawing had little or no effect on the serotonin-dependent stimulation of adenylate cyclase. An Eadie—Scatchard plot (Fig. 1, upper inset) was linear and the Kcr for serotonin was cal- culated to be 530 nM. From a Hill plot (Fig. 1, lower inset), the interaction coefficient (n) was found to be 1.0, revealing no cooperativity in the serotonin-dependent activation of ade- nylate cyclase. [Ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA; 1-10,000 uM) had no effect on the stimulation of adenylate cyclase activity by serotonin, which suggests the ef- fect of serotonin is not dependent on Ca?* ions (data not shown). Adenylate cyclase also was stimulated by LSD (Kact = 12 nM) and the LSD analogs metergoline (Kact = 250 nM) or methy- sergide (Kact = 620 nM) (Fig. 2A). The maximum stimulations of adenylate cyclase activity by LSD or LSD analogs were less than those obtained in the presence of serotonin (Fig. 1). The synthesis of [82P]cAMP in the presence or absence of 10 uM Proc. Natl. Acad. Sci. USA 76 (1979) 10 -—4                   L 6 2 F § 9+ = ae 4 2 ale a 4 z 2 an L — - z 8 5 0 a £ 7F 4 £ Io. 2 - 4 6 — + Ve 4 @ o4 4 1 4 a 5 P1012 3 log [SHT], uM 4 k I 1 1 o 107 106 105 io [Serotonin], M Fic. 1. Serotonin-dependent increase in adenylate cyclase ac- tivity in a homogenate of NCB-20 hybrid cells. Results show the means (+SD) of triplicate determinations of adenylate cyclase activity as a function of serotonin concentration. (U/pper Inset) Eadie- Scatchard plot; \\\\V S5HT is the serotonin-dependent increase in [32P]cAMP synthesis (pmol/min per mg of protein). (Lower Inset) Hill plot; “r” is the increase in adenylate cyclase activity at each se- rotonin concentration and “R” is the maximum increase. serotonin or 0.1 uM LSD was linear with respect to time and did not desensitize within 16 min (Fig. 2B). In another exper- iment, no desensitization to serotonin was detected in 30 min (not shown). Adenylate cyclase activity also was increased by the following analogs of serotonin: tryptamine, bufotenine, 5-methoxytryp- tamine, and N,N’-dimethyl-5-methoxytryptamine (Fig. 3A). The Kac: values for these compounds were 400-570 nM, and maximum stimulations of adenylate cyclase activity were similar to that of serotonin (Table 1). The serotonin receptor antagonists, mianserin or cyprohep- tadine, inhibited the stimulation of adenylate cyclase by 10 uM serotonin (Fig. 3B). The K; values were 43 nM for mianserin and 95 nM for cyproheptadine. Mianserin and cyproheptadine also inhibited the stimulation of adenylate cyclase by LSD (Ki = 100 nM and 64 mM, respectively). The dopamine receptor antagonists, fluphenazine or pimozide, inhibited serotonin- dependent stimulation of adenylate cyclase (Ki = 47 nM and         £23 3 j c ms i — = Qa 4 : g n- a yf ° E x AK a a * 1x oe . “ 2 a k mt Ay £ e i » wo a YY o> a € nt r aa 7 = a 5A hye Ue : a k ~ 2 8 = : xy pe Pd So G Je US o t “=n we E 3 ; ‘ - : | eae [Activator], M , Minutes FIG. 2. Activation of adenylate cyclase in homogenates of NCB-20 hybrid ceils as a function of concentration of lysergic acid derivatives (A) or by LSD and serotonin as a function of incubation time (B). (A) Values are the means (+SD) of triplicate determina tions: O, LSD; A, metergoline; and O, methysergide. (B) Volume of each reaction mixture was 2 ml; concentrations of reagents are give” in Materials and Methods. At each time indicated, a 100-yl aliquot was removed and the reaction was terminated. Adenylate cyclasé activity was determined in the presence of water(O); 0.1 uM LSD (4\": 10 uM serotonin (DB).   Biochemistry: MacDermot et al.       c — r& 3 se * a| & 5 ; : Ze f° aa vy 2 2 i . E B / \\\\ o s ga \\\\\\\\ a a é N ¢ x - y % 6s 3 a E Vs ie a ° 4 5% 7l 3 € - z + aa < = oS 40 o ice Co ic? . . [Activator], M [Antagonist], M Fic. 3. Activation of adenylate cyclase by tryptamine derivatives (A) and the inhibition of serotonin-stimulated adenylate cyclase ac- tivity in homogenates of NCB-20 hybrid cells by mianserin and cyproheptadine (B). (A) Values are the means of triplicate determi- nations of adenylate cyclase activity in the presence of increasing concentrations of tryptamine derivatives: @, serotonin {3.89}; O, tryptamine [3.90]; 4, bufotenine [2.75]; 0, 5-methoxytryptamine [3.98]; or X, N.N’-dimethyl-5-methoxytryptamine [3.51]. (Values shown in brackets are the pmol of cAMP/min per mg of protein that correspond to 100% on the ordinate.) (B) Results show the means (+SD) of triplicate determinations of adenylate cyclase activity in the presence of 10 uM serotonin and increasing concentrations of ei- ther mianserin (A) or cyproheptadine (0). Basal enzyme activity was 4.93 (£0.27) pmol of cAMP/min per mg of protein. 250 nM, respectively). These K; values are greater than those reported for the inhibition of [2H )haloperidol binding to do- pamine receptors in calf brain membranes (0.88 nM and 0.81 nM, respectively) (20). The a- or 8-adrenergic receptor an- tagonists, phentolamine or propranolol, respectively, had little or no effect on the stimulation of adenylate cyclase by serotonin. Results are summarized in Table 2. The effect of serotonin concentration on adenylate cyclase activity in the presence or absence of a saturating concentration of LSD (10 uM) is shown in Fig. 4A. LSD stimulated adenylate cyclase and also partially inhibited the serotonin-dependent increase in enzyme activity. The effect of LSD concentration on adenylate cyclase activity was determined by using a dif- ferent homogenate in the presence or absence of 10 uM sero- tonin (Fig. 4B). LSD increased adenylate cyclase activity (Kact = 12 nM), as shown previously, and also partially inhibited the serotonin-dependent activation of adenylate cyclase (Kj = 10 nM). Serotonin stimulated adenylate cyclase activity only slightly Table 1. Specificity of serotonin receptors coupled to adenylate cyclase of NCB-20 hybrid cells   Maximum activation, Additions Koc, nM % Tryptamine derivatives Serotonin 530 80 5-Methoxytryptamine 570 78 Tryptamine 400 77 N,N’-Dimethyl-5-methoxytryptamine 410 73 Bufotenine 520 60 Lysergic acid derivatives LSD 12 41 Metergoline 250 45 Methysergide 620 32   The activation constant (Kae) for each compound is the concen- tration required for half-maximal stimulation of adenylate cyclase activity. The salts of the compounds tested are given in Materials and Methods. Proc. Natl. Acad. Sci. USA 76(1979) 1187 Table 2. Effects of receptor antagonists on serotonin- or LSD-activated adenylate cyclase of NCB-20 hybrid cells   Adenylate cyclase, Kj (nM)     Additions 5HT-activated LSD-activated Mianserin 43 100 Cyproheptadine “95 64 LSD (partial agonist) 10 Fluphenazine 47 Pimozide 250 Phentolamine >10,000 Propranolol > 10,000   The K; values were calculated from the following equation: Ki = ICso/1 + (factivator]/K act of activator), where ICso is the concentration of antagonist producing half-maximal inhibition. 5HT, serotonin (5-hydroxytryptamine). in homogenates prepared from NG108-15, NBr-10A, NBr-20A hybrid, or parental N18TG2 neuroblastoma cells (Table 3). Treatment of intact NG108-15 or NIE-115 cells with 10 uM serotonin for 0.5, 1.0, 1.5, 2.0, 2.5, 5.0, and 10 min had little or no effect on intracellular levels of cAMP or cGMP (not shown). As shown in Table 4, GTP increased basal adenylate cyclase activity by 31% and increased serotonin-dependent activation by 265%. Sodium fluoride stimulated adenylate cyclase activity 850% and completely blocked the effect of serotonin. Gpp(NH)p also stimulated adenylate cyclase activity 125%; however, the stimulatory effects of Gpp(NH)p and serotonin were additive. PGE, activated adenylate cyclase, and serotonin reduced by 13% the PGE -dependent activation of the enzyme. Sodium fiuoride or Gpp(NH)p inhibited PGE)-stimulated adenylate cyclase activity, and serotonin further reduced the activity in the presence of PGE and Gpp(NH)p. These results suggest that activation of adenylate cyclase by serotonin re- quires GTP and reveals no unusual interaction with sodium fluoride or PGEj, with respect to the coupling of serotonin re- ceptors to the adenylate cyclase complex. However, the additive rather than synergistic effects of serotonin and Gpp(NH)z may indicate a difference in the mechanism of coupling of serotonin receptors to adenylate cyclase compared to other species of receptors. Responses of a NG108-15 hybrid cell to serotonin applied iontophoretically are presented in Fig. 5, which are represen- tative of the responses of NCB-20 and N1E-115 cells. Serotonin application resulted in two action potentials followed by cell depolarization for more than | sec (Fig. 5A), confirming pre- vious findings (19). Repetitive application of serotonin, at a 18-7 T T het ot 710 - \\\\°   a     pmol cAMP/min per mg protein pmol cAMP/min per mg protein gu Li pel pul siig 10-7 10-6 10-51040 10-8 10-7 10-6 105 {Serotonin}, M [LSD),M Fic. 4. Effect of LSD and serotonin on adenylate cyclase activity in homogenates of NCB-20 hybrid cells. (A) Increase in adenylate cyclase activity in response to increasing concentrations of serotonin in the absence (Q) or presence (@) of 10 »M LSD. (B) Response to increasing concentrations of LSD in the absence (A) or presence (4) of 10 uM serotonin. Oo 11388 Biochemistry: MacDermot et al. Table 3. Effect of serotonin (5HT) on adenylate cyclase activity in homogenates of neuroblastoma N18T62 or hybrid cell lines   pmol cAMP/min per mg protein   Activation, Cell line Basal 10 uM 5HT % NCB-20 7.03 14.8 111 NBr-20A 7.08 8.36 18 NG108-15 14.2 16.5 16 N18TG2 4.09 4.24 4 NBr-10A 48.7 50.0 3   frequency of 0.51 Hz, almost completely desensitized the re- sponse to serotonin (Fig. 5B). Responsiveness to serotonin re- covered within a few minutes in the absence of serotonin. LSD did not inhibit serotonin-dependent cell depolarization (Fig. 5C), and furthermore addition of LSD did not depolarize the cell (not shown). Application of PGE), which markedly in- creases adenylate cyclase activity (Table 4), had little or no effect on cell membrane potential (unpublished results) or acetylcholine release (21) from NG108-15 cells. NG108-15 (18) and NCB-20 (unpublished results) hybrid cells form synapses with rat striated muscle cells in vitro. Se- rotonin-dependent release of acetylcholine from an NG108-15 hybrid cell at a synapse was measured by intracellular recording of synaptic potentials in a rat myotube (Fig. 6A). Application of serotonin to the hybrid cell resulted in an increase in ace- tylcholine secretion from the NG108-15 hybrid cell and a greater than 50-fold increase in the frequency of muscle syn- aptic responses. LSD neither mimicked nor inhibited seroto- nin-dependent synaptic responses of the muscle cell (not shown). Serotonin had no effect on the membrane potential of muscle cells that were not innervated by NG 108-15 or NCB-20 cells. The effect of serotonin on [2H]acetylcholine release from NG108-15 cells into the medium was measured. Muscle cells were not present. Cells were incubated with [SH]choline and washed and [°H]acetylcholine released into the medium was separated from [H]choline and assayed (Fig. 6B) (21). Sero- tonin stimulated acetylcholine release from cells as shown (21), and acetylcholine release was not inhibited by 50-500 nM LSD. The same concentrations of LSD had little or no stimulatory effect on acetylcholine release. Table 4. Effect of GTP, sodium fluoride, Gpp(NH)p, PGE), and serotonin on NCB-20 adenylate cyclase activity   pmol cAMP/min per mg protein Addition Basal 10 uM 5HT Exp. A Control 16.3 20.0 1M GTP 21.4 31.2 Exp. B Control 5.06 8.88 20 mM NaF 43.5 42.6 10 nM Gpp(NH)p 11.4 16.6 10 uM PGE, 90.2 78.2 10 uM PGE, + 20 mM NaF 58.1 55.0 10 nM PGE, + 10 uM Gpp(NH)p 86.4 75.7   In Exp. A, each reaction mixture contained 81.6 ug of protein of a 30,000 X g particulate fraction, washed three times with 25 mM Tris-HCI (pH 7.4), recovered by centrifugation (20 min). In Exp. B, adenylate cyclase activity of an unfractionated NCB-20 homogenate is shown. Each value is the mean of triplicate determinations. 5HT, serotonin. Proc. Natl. Acad. Sci. USA 76 (1979)   Seconds 9 20 50190190 SuT A |5HT \"LSD C       TTT TT           Seconds Fic. 5. Serotonin (SHT)-dependent regulation of ionophore activity in NG108-15 hybrid cells. (A) Iontophoretic application of serotonin to a hybrid cell. The iontophoretic pipette contained a so- lution of 25 mM serotonin in water; a current of 100 nA was passed for 1 msec (shown in the trace at the bottom of each panel). (B) Re- petitive serotonin application at a frequency of 0.51 Hz, which results in desensitization. (C) Effect of LSD on the depolarizing response was determined with repeated iontophoretic applications of serotonin in the absence of LSD and then after addition of 20 ul of 100 «M LSD in 150 mM NaCl. Prior to each pulse, a voltage calibration of 10 mV was passed for 10 msec. DISCUSSION Activation of serotonin receptors of NG108-15 (19, 21) or NCB-20 hybrid cells results in cell depolarization, action po- tentials, and release of acetvlcholine into the medium. These responses desensitize in less than 15 sec and are not inhibited nn   frnot [*H] ACh released/mg protein   a { &e 2 Of 4250 Z [3 | e207 4 Q % & 4209 a 10 : *y 3 : : 2 i ° 4 [ i = QO O8 es _ 9g ees 1 150 “AO oS G eC 20 Pes are 35 Seconds Minutes Fic. 6. Serotonin-dependent release of acetylcholine from NG108-15 hybrid cells. (A) Depolarizing responses of a rat striated muscle cell to acetylcholine released from a N6108-15 cell in response to serotonin (5HT) at a synapse; recording obtained with an intra- cellular microelectrode. Two microliters of a solution containing 10 uM serotonin and 150 mM NaC! was applied to the hybrid cell (arrow). (Top) Trace of an AC recording at high gain; (Middle) trace of a DC recording at low gain; (Lower), the number of spontaneous and serotonin-evoked muscle responses per sec. (B) [®H]Acetylcholine ([]H}ACh) release from hybrid cells into the medium. Cells were grown in the presence of 1 mM BtgcAMP for 17 days and then transferred to capillary pipettes (21) and grown for an additional 2 days. Cells then were incubated in 25 uM [methyl-?H] choline (4.2 Ci/mmol) for 45 min and washed by perfusion (0.4 ml/min) for 17.5 min before 1-ml fractions were collected. [7H}Acetylcholine in the perfusate was separated from |methyl-*H}choline and measured (21). Cells were exposed to 10 uM>serotonin (5HT) with 50 nM (cd), 500 nM (A), or no (O) LSD at the times shown, Biochemistry: MacDermot et al. or mimicked by LSD. Serotonin also stimulates adenylate cy- clase activity of NCB-20 hybrid cells, and in contrast these re- ceptors do not desensitize. In addition, LSD activates adenylate cyclase and partially inhibits activation of the enzyme by se- rotonin. The results suggest that cell depolarization and acti- vation of adenylate cyclase are mediated by different species of serotonin receptors. The serotonin receptors that mediate excitatory, depolarizing responses resemble M-receptors of neurons in the peripheral nervous system (6). These receptors differ from both pre- and postsynaptic serotonin receptors of mammalian brainstem (5, 7), which inhibit neuronal firing and are also activated by LSD. Inhibitory serotonin responses not affected by LSD have been found in brainstem neurons (22), the suprachiasmic nucleus (4), and half the serotonin-sensitive neurons of cat cerebral cortex (23). The specificity of serotonin receptors coupled to activation of adenylate cyclase in NCB-20 hybrid cells resembles that of serotonin receptors coupled to adenylate cyclase in mammalian brain (3). The demonstration that activation of adenylate cyclase by serotonin is stimulated by GTP, that sodium fluoride uncouples activation by serotonin, and that serotonin inhibits to a small extent the stimulation of adenylate cyclase by PGE suggests a common mechanism for the coupling of receptors for sero- tonin and other neurotransmitters to the adenylate cyclase complex. However, the additive, rather than synergistic effects of serotonin and Gpp(NH)p suggest that coupling of the sero- tonin receptors to the adenylate cyclase complex may differ from that of other neurotransmitters. Serotonin stimulates udenylate cyclase activity 50-100% in homogenates of colliculus of neonatal rat brain, which is similar to the extent of activation of adenylate cyclase by serotonin in NCB-20 homogenates. Eadie~Scatchard analysis of the activation of adenylate cy- clase by serotonin suggests a bimolecular interaction and reveals no evidence of receptor heterogeneity. The Hill interaction coefficient (n) is 1.0, indicating independent, noncooperative reactions. LSD activates adenylate cyclase (Kact = 12 nM) and inhibits the activation of the enzyme by serotonin (K; = 10 nM). In addition, mianserin and cyproheptadine inhibit serotonin activation of adenylate cyclase (Kj = 43 nM and 95 nM, re- spectively) and LSD activation of adenylate cyclase (K, = 100 nM and 64 nM, respectively). These results show that serotonin and LSD interact at a receptor site(s) that mediates activation of adenylate cyclase. Enjalbert et al. (3) have shown a complex interaction between serotonin and LSD at the level of adenylate cyclase in mammalian brain. Interactions between serotonin and LSD have also been demonstrated in binding studies (24, 25). Binding sites for (SH|LSD were detected in NCB20 homog- enates (unpublished results); the Kp,,, was 36 nM, the Hill coefficient was 1.0, and the receptor concentration was 385 {mol/mg of protein. [7H|LSD was displaced by serotonin (K; = 110-180 nM). These results agree well with those presented for the stimulation of adenylate cyclase activity by a serotonin receptor that is also responsive to LSD. Two binding sites for [2H serotonin were detected in NCB-20 homogenates [KDiyp = 200 nM and 3750 nM] and serotonin and LSD interactions Proc. Natl. Acad. Sci. USA 76 (1979) 1139 also were shown. LSD does not displace serotonin from the low-affinity serotonin site, which suggests that these molecules may function as receptors mediating cell depolarization and acetylcholine release. We conclude that NCB-20 hybrid cells possess two species of serotonin receptors, that activation of adenylate cyclase does not affect the rate of acetylcholine release, and, conversely, that serotonin-dependent cell depolarization does not affect intra- cellular levels of cAMP or cGMP in the hybrid cells tested. 1. Von Hungen, K., Roberts, S. & Hill, D. F. (1975) Brain Res. 84, 257-267. 2. Enjalbert, A., Bourgoin, S., Hamon, M., Adrien, J. & Bockaert, J. (1978) Mol. Pharmacol. 14, 2-10. 3. Enjalbert, A., Hamon, M., Bourgoin, S. & Bockaert, J. (1978) Mol. Pharmacol. 14, 11-23. 4. Bloom, F. E., Hoffer, B. J., Siggins, G. R., Barker, J. L. & Nicoll, R. A. (1972) Fed. Proc. Fed. Am. Soc. Exp. Biol. 31, 97-106. 5. Haigler, H. J. & Aghajanian, G. K. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, 2159-2164. 6. Gaddum, J. H. & Picarelli, Z. P. (1957) Br. J. Pharmacol. 12, 323-328. 7. Aghajanian, G. K. & Haigler, H. J. (1975) Psychopharmacol. Commun. 1, 619-629. 8. Gerschenfeld, H. M. & Paupardin-Tritsch, D. (1974) J. Physiol. 243, 427-456. 9. Amano, T., Richelson, E. & Nirenberg, M. (1972) Proc. Natl. Acad. Sci. USA 69, 258-263. 10. Minna, J., Glazer, D. & Nirenberg, M. (1972) Nature (London) New Biol. 235, 225-231. 11. Amano, T., Hamprecht, B. & Kemper, W. (1974) Exp. Cell Res. 85, 399-408. 12. Minna, J. D., Yavelow, J. & Coon, H. G. (1975) Genetics, Suppl. 79, 373-383. 13. Klee, W. A. & Nirenberg, M. (1974) Proc. Natl. Acad. Sci. USA 71, 3474-3477. 14. Sharma, S. K., Nirenberg, M. & Klee, W. A. (1975) Proc. Nail. Acad. Sci. USA 72, 590-594. 15. Salomon, Y., Londos, C. & Rodbell, M. (1974) Anal. Biochem. 58, 541-548. 16. Matsuzawa, H. & Nirenberg, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3472-3476. 17. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275, 18. Nelson, P., Christian, C. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 123-127. 19. Christian, C. N., Nelson, P. G., Bullock, P., Mullinax, D. & Nirenberg, M. (1978) Brain Res. 147, 261-276. 20. Burt, D. R., Creese, I. & Snyder, S. H. (1976) Mol. Pharmacol. 12, 800-812. 21. McGee, R., Simpson, P., Christian, C., Mata, M., Nelson, P. & Nirenberg, M. (1978) Proc. Natl. Acad. Sci. USA 75, 1814- 1318. 22. Boakes, R. J., Bradley, P. B., Briggs, I. & Dray, A. (1969) Brain Res. 15, 529-531. 23. Roberts, M. H. T. & Straughan, D. W. (1967) J. Physiol. 193, 269-294. 24. Bennett, J. P., Jr. & Snyder, S. H. (1976) Mol. Pharmacol. 12, 373-389. 25. Fillion, G. M. B., Rousselle, J. C., Fillion, M. P., Beaudoin, D. M., Goiny, M. R., Deniau, J. M. & Jacob, J. J. (1978) Mol. Pharmacol. 14, 50-59.", "Nirenberg, Marshall W. ; Minna, John, 1941- ; Wilson, Steven P. ; Higashida, Haruhiro ; MacDermot, John ; Matsuzawa, Hiroshi", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9rqh_qdaz~7pt2", "00000000-0000-0000-9DCA-ED7469AD29E4", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Monoclonal Antibody to a Plasma Membrane Antigen of Neurons", "101584910X67", null, "1979", "October 1979", "In the late 1970s and early 1980s, the biochemical lab at NIH undertook a series of studies with clonal and hybrid cell lines that produced antibody, which bound to certain neural tissues but not others.  This article discusses a hybrid cell line that synthesizes an antibody, suggesting the need for studies of the synthesis, localization, and function of that antigen.", "Articles", "Antibodies,Antigens,Cell Membrane,Antibody Formation,Neurons", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 76, No. 10, pp. 4913-4917, October 1979 Biochemistry Monoclonal antibody to a plasma membrane antigen of neurons (lymphocyte hybridoma cells/ganglioside/retina/brain) GEORGE S. EISENBARTH*, FRANK S. WALSH, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205 Contributed by Marshall Warren Nirenberg, July 2, 1979 ABSTRACT Fusion of spleen cells from a mouse immunized with chicken embryo retina cells with clonal mouse myeloma cells yielded a lymphocyte hybrid cell line that produced anti- body that bound to neural tissue such as retina, brain, spinal cord, and dorsal root ganglia but not to other tissues tested. The antigen was shown by indirect immunofluorescence to be as- sociated with plasma membranes of most, or all, neuron cell bodies in chicken retina, but little or no antigen was detected on axons or dendrites, Miiller cells, or retina pigment cells. The activity of antigen A2B5 is relatively stable at 100°C, is insen- sitive to trypsin, exhibits the solubility properties of a ganglio- side, and is destroyed by neuraminidase. Antibody A2B5 cyto- toxicity against retina cells is inhibited by a GQ ganglioside fraction from bovine brain (estimated half-maximal inhibition at 0.2 4M) or by N-acetylneuraminic acid (half-maximal inhi- bition at 5000 4M) but not by other purified gangliosides tested. These results suggest that the antigen is a complex ganglioside in plasma membranes of retina neuron cell bodies but not axons or dendrites.   Chicken embryo retina cells have been used to study biological processes that require interactions between neurons, such as cell aggregation (1, 2), adhesiveness (3), and synapse formation (4-7). To define surface molecules of retina neurons, we have used the technique, introduced by Milstein and coworkers (8-10), of antibody production by hybrid cells formed by fusion of mouse myeloma cells with spleen cells from mice immunized with chicken embryo retina cells. This approach is useful be- cause lymphocyte hybridomas can synthesize large quantities of monoclonal antibody with specificity for a single antigen determinant. Hybridoma cell lines have been reported which synthesize antibodies specific for cell surface antigens as diverse as the Forsmann antigen (11), HLA determinants (12), tumor-specific antigens (13), and an antigen detected on human neuroblastoma cells and fetal brain (14) (also see ref. 15). Other techniques also have been used to produce antisera against nervous system antigens. These include xenogenic im- munization followed by extensive absorption with non-neuronal tissues (16, 17) and immunization with neural cell lines (18-20), partially purified synaptosomes, plasma membranes, and protein fractions (21, 22). In this report we describe the characterization of a mono- specific antibody synthesized by lymphocyte hybrid A2B5 cells that recognizes a surface antigen restricted to cell bodies of most, or all, retina neurons. METHODS AND MATERIALS Immunization of Mice and Production of Hybrid Cells. Female BALB/c mice were immunized by intraperitoneal injections at zero time and 7 days with 1 X 107 8-day-chicken embryo retina cells [dissociated with 0.2 mM sodium ethylene glycol bis(G-aminoethyl ether)-N,N,N’,N’-tetraacetate and   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “ad- vertisement” in accordance with 18 U.S. C. $1734 solely to indicate this fact. 4913 fixed with 0.1% glutaraldehyde in Dulbecco’s phosphate-buf- fered saline without Ca?* or Mg?+ (P;/NaCl) for 5 min, sus- pended in 0.25 ml of complete Freund’s adjuvant, followed at 37 days by intravenous and intraperitoneal injections of 5 X 106 cells (each) without adjuvant, On day 41, two spleens were re- moved, the cells were dissociated mechanically, and 2 x 108 spleen cells were fused with 2 X 107 P3X63 Ag8 mouse mye- loma cells (8) [obtained from John Minna] in the presence of 0.8 ml of 50% polyethylene glycol 1000 (Baker) according to the methods of Galfre et al. (23). After fusion, cells were sus- pended in medium A [Dulbecco's modification of Eagle’s minimal essential medium containing 100 4M hypoxanthine, 1 4M aminopterin, 16 uM thymidine, and 20% fetal bovine serum (heat inactivated at 56°C for 30 min)] and inoculated into 92 wells (2.2 X 10° cells per ml of medium per well per 2-cm? surface area) of Costar 3524 multiwell plates. A2B5 hy- brid cells (also termed F12A2B5) also were grown as ascites tumors in BALB/c mice that had been injected intraperitone- ally with 0.5 ml of pristane (Aldrich) 7 days prior to injection of 1-10 X 108 cells, Cytotoxicity and Absorption Assays. Synthesis of anti-retina antibodies by hybridoma A2B5 cells was measured by com- plement-dependent cytoxicity. Retinas obtained from White Leghorn chicken 8-day embryos (Truslow Farms, Chestertown, MD) were dissociated by incubation with 0.5% trypsin (crys- tallized 8 times, Worthington) for 5 min and washed by cen- trifugation. Approximately 1.2-1.6 X 108 cells were suspended in 4 ml of Eagle's minimal essential medium supplemented with 10% fetal bovine serum containing 400 wCi of Na®!CrO, (4.8 HM; 1.5 X 10\" becquerels) incubated for 40 min at 37°C in an atmosphere of 95% air/5% COs. Cells were washed twice with P;/NaCl, incubated at 4°C for 30 min in Pi/NaCl, washed three times with P;/NaCl, and then suspended in P;/NaCl. 5!Cr- Labeled retina cells (1 X 106 cells, approximately 2000- 10,000 cpm) were incubated in a mixture of 95 yl of P;/NaCl, 5 ul of guinea pig serum as a source of complement, and 50 ul of cul- ture medium or diluted antibody in microtiter U plates (catalog no. 76-205-042, Flow General, Hamden, CT). The cell sus- pension was incubated for 30 min at 37°C; cells then were sedimented at 1000 X g; the supernatant solution was harvested by using Titertek harvesting filters (Flow General, catalog no. 78-210-05) and the radioactivity in the supernatant solutions that was absorbed by the filters was determined. Saturating concentrations of antibody A2B5 in the presence of complement released 50-60% of radioactive material from cells compared to that released by 0.3% Triton X-100. In the absence of com- plement, <5% of the radioactive material was released.   Abbreviation: P;/NaCl, Dulbecco's phosphate-buffered saline without Ca?* or Mg*+. * Present address: Box 3021, Department of Medicine, Duke Univer- sity, Durham, NC 27710. * Present address: Muscular Dystrophy Laboratories, Institute of Neurology, National Hospital, Queen Square, London WCI N 3BG, England. AG34 Biochemistry: Eisenbarth e¢ at. Cells used for absorption of antibody were prepared by mincing the embryonic tissues into 1-mm pieces, triturating the suspension with a 10-ml pipette, and forcing the cells through a Nitex 130 nylon filter. Various concentrations of cells suspended in 100 yl of P,/NaCl were incubated with 0.4 yg of antibody {antibody concentration determined by assay with 125]_labeled protein A (24)] for 30 min at room temperature. Cells were removed by centrifugation at 8000 X g for 2 min, and 50 yl of the supernatant solution (absorbed antibody) was added to each well to assay cytotoxicity against 8-day-embryo retina cells. Antigen Characterization. White Leghorn chicken eggs fertilized 8 days previously were obtained from Truslow Farms. Ten 8-day-embryo retinas and three 8-day-embryo brains were homogenized separately in 3.3 ml of methanol /chloroform/ water, 2:1:0.3, (vol/vol), in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 1000 X g for 20 min at 4°C, the supernatant fraction was removed, and the pellet was extracted with 3.8 ml of methanol/chloroform/water, 2:1:0.8 (vol/vol). After centrifugation (1000 X g for 20 min at 4°C), the supernatant fractions were combined (fraction A). Two milliliters of chloroform and 2 ml of HzO were added to the pooled supernatant fraction, the mixture was centrifuged at 1000 X g for 20 min, and the lower chloroform phase (fraction B) and upper methanol/H,O phase were separated. Methanol was removed from the upper phase by flash evaporation (fraction C), and the residue was tested for ability to inhibit antibody A2B5-dependent cytotoxicity. In other experiments, the upper phase was dialyzed overnight at 4°C against 50 vol of H2O (changed three times) and lyophilized, and the residue was dissolved in chloroform/methanol/H,20, 10:5:1 (vol/vol) (fraction D). Solvents were removed from fractions by evapo- ration under a stream of nitrogen or air. The residues were dissolved in HO and assayed for inhibition of cytotoxicity. GM,, GD), and GT, were obtained from Supelco; GT; and GQ fractions were kindly provided by Peter Fishman (Labo- ratory of Neurochemistry, National Institutes of Health). The GQ fraction purified by DEAE column chromatography and silicic acid column chromatography (25) was approximately 1000-fold enriched in GQ compared to unfractioned bovine brain gangliosides. Indirect Immunofluorescence. Chicken neural retina was fixed with 3% formaldehyde in P,/NaCl for 30 min, washed with P,/NaCl, immersed in liquid nitrogen, and then sectioned (16 zm). Sections were incubated with 50-100 j] of A2B5 as- cites fluid diluted 1:100 (2-4 ug of antibody) for 45 min at room temperature and then washed with P;/NaCl for 45 min. For immunofluorescence of monolayer cultures, cells were disso- ciated from 8-day-embryo retina (0.5% trypsin for 5 min at 37°C) and cultured for 24 hr in 35-mm petri dishes (1 X 10® cells per dish) in 90% Eagle’s minimal essential medium (GIBCO)/10% fetal bovine serum. The cells were fixed as de- scribed above, washed, and incubated with 4 ug of antibody in 100 yl of P,/NaCl for 45 min. Cultured retina cells or sections were incubated with fluorescein-labeled rabbit antibody di- rected against mouse IgG for 30 min at room temperature and then were washed with P,/NaCl. RESULTS Spleen cells from a mouse immunized with chicken retina cells from 8-day embryos and P3X63 Ag8 myeloma cells which lack hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) (8) were fused in the presence of polyethylene glycol. Two weeks after fusion, colonies were present in all wells. Part of the medium was removed and assayed (i) for antibody directed against retina cells by assaying binding of !*°I-labeled protein EPG. Neat SO A to 8 day embryo retina cells with bound immunoglobulin (26) and (ii) for complement-dependent cytotoxicity by using 8- day-embryo retina cells labeled with 5!Cr as targets. Media harvested from 45 of the 92 wells assayed contained antibodies that bound to retina cells and to protein A. Cytotoxic antibodies were detected in three wells and the properties of one, A2B5, is described in this communication. Antibody was obtained by harvesting the medium from cultured cells or ascites fluid from BALB/c mice with A2B5 ascites tumors. The ascites fluid was cytotoxic, at a dilution of 1:25,000, to 8-day-embryo retina cells and was used for most of the studies described. A2B5 cells were cloned six times by dilution (0.8 cell /well) in multiwell plates. After 2 weeks of incubation for each cloning cycle, approxi- mately 30% of the wells contained colonies. Initially, both positive and negative sublines of A2B5 were obtained with re- spect to the synthesis of antibody directed against chick retina; however, cycle 5 of cloning resulted ina subline (A2B5 clone 105) that, when cloned again, gave rise only to positive colonies. Ascites fluid from mice bearing subline A2B5 clone 105 was cytotoxic, at a dilution of 1:300,000, to 8-day-embryo retina cells. Different tissues were assayed for antigen A2B5 by incu- bating the antibody with the cells and, after absorption, de- termining the remaining cytotoxic activity against 8-day- embryo retina cells. The cytotoxic activity of antibody A2B5 was removed by 8- and 16-day-embryo retina or brain cells but not by muscle, heart, kidney, or liver cells or erythrocytes (Fig. 1). The antigen also was detected in human and bovine brain and chicken dorsal root ganglion (data not shown). The neural retina of the chicken 16-day embryo consists of three layers of cell bodies separated by two layers of axons and dendrites connected by synapses. The distribution of antigen A2B5 in sections of 16- and 8-day-embryo retina and adult T T T TT TTT J —— T ror ry        8 60 40 - \"I | OWS 10 NUMBER OF CELLS USED FOR ABSORPTION Fic. 1. Residual cytotoxicity of antibody A2B5 after absorption with different concentrations of cells from 8- or 16-day-embryoe tissues: @, 8-day, brain; O, 16-day, brain; m, 8-day, retina; 0, 16-day. retina: #, 8-day, liver; A, 16-day, liver; &, 16-day, muscle; @, 16-day. heart; &, 16-day, kidney; @, 16-day, erythrocytes. Residual cytotox- icity = (cpm released due to antibody A2B5 after absorption/epm released due to unabsorbed antibody A2B5) x 100. One hundred percent residual cytotoxicity corresponds to the release of 785 cpm from 8-day-embryo cells labeled with °!Cr, in response to 0.4 wg of antibody A2B5. RESIDUAL CYTOTOXICITY (percent)   Biochemistry: Eisenbarth et al.   retina, determined by indirect immunofluorescent staining, is shown in Fig. 2. All regions of the 16-day-embryo retina oc- cupied by cell bodies bound antibody A2B5, but the antigen was not detected on axons or dendrites of neurons in the inner or outer synaptic layers of the retina or on axons of ganglion neurons (Fig. 2A). Layers of cell bodies separated from pro- cesses are not present in 8-day-embryo retina, and antibody A2B5 bound to cells in all regions of 8-day-embryo retina (Fig. 2B). In adult chicken retina (Fig. 2C), antibody A2B5 bound to cell bodies of photoreceptor cells, amacrine neurons, hori- zontal neurons, bipolar neurons, and ganglion neurons but not to cell processes in the inner and outer synaptic layers of the retina. In this and other retina sections, the inner segments of photoreceptor cells were intensely fluorescent and fluorescence     Proc. Natl. Acad. Sci. USA 76 (1979) 4915 FIG. 2. Distribution of antigen A2B5 in sections of chicken retina from 16-day embryo (A), 8-day embryo (B), and adult (C). Letters refer to retina layers: R, photore- ceptor cell layer; O, outer synaptic layer; IN, inner nuclear layer con- taining cell bodies of horizontal, bipolar, and amacrine neurons and Miller cells; IS, inner synaptic layer comprised of processes and synapses of amacrine, bipolar, and vanglion neurons; G, ganglion neuron cell bodies; A, ganglion neuron axons, (Bar, 4 um.) was detected both within the cells and on the surface mem- brane. Thus, the antibody may be reacting with both plasma membrane and cytoplasmic antigens. Less antibody A2B5 bound to the central part of the inner nuclear layer which contains Miller cell bodies (retina glial cells) than to other areas of the retina with cell bodies. Antibody A2B5 also did not bind to processes of Miller cells which traverse the inner and outer synaptic layers. In contrast to the pattern of fluorescence with antibody A2B5, antibodies produced by other hybrid cell lines and rabbit anti-retina antibodies bound to all layers of the retina {data not shown). The distribution of antigen A2B5 on retina cells dissociated from 8-day-embryo and cultured for 24 hr also was determined. These retina cells adhered to the substratum, extended pro- FIG. 3. Distribution of antigen A2B5 on chicken retina cells dis- sociated from 8-day-embryo and cultured for 1 day, detected by in- direct. immunofluorescence. (A) Bright-field view of the cultured cells showing cell bodies and pro- cesses (denoted by arrows). (8) Same field showing fluorescent cell bodies and cell processes without fluorescence. (Bar, 20 um.) 4916 Biochemistry: Eisenbarth et al. Table 1. Properties of retina antigen A2BS in 100,000 X ¢ pellet fraction   Residual cytotoxicity, Antibody A2B5 plus: % No addition 100 + 5 Pellet 445 Pellet, 3 min at 100°C 2345 Pellet, 30 min at 100°C 4044 Pellet, 5 min with trypsin 643 Pellet, 30 min with trypsin 38+6 Pellet extracted with chloroform/methanol, 2:1 87 +18 Material extracted from 100,000 X g pellet into chloroform/methanol, 2:1 38849   Pellet fraction corresponded to 44 pg of unfractionated retina protein; antibody A2B5 was 0.4 wg per well. Release of *!Cr from 8- day-embryo retina cells was determined. Where specified, the pellet traction was incubated with 0.8 mg of trypsin in 400 yl at 37°C for 3 or 30 min: then 0.8 mg of soybean trypsin inhibitor was added and the effect on cytotoxicity was determined. Residual cytotoxicity is defined in the legend to Fig. 1. One hundred percent residual cytotoxicity corresponded to 1515 cpm released from 8-day-embryo retina cells labeled with *!Cr. Each value is the mean + SEM of three determi- nations, cesses, and formed aggregates (Fig. 3). Antibody A2B5 bound to >60% of the cell bodies but little or no antigen was detected on cell processes. The antibody did not bind to retina pigment cells or to cells with the morphology of fibroblasts or epithelial cells (these comprise <20% of the cell population). The re- maining cells that lack antigen A2B5 may be Miller cells. An- tigen A2B5 was detected on the soma and on the processes of cultured chicken embryo dorsal root ganglion cells; thus, lo- calization to the cell soma is not an invariant property of the antigen. Antigen A2B5 was assayed by inhibition of antibody A2B5- dependent cytotoxicity (Table 1). The antigen was inactivated only 36% on incubation at 100°C for 30 min, was insensitive to trypsin, was sedimented by centrifugation at 100,000 x g for 30 min, and was extracted from the pellet into chloroform/           > T y T y T J T 5 CHLOROFORM PHASE METHANOL: H,O PHASE x 1 Fae © E NO oO EXTRACT E > oO oO RETINA LL O Lu oO wn 7 BRAIN oo \\\\ 1 Ll ! \\\\ 1500 1:1000 1:2000 NoAb 1:00 1:1000 NoAb ANTIBODY A2B5 TITER FIG. 4. Inhibition of antibody A2B5-dependent cytotoxicity by molecules extracted from 8-day-embryo chicken brain or retina into chloroform/methanol/H20, 3:1:0:3. Antigenic activities (corre- sponding to antigen extracted from 3.9 mg of brain or 1.0 mg of retina, wet weight) in the chloroform phase (fraction B) and in the metha- nol/H2O phase (fraction C) are shown in A and B, respectively.                Proc, Natl. Aead. Sci. USA 6 ddEY     Table 2. Effect of neuraminidase on anugen A2BS Residual cytotoxicity, Additions © None 0 Antibody A2B5 100 + 2 Antibody A2B5 + antigen A2B5 5344 Antibody A2Ba + neuraminidase 9246 Antibody A2B5 + antigen A2B5 + neuraminidase 86+3 Antibody A2B5 + antigen A2B5 + boiled neuraminidase 4342   Antigen A2B5 extracted from 7 mg (wet weight) of chicken 8-dav- embryo brain (fraction D) was incubated at 37°C for 60 min in a re- action mixture (final volume 320 yl) containing 30 mM sodium acetate (pH 5.5), 0.43 wg of neuraminidase protein from Clostridium per- fringens purified by affinity chromatography (47 units/mg protein. Sigma), or heat-inactivated neuraminidase (100°C, 10 min). Then. 20 pl of the reaction mixture containing antigen A2B5 extracted from 440 pg (wet weight} of brain and 0.027 wg of neuraminidase or heat- inactivated neuraminidase protein was added to each well assaved for cytotoxicity (0.4 wg of antibody A2B5 per well). Residual cyto- toxicity is defined in the legend of Fig. 1. One hundred percent re- sidual cytotoxicity corresponded to the release of 196 cpm. Each value is the mean + SEM of three determinations. methanol, 2:] (vol/vol). Antigen A2B5 was solubilized by ex- traction of 8-day-embryo chicken brain or retina with metha- nol/chloroform/H,20, 2:1:0.3 (vol / vol). Two additional volumes of chloroform and 2 vol of HO were added. The lower chlo- roform phase and the upper methanol/H20 phase were sepa- rated, taken to dryness, and assayed for inhibition of antibody A2B5-dependent cytotoxicity (Fig. 4). All of the antigen ex tracted from brain or retina partitioned into the methanol/H20 phase and thus exhibited the solubility of a ganglioside. A2B5 antibody-dependent cytotoxicity was inhibited by compounds in the methanol /H2O phase (Fig. 4B) but not by material in the chloroform phase (Fig. 4A). When material in the methanol/ H2O phase was fractionated by thin-layer chromatography {silica gel plates; propanol/H2O, 70:30 (vol/vol)|, antigenic activity remained close to the origin (Rr = 0.02), with GQ gangliosides. Because the antigenic material exhibited the properties ol a ganglioside, the effect of neuraminidase (purified by affinity   GLUCOSAMINIC ACID          N-ACETYL NEURAMINIC | ACID 20 UNFRACTIONATED GANGLIOSIDES RESIDUAL CYTOTOXICITY (percent) @ So Ga 0 pot — 7 6 5 4 3 2 CONCENTRATION —Log (MOLARITY) Fic. 5. Inhibition of antibody A2B5-mediated cytotoxicitY hs purified ganglioside preparations and saccharides. Each point re? resents the mean of three determinations. The effects of gangliosid” GQ (®), GM; (m), GD, (4), and GT; (@), bovine gangliosides (0” N-acetylneuraminic acid (4), and glucosaminic acid (DB) were studie at the concentrations shown.   Biochemistry: Eisenbarth et al. column chromatography) on the antigenic activity was deter- mined (Table 2). Incubation with neuraminidase almost com- pletely blocked the inhibitory effect of antigen A2B5 on anti- body-dependent cytotoxicity. The effects of partially purified ganglioside fractions and several monosaccharides on release of 5!Cr from 8-day-embryo retina cells due to antibody A2B5 cytotoxicity are shown in Fig. 5. A GQ ganglioside fraction (ganglioside molecules with four N-acetylneuraminic acid residues) purified approximately 1000-fold inhibited cytotoxicity 50% at an estimated concen- tration of 0.2 uM. A crude bovine ganglioside fraction and N-acetylneuraminic acid also inhibited the cytotoxic effect of antibody A2B5 with half-maximal inhibitions estimated to be 600 and 5000 uM, respectively. Gangliosides GT,, GD, and GM, and glucosaminic acid had little or no effect on cytotox- icity. These results suggest that antibody A2B5 recognizes an antigen with the properties of a complex ganglioside such as a GQ ganglioside. Further work is needed to define the structure of the antigen. Ina preliminary study, antibody A2B5 was used to explore the possible role of the antigen in synapse formation. Saturating concentrations of antibody A2B5 did not inhibit the formation of synapses between dissociated 8-day-embryo chicken retina cells and rat myotubes or the transmission across synapses. DISCUSSION A hybrid cell line that synthesizes a cytotoxic antibody directed against chicken embryo retina neurons was obtained by fusion of mouse myeloma cells with spleen cells of mice immunized with retina cells. The antigen also was found in chicken embryo brain, spinal cord, and dorsal root ganglion neurons and in bovine and human brain but was not detected in heart, muscle, liver, kidney, or erythrocytes of chicken embryos. The antigen of chicken retina was shown by indirect immunofluorescence to be associated with plasma membranes of most, or all, neuron cell bodies including photoreceptor cells, horizontal neurons, amacrine neurons, bipolar neurons, ganglion neurons, and neuroblasts. The antigen was not detected on axons or dendrites in the synaptic layers of the retina, even though the synaptic layers contain more plasma membrane than equivalent areas of the retina composed of cell bodies. Bipolar neurons may contain less antigen A2B5 because they fluoresced less intensely than did other retina neurons. In addition, inner segments of photoreceptor cells fluoresced more intensely than outer seg- ments. Antigen A2B5 was not detected on retina Miller cells, pigment cells, or cells with the morphology of fibroblasts or epithelial cells. Molecules in chicken retina with a distribution complementary to that of antigen A2B5 (ie., preferentially localized to axons or dendrite plasma membranes) have been reported, such as cell adhesion molecules (27), acetylcholines- terase (EC 3.1.1.7) (28), nicotinic acetylcholine receptors (29), and muscarinic acetylcholine receptors (30). A2B5 antigenicity was not destroyed by trypsin or by incu- bation at 100°C but was inactivated during incubation with neuraminidase. The antigen sedimented at 100,000 X g and was soluble in chloroform/methanol (2:1). A GQ ganglioside fraction from bovine brain purified 1000-fold inhibited the cytotoxic effect of antibody A2B5 on retina cells. The concen- tration for half-maximal inhibition was estimated to be 0.2 uM. In contrast gangliosides with three or less N-acetylneuraminic acid residues that were tested had no effect on cytotoxicity at concentrations of 1 mM. Antibody A2B5 may not interact with all molecules in the GQ ganglioside fraction; thus, the observed half-maximal inhibition of cytotoxicity may be a minimal es- timate of the affinity of the antigen for the antibody. Proc. Natl. Acad. Sci. USA 76 (1979) 4917 The localization of antigen A2B5 on the exterior surface of cell bodies of neurons raises questions concerning the function of the antigen. Whether the antigen is involved in the sorting out of neurites from cell bodies or in determining the relative positions of neuronal soma in the retina is unknown. However, the availability of large quantities of monoclonal antibody should be useful for further purification of the antigen, for separating populations of neurons that possess the antigen from other cell types, and for studies on the synthesis, localization, and function of the antigen. . We thank Dr. Peter Fishman for generously supplying us with a purified GQ ganglioside fraction and Dr. Roscoe Brady for helpful suggestions concerning fractionation of chicken brain gangliosides. 1. Hausman, R. E. & Moscona, A. A. (1976) Proc. Natl. Acad. Sci. USA 73, 3594-3598. 2. Sheffield, J. B. & Moscona, A. A. (1970) Dev. Biol. 23, 36-61. 3. Thiery, J. P., Brackenbury, R., Rutishauser, U. & Edelman, G. M. (1977) J. Biol. Chem. 252, 6841-6845. 4. Vogel, Z., Daniels, M. P. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 2370-2374. 5. Stefanelli, A., Zacchei, A. M., Caravita, S., Cataldi, A. & leradi, L. A. (1967) Experientia 23, 199-200. 6. Puro, D. G., DeMello, F. G. & Nirenberg, M. (1977) Proc. Natl. Acad. Sci. USA 74, 4977-4981. 7. Ruffolo, R. R., Eisenbarth, G. S., Thompson, J. M. & Nirenberg, M. (1978) Proc. Natl. Acad. Sci. USA 75, 2281-2285. 8. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495- 497. 9. Williams, A. F., Galfre, G. & Milstein, C. (1977) Cell 12, 663- 673. 10. Pearson, T., Galfre, G., Ziegler, A. & Milstein, C. (1977) Eur. J. Immunol. 7, 684-690. 11. Stern, P. L., Willison, K. R., Lennox, E., Galfre, G., Milstein, C. Secher, D. & Ziegler, A. (1978) Cell 14, 775- 783. 12. Lampson, L., Levy, R., Grumet, F., Ness, D. & Pious, D. (1978) Nature (London) 271, 461-462. 13. Koprowski, H., Steplewski, Z., Herlyn, D. & Herlyn, M. (1978) Proc. Natl. Acad. Sci. USA 75, 3405-3409. 14. Kennett, R. & Gilbert, F. (1979) Science 203, 1120-1121. 15. Melchers, F., Potter, M. & Warner, N. L., eds. (1978) Curr. Top. Microbiol. Immunol. 81. 16. Goldschneider, I. & Moscona, A. A. (1972) J. Cell Biol. 53, 435-449. 17. Schachner, M., Wortham, K. & Kincade, P. (1976) Cell. Immu- nol. 22, 369-374. 18. Akeson, R. & Herschman, H. R. (1974) Proc. Natl. Acad. Sci. USA 71, 187-191. 19. Martin, S. E. (1974) Nature (London) 249, 71-78. 20. Fields, K. L., Gosling, C., Megson, M. & Stern, P. L. (1975) Proc. Nail. Acad. Sci. USA 72, 1296-1300. 21. Bock, E., Jorgensen, O. S. & Morris, S. J. (1974) J. Neurochem. 22, 1013-1017. 22. Mikoshiba, K., Huchet, M. & Changeaux, J. P. (1977) Proceed- ings, Sixth International Congress of Neurochemistry, Co- penhagen, p. 278. 23. Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W. & Howard, J. C. (1977) Nature (London) 266, 550-552. 24. Langone, J. J., Boyle, M. D. & Borsos, T. (1977) J. Immunol. Methods 18, 281-288. 25. Ledeen, R. W. & Yu, R. K. (1978) Res. Methods Neurochem. 4, 371-410. 26. Schneider, M. & Eisenbarth, G. (1979) J. Immunol. Methods, in press. 27. Rutishauser, U., Thiery, J., Brackenbury, R. & Edelman, G. (1978) J. Cell Biol. 79, 371-381. 28. Shen, S. C., Greenfield, P. & Boel, E. J. (1956) J. Comp. Neurol. 106, 433-440. 29. Koelle, G. B., ed. (1963) Handbook of Experimental Pharma- cology (Springer, New York), Vol. 15. 80. Vogel, Z. & Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 1806-1810. 31. Sugiyama, H., Daniels, M. P. & Nirenberg, M. (1977) Proc. Natl. Acad. Sci. USA 74, 5524-5528.", "Eisenbarth, George S. ; Nirenberg, Marshall W. ; Walsh, Frank (Frank S.), 1953-", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-n9h6-xj53_c3kg", "00000000-0000-0000-6781-B5174CA67598", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "A Topographic Gradient of Molecules in Retina Can be Used to Identify Neuron Position", "101584910X68", null, "1981", "April 1981", "As part of Marshall Nirenberg's continued study of retinal gene expression, the objective of this study is to identify the role of certain molecules in the coding of information in the retina based on their location and ability to facilitate neural networks.", "Articles", "Nerve Tissue Proteins,Antibodies,Epitopes,Neurons", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "proc. Natl. Acad. Sci. USA Vol. 78, No. 4, pp. 2145-2149, April 1981. Biochemistry A topographic gradient of molecules in retina can be used to identify neuron position (cell membrane/antigen/embryo/synapses/hybrid cells) CG. Davip TRISLER, MICHAEL D. SCHNEIDER, AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205 Contributed by Marshall Warren Nirenberg, December 24, 1980 ABSTRACT A monoclonal antibody was obtained that binds to cell membrane molecules distributed in a topographic gradient in avian retina. Thirty-five-fold more antigen was detected in dor- soposterior retina than in ventroanterior retina. Most of the an- tigen was associated with the synaptic layers of the retina. Less antigen was detected in cerebrum, thalamus, cerebellum, and optic tectum, but little or none was found in non-neural tissues tested. The antigen was found on most or all cell types in retina, and the concentration of antigen found is a function of the square of the circumferential distance from the ventroanterior pole of the gradient toward the dorsoposterior pole. Thus, the antigen can be used as a marker of cell position along the ventroanter- ior-dorsoposterior axis of the retina.   Topographic relationships between retina ganglion neurons are conserved, forming point-to-point representations of the retina, when ganglion neurons synapse in tectum or certain other re- gions of brain. Thus, the retina is a favorable model system for studying the formation of synaptic circuits. However, the mo- lecular basis for spatial order has not been defined. Sperry (1) hypothesized that two orthogonal gradients of molecules on ret- ina ganglion neurons and corresponding gradients of compli- mentary molecules in the optic tectum might determine the specificity of synaptic connections between retina and tectum neurons. Other mechanisms proposed include adhesive inter- actions between migrating neurites, myelination of bundles of axons, and formation of extracellular channels by glia to guide axons in appropriate directions (2). Antibodies provide a means of identifying surface molecules and reactions required for neural function (3-7). Rabbit anti- serum to clonal retina hybrid cells was used to detect an antigen with a restricted domain in retina (8). Our objective was to detect cell surface molecules with topo- graphic specificity in the retina, as candidates for neuronal rec- ognition molecules. Monoclonal antibodies were obtained by fusing P3X63 Ag8 mouse myeloma cells (9) with spleen cells from mice immunized with small portions of dorsoposterior or ventral chicken embryo retina. A hybridoma antibody was ob- tained that binds to cell membrane molecules that are distrib- uted in a dorsoposterior > ventroanterior gradient in retina. RESULTS The rationale for the experiments is shown in Fig. 1. Spleen cells from mice immunized with small portions of dorsoposterior or ventral neural retina from the left eyes of 14-day chicken embryos were fused with P3X63 Ag8 mouse myeloma cells, and the binding of hybridoma antibody to cultured cells from the sector of the retina used for immunization was compared with   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U. §. C. §1734 solely to indicate this fact. 2145     MOUSE IMMUNIZATION WITH CHICK RETINA SEGMENTS , —— ra N, . Ai P Ai iP Ye lA Vv v HYBRIDOMA ANTIBODY SPECIFICITY DD - D f f a x At iP t 1 A P 4 i ‘ i Vv NUMBER v NUMBER OF OF HYBRIDOMA HYBRIDOMA COLONIES COLONIES tee t + 1 + + 5 + + 13 + + 3 — — 54 — ~ 101         Fic. 1. (Upper) Hybridoma cell lines were derived from mice im- munized with cells mechanically dissociated from dorsoposterior (Left) or ventral (Right) retina from the left eyes of 14-day White Leghorn chicken embryos (Gallus gallus) (sections labeled 4 and 1, respectively, in Fig. 2). Symbols, A, D, P, and V correspond to anterior, dorsal, pos- terior, and ventral, respectively. The choroid fissure, through which axons exit or enter the retina, shown extending from the ventroanteri- or margin of the retina, was used as a landmark for dissection. Female BALB/c mice were injected intraperitoneally at 0,7, and 14 days with 8 x 10° retina cells in 0.4 ml of Dulbecco’s phosphate-buffered saline and intravenously with 2 x 10° cells in 0.1 ml of the saline. On day 17, 108 dissociated spleen cells from each mouse were fused (10) with 2 x 107 P3X63 Ag8 mouse myeloma cells. After fusion, the cells were suspended with 5 x 10° spleen cells from a mouse that had not been immunized in 50 ml of medium A. [Medium A is selective medium D20SHAT of Berzofsky et ai. (11) except that it contains 10% fetal bo- vine serum, 1 «M aminopterin, 2 milliunits of insulin per ml, and each nonessential amino acid at 0.1 mM.] Cells were added to 96-well plates (Falcon) (186,000 cells per 74 j:1 of medium A per well), incubated at 37°C in a humidified atmosphere of 10% CO,/90% air, and fed addi- tional medium A (0.1 ml per well) on the 5th and 10th days of incu- bation. (Lower) To identify hybridomas synthesizing antibodies to re- gional antigens in retina, cells from the portion of 12-day chicken embryo retina (left eye) used for mouse immunization, or the rest of the retina, were dissociated with trypsin and collagenase, and transfer plates (12) were inoculated with 1.2 x 10° of the cells per well. Cells were cultured for 2 days in 90% Eagle’s minimal essential medium/ 10% fetal bovine serum; the medium was replaced with 50 yl of me- dium conditioned by hybridoma cells, and plates were incubated for 30 min at 37°C. Each well was washed three times with 150 j] of solution B (1 mg of pigskin gelatin per ml of phosphate-buffered saline) at 4°C. Each cell monolayer was incubated for 30 min at 37°C with 50 yl of solution B containing 1.6 nM rabbit '*°I-labeled F(ab’), antibody frag- ment [?25]-F(ab’))] directed against mouse IgG heavy and light chains (9799 cpm) and 500 xg of bovine serum albumin. Cells were washed as above, and bound radioactivity was determined. F(ab’), (Cappel) was purified on a mouse IgG-Sepharose 4B column and iodinated with mono-!“] Bolton—-Hunter reagent (Amersham) (13). binding to cells from the remainder of the retina. Cultured ret- ina cells were used to increase the probability of detecting an- tibodies directed against cell surface molecules. Sixty-eight of 2146 — Biochemistry: Trisler et al. 672 wells inoculated contained hybridoma colonies derived from a mouse immunized with dorsoposterior retina cells. One cell line (14H3) synthesized antibody that bound more to dor- soposterior retina cells than to cells from the rest of the retina, whereas 13 hybridoma antibodies bound equally to cells from both portions of retina. Antibodies to retina were not detected with 54 additional cell lines. Hybridoma colonies derived from a mouse immunized with ventral retina cells were detected in 109 of the 672 wells plated. Five antibodies of the 87 tested bound somewhat more to cells from ventral retina than to cells from the rest of the retina, and 3 antibodies bound equally. Thus, 14% of the 155 hybridoma cell lines examined synthesize antibodies to retina; however, only one antigen was detected, termed TOP for toponymic (i.e., a marker of position), with an asymmetric distribution, more abundant in dorsoposterior ret- ina than in the remainder of the retina. Distribution of TOP Molecules in Retina. Retinas from right or left eyes of chicken embryos were cut into eight sections as shown in Fig. 2, and cells from each section were assayed for {!*1-F(ab’),‘anti-TOP antibody‘TOP antigen] complexes. Bi- laterally symmetrical gradients of TOP were found in retina from right and left eyes. The highest concentrations of antigen detected were in dorsoposterior or dorsal retina and the lowest, in ventroanterior or ventral retina. Little }°I-F(ab’), bound to   VW toot tot TOPOGRAPHIC GRADIENT OF ANTIGEN         DORSAL DORSAL   VENTRAL RIGHT RETINA VENTRAL LEFT RETINA pMOL 1251-F(ab‘lg BOUND/mg PROTEIN       Fic. 2. Gradients of TOP molecules in retina from right (e) and left (a) eyes of 14-day chicken embryos. e———-@ and a——a, anti-TOP antibody; @----@ and a----4, P3X63 Ag8 antibody; o——o and A——A, buffer B without antibody to TOP. Values shown within appropriate sections of retina are pmol of '”*I-F(ab’), specifically bound per mg protein (i.e., pmol of !7°I-F(ab’), bound in the presence of anti- TOP antibody minus pmol of }”°I-F(ab’), bound in the presence of P3X63 Ag8 antibody). The assay conditions for TOP antigen used in this and subsequent experiments, except where stated, were as follows. Each retina was cut into eight sections as shown. Retina cells were mechanically dissociated. in phosphate-buffered saline at 4°C by trit- uration (10 times) with a 200 yz] micropipette tip (Medical Laboratory Automation), and 100 j] of a cell suspension (usually 160 yg of protein; range, 50-250 yg) was added to each well of polyvinyl chloride 96-well V-bottom plates (Dynatech) treated with solution B. Cells were cen- trifuged at 1300 x g for 5 min at 4°C and supernatant solutions were decanted. Each pellet was washed three times in solution B at 4°C (150 pl each wash), suspended in 50 yl of an antibody solution containing 1.0 yl of ascites fluid in solution B (except where specified), and in- cubated for 30 min at 4°C. Retina cells were washed three times as above, suspended in 50 yl of solution B containing 440 nM rabbit }751- F(ab’), (5-10 x 10* cpm) and 500 yg of bovine serum albumin, and incubated at 4°C for 30 min, unless otherwise specified. Pellets were . washed three times as described above, wells were separated, and ra- dioactivity was determined. Each value shown is the mean of two or three determinations. Protein was determined by a modification of the method of Lowry et al. (14). Proc. Natl. Acad. Sci. USA 78 (1981)                                             A. PROTEIN = [|B MONOCLONAL ANTI- 2 [0.5251 Ftab’i2 ANTI. | o CONCENTRATION & gi BODY CONCEN- i | MOUSE IgG CONCEN- 26 TOTAL, | @ | TRATION 230; TRATIO | g : >, / Re a a a £ 5 hit ; | : 4 a 3° Z 2% 7 434 3 | wm m | a = 2 7 2 NONSPECIFIC 3 f ‘ol won. ¢ VENTRAL ig! HIE g Lz RETINA | 8 Bi j a NONSPECIFIC | = At = -O-6 18 a 0 200 400 6007 00.0010.010.1 127, 4 ug RETINA PROTEIN $ wi ASCITES FLUID 3 uM 1251-Flab‘)2 a a Fic. 3. (A) Effect of retina protein concentration on ?”°I-F(ab’), binding in the presence of anti-TOP antibody (A) or P3X63 Ag8 anti- body (0). @, }”°I-F(ab’}, bound specifically. (B) Effect on hybridoma antibody (ascites fluid) concentration on )7°I-F(ab’), binding. Solid symbols, specific binding of ‘”5I-F(ab’)) due to anti-TOP antibody; open symbols, nonspecific binding with P3X63 Ag8 antibody. @, 0, Dorsal retina, sections 4 and 5; a, 4, ventral retina, sections 1 and 8. Reaction mixtures contained 220 nM '*]-F(ab’), (3.68 x 107° uCi/pmol). (C) Effect of concentration of rabbit '7*]-F(ab’), anti-mouse IgG. Solid symbols, specific '**I-F(ab’), binding; open symbols, nonspecific bind- ing to cells from dorsal retina (@, C) or ventral retina (m, 0, a, A). retina cells when antibody to TOP was omitted or was replaced with P3X63 Ag8 antibody. Other hybridoma antibodies includ- ing A2B5 (6) bound equally to cells from different regions of retina (not shown). Assay Conditions. The effects of varying retina protein, hy- bridoma antibody, or }*1-F(ab’), anti-mouse IgG are shown in Fig. 3. Specific binding of !I-F(ab’), was proportional to retina protein in the range 65-300 yg of protein (Fig. 3A). Half-max- imal and maximal specific binding of '!*I-F(ab'), to dorsopos- terior retina cells were obtained with 1:1350 and 1:100 dilutions of ascites antibody to TOP, respectively; higher concentrations of antibody to TOP reduced specific !I-F(ab'), binding (Fig. 3B). Specific !I-F(ab’), binding to ventral retina was low; how- ever, the concentrations of anti-TOP antibody required for half- maximal and maximal specific binding did not differ greatly from those found with dorsal retina. Thus, no obvious difference was detected in the affinity of the antibody for antigen in dorsal and ventral retina. Nonspecific binding of !*I-F(ab’), to retina cells was low at all concentrations of P3X63 Ag8 antibody tested. The antibody to TOP was identified as an IgG] with « light chains (not shown). Half-maximal and maximal specific binding of }°1- F(ab’), were obtained with approximately 0.5 and 2 uM )I- F(ab’),, respectively, both to dorsal and ventral retina cells (Fig. 3C). Nonspecific ‘*I-F(ab’), binding in the presence of P3X63 Ag8 ascites antibody was proportional to ‘I-F(ab’), concen- tration and was not saturating at 3.52 4M, the highest '”I- F(ab’), concentration tested. Geometry of the Gradient. The retina grows by accretion of concentric rings of neurons at the periphery; thus, central retina is the oldest portion of the retina and peripheral retina is the youngest. To determine whether the antigen gradient is a polar gradient that rotates around the center of the retina with uni- form antigen concentration along any arc from center to pe- riphery or is a circumferential gradient extending from dorso- posterior to ventroanterior retina, left retinas of 14-day chicken embryos were cut into eight central and eight peripheral sec- tions (Fig. 4A) which were assayed for TOP. A 35-fold gradient of antigen was found extending from dorsoposterior to ventroan- terior margins of the retina aligned parallel to the long axis of the choroid fissure. As shown in Fig. 4B, strips of retina extending from the dor- soposterior to ventroanterior margins, or perpendicular to this axis from anterior to posterior margins, were removed and each Biochemistry: Trisler e¢ al.            ia Zz Z © QUTER i of 8 & & 1) a é E a 8 z 3 3 8 ! o a = 7 e 7 270 J Q1r27345 678 g 5 10 50 = RETINA SECTION = ” PERCENT OF MAXIMAL DISTANCE Fic. 4. Geometry of the TOP gradient in 14-day chicken embryo retina. Specifically bound !”51-F(ab’), (pmol/mg of protein) is shown on the ordinate in A and B and within the appropriate segment of ret- ina tested in A. (A) Each retina (left eye) was cut into eight 45° sections (7.25 mm in length from the center to periphery ofretina) and each was divided into central (4.9 mm) and outer (2.35 mm) segments. (B) Dem- onstration that TOP concentration detected depends on the square of distance from the ventroanterior margin of the retina. Percentage of maximal circumferential distance is shown on the abscissa; 100% cor- responds to 14.5 mm. 4, Strips of retina from ventroanterior (0%) to dorsoposterior (100%) retina margins, 14.5 x 2.5 mm, were removed from eight retinas (left eyes), and each was cut into nine segments (1.6 x 2.5 mm) as shown; each segment was assayed for TOP antigen. 5, Strips of retina from dorsoanterior (0%) to posterior (100%) margins of the retina perpendicular to the choroid fissure were prepared and assayed as above. ©, Data from A. The length of the arc from the ventral pole of the gradient to the center of each segment was calculated by assuming the retina to be a hemisphere and using equations for spher- ical triangles. was cut into nine pieces which were assayed for antigen. The concentration of TOP molecules detected varied continuously and logarithmically with the logarithm of distance along the circumference of the retina from the ventroanterior pole of the gradient to the dorsoposterior pole, with a slope of 2. In con- trast, little or no change was detected in retina cells along a perpendicular axis from anterior to posterior margins of the ret- ina. The data are described somewhat better by a power func- tion than by a logarithmic function, but a logarithmic function has not been ruled out.   Fic. 5. Autoradiographs of 14-day chicken embryo retina. (A and B) D silver grains over cell soma in the inner nuclear layer appear dim due to s Proc. Natl. Acad. Sci. USA 78 (1981) 2147 The concentration of [!25-F(ab’),‘anti-TOP antibody-TOP antigen] complex detected is described by the relationship: [TOP complex] = distance’. Thus, cell position along a ventroanterior—dorsoposterior axis of retina can be identified by the concentration of TOP detected: in which [TOP complex] is the fraction of maximal pmol of !”I- F(ab’), specifically bound per mg of protein, F,/F yy, and dis- tance is the fraction of maximal circumferential distance from the ventroanterior to the dorsoposterior poles of the gradient, D,/Dmax: Under the conditions used, Finx is 20 pmol of 125] F(ab’), bound specifically per mg of protein and D,,,, is 14.5 mm. Thus, the calculated mean position in retina of cells that bind 5 pmol !”I-F(ab’), specifically per mg protein is 7.25 mm from the ventroanterior pole of the gradient, which agrees well with the experimental values. Autoradiography and Immunofluorescence. Autoradiogra- phy revealed most antigen in dorsoposterior chicken embryo retina in the inner and outer synaptic layers of the retina (Fig. 5 A and B). The antigen was detected in lesser amounts on the soma of most, or all, cell types in dorsoposterior retina. Little antigen was detected in ventroanterior retina (Fig. 5 C and D). Similar results were obtained by immunofluorescence (not shown). All cells mechanically dissociated from 8-day chicken embryo dorsoposterior retina exhibited punctate ring fluores- cence. All cells from middle retina also were fluorescent, but less intensely than dorsoposterior retina cells. At each location, no obvious heterogeneity in cell population was seen. No flu- orescent cells were detected in ventroanterior retina, although low levels of TOP were detected in ventroanterior retina by'”I- F(ab’), binding. Expression of Antigen During Development. The concen- tration of TOP antigen detected was higher in dorsoposterior retina than in ventroanterior retina at every age tested from the 4-day embryo through the adult (Fig. 6A Inset), and the axis and polarity of the gradient did not change during development (Fig. 6A). The concentration of antigen detected in the dorsal half of the retina increased 3-fold between the 4th and 12th days of embryonic development and then decreased slightly the adult. Antigen concentration detected in ventral retina did not vary with development. The amount of protein per retina and   ark-field (A) and phase-contrast (B) views of dorsal retina. In A, some taining of cells by toluidine blue. (C and D) Dark-field (C) and phase- contrast (D) views of ventral retina. R, photoreceptor layer; OS, outer synaptic layer; IN, inner nuclear layer; IS, inner synaptic layer; G, ganglion cell layer; A, ganglion cell axon layer. (x 630.) Outer halves of 14-day chicken embryo dorsal or ventral retina were immersed in liquid Freon and then in liquid nitrogen and were cut in sections 16 wm thick. Each se fluid diluted 1:50 with solution B) for 45 min at 4°C, washed six times B containing 20 yg of fluorescein conjugate of rabbit IgG anti-mouse I each section was incubated with 50 yl of solution B containing 440 mM ction was incubated with antibody to TOP or P3X63 Ag8 antibody (ascites (15 min) with solution B (50 yl per wash), incubated with 50 yl of solution gG (Cappel) for 45 min at 4°C, and washed as above. For autoradiography, 125]. F(ab’), (L x 10° cpm) and 500 yg of bovine serum albumin for 30 min at 4°C, washed as above, and coated with NTB-2 nuclear track emulsion (Kodak). Slides were exposed in the dark (22 days) at 4°C in the presence of a dessicant and stained with 0.02% toluidine blue.                       2148 Biochemistry: Trisler et al. z , 2 | rd T T T T Q A. TOPOGRAPHIC GRADIENT | 2 B. ANTIGEN AND x VS. DEVELOPMENTAL AGE ti PROTEIN/RETINA 2 20b 16 = 100 100 E g 1251.Ftab’ Bo s [-Fiab’}o z fo} 3 2 < © 4 a 10 2 > z 3 « = G PROTEIN i 5 10 2 1.0 WF f S & a Ba a a) 2 z 5 z : g oO 0.1 8 4 2 $ 2 a 0.01 L l \\\\ iy10.01 z 4 8 12 16 20 ADULT DAYS AFTER FERTILIZATION Fic. 6. TOP antigen in chick retina as a function of developmental age. (A) Each retina (left eye) was cut into eight sections as shown in Fig. 2. Symbols and days in ovo are: 0, 8; A, 10;5, 12; , 14; v, 16; @, 18; and m, adult. (Inset) }°1-F(ab’), bound specifically is shown on the ordinate; days in ovo and adult (AD) are shown on the abscissa. ©, dorsal half of retina (sections 3-6); a, ventral half of retina (sections 1, 2, 7, and 8). Data for retina from 4- and 6-day embryos are shown only in Inset. (B) 0, '*I-F(ab’), bound specifically per retina; 4, protein per retina; @, ordinate represents pmol **I-F(ab’), bound specifically per mg of protein. TOP antigen detected per retina increased 470- and 620-fold, respectively, between the 4-day embryo and the adult; the amount of antigen detected per mg of protein remained rela- tively constant (Fig. 6B). These results show that a gradient of TOP molecules is formed early in retina development, during the period of active neuroblast proliferation and neuron genesis, and that the gradient is maintained after neuron genesis ceases. Tissue Specificity. Highest concentrations of TOP antigen detected were in regions of the nervous system derived from prosencephalon (forebrain): retina > cerebrum > thalamus (Table 1). Low levels of antigen were found in dorsal and ventral retina pigment epithelium, optic nerve, optic tectum, and cer- ebellum; little or no antigen was detected in heart, liver, kid- ney, or cells from blood. Chicken Embryo with an Ectopic Eye. During the course of these studies, a chicken embryo with three eyes was found (Fig. 7). The third eye was situated in the middle of the fore- head, facing in a dorsoanterior direction. Retinas from right, middle, and left eyes contained gradients of TOP molecules with normal polarity and alignment with the choroid fissure. Table 1. Distribution of TOP antigen in chicken tissues '25].F(ab’), specifically bound, pmol/mg protein   14-day Tissue embryo Adult Dorsal neural retina 12.0 7.70 Ventral neural retina 1.08 2.70 Cerebrum 3.30 3.12 Thalamus 2.13 _ Optic nerve _— 0.58 Optic tectum 0.15 _ Cerebellum 0.23 0.46 Dorsal retina pigment epithelium 0.26 _ Ventral retina pigment epithelium 0.25 _ Heart, liver, kidney, or blood cells 0.002-0.055   Proc. Natl. Acad. Sci. USA 78 (1981)   3 ToT A.      BEAKS   #MOL 1251-F(ab'}2 BOUND/RETINA SECTION   Poppy 12345 67 8 RETINA SECTION Fic. 7. (A) TOP antigen gradients in retinas from the right (0), middle (2), and left (a) eyes of a 14-day chicken embryo with three eyes, Total ‘*°1-F(ab'), bound per retina section is shown. Reaction mixtures contained 2.38 nM '°1-F(ab’)s. (B) Frontal view of head of embryo. The third eye is situated on the forehead facing in a dorsoanterior direction. The embryo had two pairs of beaks, two brains in one head, and one body. Thus, a gradient of TOP antigen was generated with normal orientation in the supernumerary eye despite the abnormal orientation of the eye in the embryo. Species Specificity. Gradients of TOP molecules with similar opientation and symmetry were detected in turkey, quail, and duck embryo retina, 17, 15, and 16 days after fertilization, re- spectively (Fig. 8). '°I-F(ab’), concentrations in reaction mix- tures were low (0.074-0.26 nM); thus, bound !*]-F(ab’), also was low. With 440 nM ’”°1-F(ab’),, 7.5 pmol of !*I-F(ab’), bound specifically per mg of dorsoposterior quail retina protein, comparable to chicken retina. The antigen was not detected in retina of goldfish, Xenopus laevis, Rana pipiens, or Fisher rats (not shown). Properties of TOP Antigen. A dorsal—ventral gradient of GM, ganglioside molecules (II’NeuAc-G,Ose,Cer) in chicken embryo retina has been postulated but not detected (15). Bovine brain gangliosides (10-10,000 4M) did not inhibit the binding of anti-TOP antibody to retina (not shown). Rabbit antisera to mono-, di-, and trisialogangliosides (gift of C. Alving) bound to retina; however, a ganglioside gradient was not detected.       12 T T T T T T T T TURKEY 3 @ Q2 rar Be Se a ge get gs - > 7 Oo 4 oo x = a oO 0 Ly 12345 67 8     RETINA SECTION Fic. 8. TOP gradients in retina from Japanese quail (0), Coturnix coturnix japonica (15-day embryo); White Pekin duck (a), Anas pla- tyrhynchos (16-day embryo); and turkey (9), Meleagris gallopavo (17- day embryo). The eggs hatch 17, 28, and 28 days after fertilization, respectively. }”°I-F(ab’), concentrations and ,.Ci/pmol were as follows: quail, 0.26 nM, 0.90 wCi/pmol; duck, 0.074 nM, 2.92 uCi/pmol; and turkey, 0.078 nM, 2.38 uCi/pmol. Biochemistry: Trisler et al. Table 2. Effect of trypsin or heat on TOP antigenicity       1251 -F(ab‘)o bound specifically to retina Treatment of retina cells cells Exp. 0-30 min 30—40 min cpm % 1 Control + Trypsin inhibitor 1700 100 Trypsin + Trypsin inhibitor 93 «6 Trypsin + trypsin inhibitor — 1803 106 2 4°C, 30 min 1607 100 100°C, 30 min 1328   Inactivation of TOP retina molecules in 14-day chicken embryo dor- gal retina by trypsin or heat. In Exp. 1, retina cells were incubated for 30 min at 37°C, in phosphate-buffered saline alone or with 11 »M tryp- sin (crystallized three times, Worthington) or with 11 »M trypsin in- activated with 12 uM soybean trypsin inhibitor (Worthington), and then for 10 min in soybean trypsin inhibitor. TOP ascites fluid was diluted 1:1000; 1.86 nM !”5]-F(ab’), (90.5 nCi/pmol) was used. In Exp. 2, TOP and P3X63 Ag8 antibodies were diluted 1:100; 1.82 nM 1257. F(ab’), (96.9 nCi/pmol) was used. TOP antigenicity was upon incubation at 100°C or by incu- bation with trypsin (Table 2). All TOP antigenicity in retina cell homogenates was recovered from the 100,000 x g particulate fraction; soluble antigen was not detected (not shown). DISCUSSION Fusion of spleen cells from a mouse immunized with dorso- posterior chicken embryo retina with P3X63 Ag8 mouse my- eloma cells yielded a line of hybridoma cells that synthesizes antibody to molecules that are distributed in a topographic gra- dient in the retina. At least 35-fold more antigen was detected in dorsoposterior than in ventroanterior retina. The antigen was found by autoradiography in highest concentration in 14-day chicken embryo retina on neurites in the inner and outer syn- aptic layers. The antigen was found by immunofluorescence on most, or all, cells from dorsoposterior and middle portions of 8- and 14-day chicken embryo retina. These results suggest that antigen molecules are distributed in the retina on the basis of cell position, rather than cell type. Neurons in dorsal and ventral retina differ in several ways. For example, Xenopus retina is composed ofat least three clonal domains (16). One cell on each side of the 16-cell embryo gives rise to dorsal retina, another cell gives rise to middle retina, and a third cell gives rise to cells that migrate across the midline of the embryo and form ventral retina on the opposite side. In addition, dorsal retina ganglion neurons synapse in ventral tec- tum, whereas ventral retina ganglion neurons synapse in dorsal tectum (1, 17, 18), establishing a continuous, point-to-point re- tino-tectal map. Neurons are generated in chicken embryo retina between the Qnd and 12th days after fertilization; central retina is the oldest portion of the retina, and peripheral retina is the youngest. The concentration of TOP molecules detected in dorsoposterior ret- ina increased as the diameter of the retina increased, and it varied with cell position at every stage tested, from the 4-day embryo through the adult, suggesting that the gradient of TOP is established as neurons and glia (and possibly their precursors) are generated in retina. Proliferation of clonal populations of cells with different genotypes in mosaic mice results in radial patterns of cells in neural retina and retina pigment epithelium (19); whether the gradient of TOP molecules is due to clonal inheritance remains to be determined. Proc. Natl. Acad. Sci. USA 78 (1981) 2149 Dorsal and ventral retina cells also differ in adhesive speci- ficity—i.e., cells from dorsal retina adhere preferentially to cells from ventral retina or tectum and vice versa (20-22). However, the properties of TOP molecules differ from those reported for preferential adhesion of dorsal retina cells and for retina adhe- sion factors such as cognin (4), CAM (5), and ligand and agglu- tinin (23). The dorsoposterior portion of chicken or pigeon retina con- tains a fovea, 3- to 5-fold more amacrine synapses than ventral retina (24), and a relatively high concentration of red droplets in photoreceptor cells and functions as a binocular visual field for pecking (25). Thus, dorsoposterior retina differs from other portions of retina in embryologic development, migration of cells and axons across the midline of the embryo, synapse spec- ificity, adhesive specificity, and function. The results suggest that a gradient of TOP molecules is formed by a gradient of cells which have different numbers of antigenic TOP molecules depending on cell position in the ret- ina. No evidence was found for topographically distributed dif- ferences in antigen affinity for antibody or in the proportion of cells expressing antigen. Variation in antigen accessibility has not been excluded. The mechanism of generating and main- taining the gradient of TOP, highly ordered with respect to the axis of the retina, remains to be determined. The function of TOP molecules has not been determined. However, since TOP antigen concentration detected is contin- uously graded and distributed on the basis of cell position not cell type, our working hypothesis is that TOP molecules play a role in the coding of positional information in the retina. Sperry, R. W. (1963) Proc. Natl. Acad. Sci. USA 50, 703-710. Silver, J. & Sidman, R. L. (1980) J. Comp. Neurol. 189, 101-111. Goldschneider, I. & Moscona, A. A. (1972) J. Cell Biol. 53, 435-449. 4. Hausman, R. E. & Moscona, A. A. (1979) Exp. Cell Res. 119, 191-204. 5. Thiery, J.-P., Brackenbury, R., Rutishauser, U. & Edelman, G. M. (1977) J. Biol. Chem. 252, 6841-6845. 6. Eisenbarth, G. S., Walsh, F. S. & Nirenberg, M. (1979) Proc. Natl. Acad. Sci. USA 76, 4913-4917. 7. Barnstable, C. J. (1980) Nature (London) 286, 231-235. 8. Trisler, G. D., Donlon, M. A., Shain, W. G. & Coon, H. G. (1979) Fed. Proc. Fed. Am. Soc. Exp. Biol. 38, 2368-2373. 9. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495-497. 10. Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W. & How- ard, J. C. (1977) Nature (London) 266, 550-352. ll. Berzofsky, J. A., Hicks, G., Fedorko, J. & Minna, J. (1980) J. Biol. Chem. 255, 11188-11191. 12. Schneider, M. D., & Eisenbarth, G. S. (1979) J. Immunol. Meth-\\", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yewk_c2a2~qqr8", "00000000-0000-0000-CD99-6C1703CF7E25", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Drosophila NK-Homeobox Genes", "101584910X69", null, "1989", "October 1989", "This article presents experimental findings of four new Drosophila homeobox genes, which encode DNA binding proteins and regulate gene expression during development or in the adult.  These newly discovered genes \"were detected with oligonucleotide probes corresponding to an amino acid sequence that is thought to be part of the nucleotide sequence recognition site of homeobox proteins.\"  These probes were also designed to provide a means of detecting additional sets of homeobox genes.", "Articles", "Drosophila,Genes, Homeobox ; DNA-Binding Proteins", "From Neuroblastoma to Homeobox Genes, 1976-1992", "5", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Proc. Natl. Acad. Sci. USA Vol. 86, pp. 7716-7720, October 1989 Biochemistry Drosophila NK-homeobox genes/ (NK-1, NK-2, NK-3, and NK-4 DNA clones/chromosome locations of genes) YONGSOK KIM AND MARSHALL NIRENBERG Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Building 36, Room 1C-06, Bethesda, MD 20892 Contributed by Marshall Nirenberg, July 6, 1989 ABSTRACT / Four Drosophila melanogaster homeobox genes were found by screening a genomic DNA library with oligodeoxynucleotides that correspond to a conserved amino acid sequence that is part of the putative site of homeobox proteins that recognizes nucleotide sequences in DNA. The amino acid sequences of NK-2, NK-3, and NK-4 homeoboxes are more closely related to one another (59-66% homology) than they are to other Drosophila homeoboxes (28-54% ho- mology), whereas the homeobox of NK-1 is most closely related, in order of decreasing homology, to muscle segment homeobox, zerknillt-1, NK-3, and distal-less homeoboxes. Three of the genes, NK-1, NK-3, and NK-4, comprise a cluster of homeobox genes located in the 93E1-5 region of the right arm of the third chromosome, whereas the fourth homeobox gene, NK-2, is located in the 1C1-5 region of the X chromosome. |   Homeobox genes encode DNA binding proteins that regulate gene expression during development or in the adult (1-4). In most cases, the similarity between different kinds of ho- meobox proteins extends only over a segment of the protein that consists of 60-61 amino acid residues, the homeo- domain, which is thought to be the portion of the protein that recognizes nucleotide sequences in DNA. Homeobox genes are particularly well expressed in nervous system, and the homeobox family of genes encodes the largest set of proteins that regulate gene expression in the nervous system that has been identified thus far (5-9). In this report, we describe four newly discovered, related Drosophila homeobox genes that were detected with oligo- nucleotide probes corresponding to an amino acid sequence that is thought to be part of the nucleotide sequence recog- nition site of homeobox proteins. METHODS AND MATERIALS Oligodeoxynucleotides. An Applied Biosystems DNA syn- thesizer 380B was used to synthesize oligodeoxynucleotides. Oligonucleotides with the trityl groups attached were purified by OPC column chromatography and trityl groups then were removed as described by Applied Biosystems. [y?*P]ATP with a specific activity of 6000 Ci/mmol (1 Ci = 37 GBq) (New England Nuclear) was used for phosphorylation of oligode- oxynucleotides catalyzed by T4 polynucleotide kinase (10). Detection and Cloning of Homeobox Genes. A Drosophila melanogaster genomic DNA library in Charon 4A (11) was obtained from the American Type Culture Collection. Recom- binant phage [48,000 plaque-forming units (pfu)] and 2 x 10° Escherichia coli KH802 cells were plated in Petri dishes (150 mm) at a concentration of 12,000 pfu per dish. Four nitrocel- lulose replica filter plaque lifts were obtained from each Petri dish, and each filter was hybridized with a different [32P]- oligodeoxynucleotide preparation [16-64 oligodeoxynucle- otide species per preparation; 1.5 x 10° cpm/ml; 120-150   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7716 fmol/ml (the sum of all species of oligodeoxynucleotides)] at 37°C overnight and washed with a solution containing tetra- methylammonium chloride at 53°C or 50°C for 30 min for 17- mers or 16-mers, respectively, as described by Wood et al. (12). DNA Sequencing.* Cloned genomic DNA fragments cleaved by restriction enzymes were subcloned into Bluescript pKS+. Both strands of the homeobox regions of the following DNA fragments were sequenced by the dideoxynucleotide chain- termination method (13) using M13 universal primers or spe- cific oligodeoxynucleotide primers and Sequenase 2 (United States Biochemical): NK-1, 1.4-kilobase (kb) EcoRI/Pst 1 DNA fragment; NK-2, 1.2-kb EcoRI/Pst I DNA fragment: NK-3, 0.7-kb Pst I DNA fragment; NK-4, 0.4-kb and 2.3-kb upstream HindIII DNA fragments. dITP was used to reduce compression of DNA bands. Locations of Genes on Chromosomes. Salivary gland poly- tene chromosomes were hybridized with EcoRI-cleaved ge- nomic DNA fragments that contained the appropriate ho- meobox region and had incorporated biotin-16 dUMP in place of some dTMP residues as described (14). Detek 1-HRP kits (Enzo Biochemicals) and the protocol supplied by the man- ufacturer were used. RESULTS AND DISCUSSION Detection of Homeobox Genes. The Drosophila genomic DNA library of Maniatis ez al. (11) in Charon 4A was screened for recombinants corresponding to homeobox genes with five [>?P]oligodeoxynucleotide probe preparations designed to hybridize to highly conserved homeobox nucle- otide sequences. The oligonucleotide preparations were 16 or 17 nucleotides long and each consisted of multiple species of oligodeoxynucleotides (described in the legend to Fig. 2). Replica filters were prepared and each filter was hybridized to a different [?*PJoligodeoxynucleotide preparation. The filters were washed under high-stringency conditions with a solution that contained tetramethylammonium chloride. which selectively binds to A:T base pairs and raises the melting temperature (,,) of A:T base pairs to that of G-C base pairs (12). The t, of each (°?P]oligodeoxynucleotide-DNA duplex then was dependent on the number of contiguous base pairs formed but was not affected by the proportion of G-C vs. A‘T base pairs (12). Consequently, all species of 17-mer oligodeoxynucleotides hybridized to DNA were washed at the same temperature (53°C) for the stringent wash, and «| 16-mers were washed at 50°C. Of the 48,000 phage plaques that were screened, ~200 clones were obtained that exhibited a positive autoradio- graphic signal with one of the five [22P]oligonucleotide probe preparations, and 7 recombinant clones were obtained that gave positive signals with two or more probe preparations. Many of the 200 clones that were detected with only one   *The sequences reported in this paper for NK-1 to NK-4 have Se deposited in the GenBank data base (accession nos. M2728. M27290, M27291, M27292, respectively). Biochemistry: Kim and Nirenberg probe were cloned, but they were not studied further. The 7 clones that were detected with two or more 32P_labeled probes were characterized by restriction site analysis and the nucleotide sequences of the homeobox regions of some of the clones were determined by using unlabeled probes as se- quencing primers. Five of the 7 clones were found to be previously unknown homeobox genes. The 2 remaining ‘Jones correspond to known homeobox genes; 1 clone con- cains zerknwllt-1 (zen-1) and zen-2 DNA (15), and the other clone corresponds to either en or inv (16) (data not shown). Characterization of Homeobox Genes. In Fig. 1 are shown partial restriction site maps of the homeobox genes NK-1, NK-2, NK-3, and NK-4, the locations of homeobox regions within the DNA inserts, and the direction of transcription. The approximate chain lengths of the cloned NK-1 and NK-2 genomic DNA fragments are 15.0 and 14.1 kb, respectively. Three of the 7 clones detected with two or more probes correspond to NK-3. Clone 6 is a 14.7-kb DNA fragment that contains the NK-3 homeobox sequence. Clones 3 and 9 contain similar or identical DNA inserts (14.6 kb) that overlap clone 6 and contain both NK-3 and NK-4 homeobox se- quences separated by ~7.8 kb. The restriction site map of NK-3 and NK-4 shown is derived from data obtained from clones 6, 3, and 9. Subcloned EcoRI DNA fragments from clone 3 were used to determine NK-3 and NK-4 nucleotide sequences. Partial Nucleotide Sequence of NK-1. The sequence of an 811-nucleotide portion of the NK-1 gene is shown in Fig. 2. The first 198 nucleotides correspond to the 3’ portion of an i:tron. Another intron, 217 nucleotides long, was found within the homeobox, between codons for homeobox amino acid residues 44 (glutamine) and 45 (valine). The intron—exon structure of the NK-1 gene was confirmed by sequencing NK-1 cDNA clones (to be described elsewhere). Three other Drosophila homeobox genes, labial (fab) (19, 20), abdominal- B (Abd-B) (21), and distal-less (DID) (22) (Brista (23)] have introns at precisely the same location within the homeobox as NK-1. The intron within the NK-1 homeobox contains a nucleotide sequence for antennapedia (Antp) protein binding (18) and one or two binding sites for zeste protein [the onsensus nucleotide sequence for zeste is TGAGYG (Y, pyrimidine) (17)]. The amino acid sequence of the initial portion of the first NK-1 exon shown in Fig. 2 is highly acidic—i.e., 12 of the first 26 amino acid residues shown are aspartyl or glutamyl residues. Twenty-five percent of the amino acid residues before the homeobox are glycine residues, which include 7 consecutive glycine residues, and 17% are serine or threonine residues.             RoR R RH R H mg (8) L I \\\\ 4. a a NK-1 it INE , ; rae (CLONE 2) R] HR RH R) Sop a | NK-2 (CLONE 5) PoP 88 P BB P P R _ \\\\ : = 8 NK-3 (CLONES) NK &NK4S igi Ne-3 NK-4 (R) (CLONE 3} NIK3&NK4 ip) NK3 NK-4 (R} (CLONE 9) Cit 8 aR aay f PEt bbe 2kb NK-3 &NK4 COMPOSITE Fic. 1. Partial restriction maps of NK-1, NK-2, NK-3, and NK-4 cloned genomic DNA fragments. Solid boxes represent homeoboxes and are not drawn to scale. Arrows indicate direction of transcrip- tion. B, BamHL- H, Hindlll: P, Pst 1, R, EcoR1: (R), EcoRI cloning site created by ligation of an EcoRI linker to genomic DNA. Proc. Natl. Acad. Sci. USA 86 (1989) 7717 Characterization of NK-2. The nucleotide sequence of the homeobox region of the NK-2 gene is shown in Fig. 3. The deduced amino acid sequence before the homeobox contains repetitive asparagine residues and a highly acidic region consisting of 14 aspartyl or glutamyl residues in a 31-amino acid segment (45% acidic amino acid residues). Twenty-five percent of the amino acid residues before the homeobox are glycine plus alanine. The carboxyl-terminal 30 amino acid residues of NK-2 are rich in histidine (20%), proline (17%), and glycine (17%). A 168-nucleotide 3’-untranslated region also is shown. Characterization of NK-3 and NK-4. The nucleotide se- quence and deduced amino acid sequence of part of the NK-3 homeobox gene are shown in Fig. 4. The initial part of the sequence consists of part of an exon that encodes 54 amino acids (17% alanine, 19% serine and threonine, and 9% asparagine), which is followed by a short, 119-nucleotide intron within the 26th codon before the homeobox. The intron—exon structure of the NK-3 gene was confirmed by sequencing NK-3 cDNA clones (K. Webber, Y.K., and M.N., unpublished data). The initial portion of the second oR 'ATGGCACCAACATGTGCCGAAAAATTCCAATTAATCGAACAATGATGCGGTGG -293 Cc CCGTGGTGATTGATTTCCGTTTTCCAATCCCCCAGGACATTGCCATITGTCTGTIGATGG -234 ATGGCCCTAGCCTGTTGACTTATGCAAAAAGAGAGACACCCGGAACTTATCGTGCCCAA -175 arcrcererrerrrrrrtrererrecact cc CAG GAT TTG AAT GAC ATG GAT -124 Gln Spi Leu Asn fisp Met [Asp] -42 CAG GAC GAT ATG TGT GAC GAT GGC AGC GAT ATC GAC GAT CCC AGC -79 Gln Met Cys Asp Aspj Gly Ser KKspl Ile Asp Asp] Pro Ser ~27 AGC GAG ACG GAC TCC AAA AAG GGA GGC AGT CGT AAT GGG GAT GGA ~-34 Ser Thr isp] Ser Lys Lys Gly Gly Ser Arg Asn Gly Asp Gly ~12 ~L]+1 RAG TCC GGA GGT GGC GGC GGA GGT GGT TCA AAG|CCT CGA CGA GCC 12 Lys Ser y Gly Gly Gly Gly Gly Gly] Ser Lys|Pro Arg Arg Ala 4     CGC ACC GCC TTC ACG TAC GAA CAA CTA GTT TCC\"CTG “GaG RAC’ AAG| 57 Arg Thr Ala,Phe Thr Tyr Glu Gln Leu Val Ser Leu Glu Asn Lys 19 hrc AAG ACC ACC AGA TAT CTC AGC GTC TGC GAG CGA CTG AAC TTG| 102 Phe Lys Thr Thr Arg Tyr Leu Ser Val Cys Glu Arg Leu Asn Leu 34 lccc CTC AGC TTG AGC CTG ACA GAG ACG caGYeTGAGCAATGATATATACT 151 Ala Leu Ser Leu Ser Leu Thr Glu Thr Gin 44 ICTATTGTTAAAGATTAAAATCCAGAGAAGTTATGTATATTTTGCAAAAAGTTGGTATAA| 210 CTATTCTCTATGCTTTTCAATTTTAATAGAAGTAATIGAGTIAABATATATTTTACTIO] 263 GAGTGACTAAATTGAAAAGBAGT IEATIACTGITTTTGAAATATTTAAATACCAATGTC| 328 ATTICTCATCATCCTITTAGRG V GIT AKA ATT TGG TIC _CAG AAC CGC CGC] 377 Val Lys Ile Trp Phe Gin Asn Arg Arg 53   CC AAG TGG AAG AAG CAG AAC/CCC GGC ATG GAT GTC AAC TCC CCC 421 ithe Lys Trp Lys Lys Gln Asn|Pro Gly Met Asp Val Asn Ser Pro 68   ACC ATC CCC CCG CCC GGC GGC GGC TCC TTC GGA CCG GG 460 Thr Ile Pro Pro Pro Gly Gly Gly Ser Phe Gly Pro Gly 81 Fic.2. Nucleotide sequence and deduced amino acid sequence of the homeobox region of the NK-1 gene. Deoxynucleotide and amino acid residues are numbered on the right; 1 corresponds to the first deoxynucleotide or amino acid residue in the homeobox, which is enclosed in a large box. The acidic amino acid region is indicated by boxed Asp or Glu residues. Repetitive Gly residues before the homeobox also are enclosed in a box. The lst and 2nd boxed nucleotide sequences in intron 2 are possible sites for binding of zeste protein to DNA (17). The 3rd site, ANNNNCATTA, is an Antp protein binding site (18). Arrowheads represent intron—exon junc- tions. Nucleotide 12 in an NK-1 genomic DNA clone was C, whereas the corresponding nucleotide residue found in an NK-1 cDNA clone was T. All oligodeoxynucleotide probes are complementary to the DNA strand shown: probe sequences, starting from the 5’-terminal nucleotide residues are as follows: —, probe 121, 24 species of 17-mers, (-)TYYTGRAACCA(T/A/G)TARAA; --, probe 125, 48 species of 17-mers, (-.AACCA(T/G/AJATYTTNACYT1G, ---, probe 126, 64 species of 17-mers, (-}AAYTCYTTYTCNAGYTC; - -, probe 127, 64 species of 17-mers, (-)-C(T/G)RTTYTCRTTRAAYTC; -:-, probe 130, 16 species of 16-mers, (-)A(C/G)T(C/G\\\\XC/G)T(T/ G)CTCCAGCTC. Y, pyrimidine; R, purine. 7718 Biochemistry: Kim and Nirenberg exon before the homeobox consists of 35% serine and 19% proline residues. The 54 amino acid residues after the ho- meobox are rich in alanyl and glycyl residues (22%) as well as leucyl residues (11%). In Fig. 5 is shown the nucleotide sequence of part of the NK-4 gene. The initial part of the sequence consists of part of an intron, which is followed by an exon that contains the homeobox domain. The carboxyl-terminal region of the de- duced NK-4 protein contains repetitive glutamine residues (M or opa repeats and a CAX repeat in the corresponding DNA). Locations of Genes on Chromosomes. The cytological lo- cations of the NK-1, NK-2, NK-3, and NK-4 genes in Drosophila third-instar larvae salivary gland polytene chro- mosomes are shown in Fig. 6. Unexpectedly, NK-1, NK-3, and NK-4 genes were found to reside in neighboring chro- mosomal bands in the right arm of chromosome 3. The NK-3 and NK-4 genes reside at 93E1-3, and the NK-1 gene resides at 93E3—5. When two probes, one for NK-1 and one for NK-3, were added to the same in situ hybridization reaction mixture, two labeled chromosomal bands were obtained at 93E1-—5 that were separated only slightly. However, the NK-2 gene resides in the 1C1—5 region of the X chromosome. In Fig. 6B, the relative positions of the NK-3/NK-4 and NK-1 genes are shown correlated with the chromosomal bands in the 93E region of chromosome 3 in Bridges’ revised map of chromosomal bands (24). These results show that NK-1, NK-3, and NK-4 comprise a cluster of homeobox genes. Either NK-1, NK-3, or NK-4 genes may be the same as torso-like, a maternal effect gene that resides at 93E and is one of the ensemble of genes that determine the anterior— posterior pattern of the embryo (2). The torso-like gene and four other genes are required for the formation of both the anterior and posterior terminal, unsegmented portions of the embryo (the acron and telson) (2). Another candidate is paired gene 9, which is thought to contain repetitive alternating codons for histidine and proline termed a paired repeat [also found in paired (25) and bicoid ACG GCC CAT GCC CTA CAC AAC AAC AAT AAT AAT ACG ACA AAC AAC -160 Thr Ala His Ala Leu His [Asn Asn Asn Asn Asp Thr Thr Asn Asn -54   AAT AAC CAC AGC CTG AAG GCC GAG GGG ATC AAC GGA GCA GGC AGT -115 n His Ser Leu Lys Ala Glu Gly Ile Asn Gly Ala Gly Ser -39 GGT CAC GAC GAT AGC CTC AAC GAA GAT GGC ATC GAG GAG GAT ATC -70 Gly His Asp Asp Ser Leu Asn [Glu Aso Gly Ile Glu G u_Asp| Ile -24 GAC GAC GTG GAC GAC GCC GAC GGC AGT GGC GGC GGG GAT GCA AAT -25 AS sp Val ASp Asp Ala (Aso Gly Ser Gly Gly Gly [Asp] Ala Asn -3     BamH aft GGA TCC GAC GGT CTG CCA AAT AAG] AAA CGG AAG CGA CGA GTC CTG 21 Gly Ser Gly Leu Pro Asn Lys|Lys Arg Lys Arg Arg Val Leu 7   TTC ACC AAG GCG CAA ACA TAT GAG CTG GAA CGT CGG TTT CGA CAA 66, Phe Thr Lys Ala Gln Thr Tyr Glu Leu Glu Arg Arg Phe Arg Gln 22 CAA CGT TAC TTG AGT GCC CCG GAA CGC GAG CAC CTG GCC AGT TTG} 111 Gin Arg Tyr Leu Ser Ala Pre Glu Arg Glu His Leu Ala Ser Leu 37 ATC CGC CTG ACG CCG ACC CAG GTG AAG ATC TGG TTT CAA AAC CAT] 156 Ile Arg Leu Thr Pro Thr Gln Val Lys Ile Trp Phe Gln Asn His 52                 AAC GAG AAG GGC 201 Asn Glu Lys Gly Tyr Glu Gly 67   CAT CCT GGT CTA CTG CAC GGC CAT GCC ACC CAT CCG CAT CAC CCC 246 His Pro! Gly Ley Leu Gis cly His Ala Thr His Pro His fils Prol 82 AGT GCC CTG CCA TCG CCC GTC GGG TAG CCGTTCCAGTTCTGGTGAGGAAC 291 Ser Ala Leu rd Ser Pra val Gly *** 90 GGAAAGCCCTGCTTGGGCGATAGTTCCAAACTGGGAGCCGACTGCGTCTCCGTGTCATC = 341 AGCCACCGCCACCGCCATGCAGAATGCCSCCGCCCATCACTTGGTTGCCCTAAATGGAG = 400 Pst | CGGCCGCCTATCAACATGCCGCTGCAG 440 Fic. 3. Nucleotide sequence and deduced amino acid sequence of the homeobox region of NK-2 genomic DNA. The homeobox domain is enclosed in a large box. Repetitive Asn residues are enclosed in a box. The acidic amino acids enclosed in boxes before the homeodomain comprise an acidic region of NK-2 protein. Proc. Natl. Acad. Sci. USA 86 (1989) (25) genes] and a homeobox, which resides at 93E1—2 and was cloned by Frigerio et al. (25). Elsewhere, we will show that NK-1 contains a paired repeat (unpublished data); however, it is not known whether NK-1 is the same as paired gene 9. It should be noted that binding sites for polycomb protein have been detected at 93E1-4 (27). One of several candidates for the NK-2 gene is twisted, discovered by Demerec er ai. (28), which is located between 1C-5 and 2C-10. The abdo- mens of adult tw mutants, viewed from behind, are rotated =30°C clockwise. However, further work is needed for the identification of NK-1, NK-2, NK-3, and NK-4 genes. Homeobox Homology. The deduced amino acid sequences of the homeobox domains of NK-1, NK-2, NK-3, and NK-4 are shown in Fig. 7 and are compared with the 23 Drosophila homeobox sequences that have been reported thus far. NK-?, NK-3, and NK-4 homeoboxes are more closely related to one another (59-66% homology) than they are to other Droso- phila homeoboxes. The maximum homology to a previously reported homeobox is to muscle segment homeobox (msh) (29) (54% homology). In order of decreasing homology, the homeobox of NK-1 is most closely related to msh, zen-/, NK-3 and Dil homeoboxes. NK-1, lab, Dil, and Abd-B genes may have originated from a common precursor because each gene contains an intron between the codons for the 44th and 45th homeobox amino acid residues. It has been suggested that homeobox proteins bind to DNA via a helix-turn-helix motif in the homeodomain and that amino acid residues 42, 43, and 47 in the third a-helix of the homeodomain interact with nucleotide residues in the major groove of DNA and determine, at least in part, the nucleotide sequence recog- nized (29-31). Since NK-1, lab, Abd-B, and Dil genes each contain an intron between the codons for the 44th and 45th homeobox amino acid residues, the part of each gene that ts thought to encode the DNA recognition site of the corre- Pst | CTG CAG TAT TAT GCG GCG GCG ATG GAC AAC AAT AAC CAC CAT CAC -31t Leu Gln Tyr Tyr Ala Ala Ala Met Asp Asn Asm Asn His His His -6€¢ CAG GCA ACG GGC ACA TCG AAC TCC AGT GCC GCC GAC TAC ATG CAG -272 Gln Ala Thr Gly Thr Ser Asn Ser Ser Ala Ala Asp Tyr Met Gln -S2 CGC AAA TTG GCC TAT TTT GGA TCS ACC CTC GCT GCT CCT TTG GAC -226 Arg Lys Leu Ala Tyr Phe Gly Ser Thr Leu Ala Ala Pro ueu Asp -5Si ATG AGA CGC TGC ACC AGC AAC GAT TCC OV orAAGTAACTCCACGARATTA “17? Met Arq Arg Cys Thr Ser Asm Aso Ser A -26 ACGCCATTCAGGCTCTAATGGACTCTGAAAAGAACGCTACTTATTCATTSGCCTTTTGT -1l8 ATAGGATGTATGOTANCTTTIGGTAATTTTCSCTTZACAGVAC TGC GAC TCA CCA obs sp Cys Asp Ser Pro -.- CCG CCA TTG AGC AGT TCC CCC TCG GAG TCG CCG CTA TCC CAC GAC aa Pro Pro Leu Ser Ser Ser Pro Ser Giu Ser Pro Leu Ser His As - ~1fe+l SGC AGT GGA TTG AGC CGC]AAG AAG CGG TCG CGT GCC GCC TTC AGC 2 ; Ser Gly Leu Ser Arg Lys Arg Ser Arg Ala Ala Phe Ser;   CAC GCC CAS GIC TTC GAG TTS GAG CGC CGC TTT GCC CAA CAG CGC] is His Ala Gin Val Phe Glu Leu Glu Arg Arg Phe Ala Gln Gin Arg ee IrAC TTG TCC GCT CCG GAA CGC ASC GAG ATG GCC AAG AGC CTG CGC 1 Tyr Leu Ser Gly Pro Glu Arg Ser Glu Met Ala Lys Ser Leu Arg leTG ACG GAG ACC CAG GTG AAG ATC TGG TTC CAA AAC CGC CGC TAC le. leu Thr Glu Thr Gln Val Lys Ile Trp Phe Gln Asn Arg Arg Tyr AAG ACC AAG CGC AAG cacl ATC CAS CAG CAC GAG GCC GCC CTT TTG Lys Thr Lys Arg Lys Gini Ile Gln Gln His Glu Ala Ala Leu Lev   GGT GCC AGC AAG AGG GTT CCC GTC CAA GTC TTG GTG CGA GAG GAT Gly Ala Ser Lys Arg Val Pro Val Gln Val Leu Val Arg Glu Asp GSC AGC ACC ACC TAC GCT CAC ATG GCT GCT CCC GGT GCT GGA CAC Gly Ser Thr Thr Tyr Ala His Met Ala Ala Pro Gly Ala Gly His Pst | ay: GGC CTC GAT CCC GCC CTG ATC AAC ATC TAC CGC CAT CAG CTG CAG os Gly Leu Asp Pro Ala Leu Ile Asn lle Tyr Arg His Gin Leu Gln Fic. 4. Nucleotide sequence and deduced amino acid sequence of the homeobox region of the NK-3 gene. The homeobox is enclosed in a box. Arrowheads represent exon-intron junctions. Biochemistry: Kim and Nirenberg sponding protein is interrupted by an intron. Further work is needed to determine whether the specificity of DNA recog- nition by NK-1 protein is altered by alternative splicing. Amino acid replacements that alter the 42nd or 43rd amino acid residues in the homeobox are of special interest since they may determine part of the nucleotide sequence that is recog- nized by the homeobox protein. The 42nd homeobox amino acid residues of NK-2 and NK-4 are proline and alanine, respectively. The unspliced form of Saccharomyces cerevi- uae mating-type factor a-1 has proline at this site (29); how- ever, neither proline nor alanine has been found at this site in any metazoan homeobox protein. A proline residue would not be expected to be part of an a-helix, unlike alanine or glutamic acid residues, which promote a-helix formation. The 43rd homeobox amino acid residue of NK-1, NK-2, NK-3, and NK-4 is threonine; however, the only other homeobox pro- teins that contain threonine at this site are msh, Dil, lab, and ro in Drosophila and Hox 1.6 and Hox 7.1 in the mouse. The amino acid sequences of most or all of these homeobox domains share other unusual features {for example, see alanine iilth amino acid residue), lysine or arginine (19th residue), glutamic acid (30th residue), tyrosine (54th residue), and serine or threonine (56th residue)]. The presence of the same or similar unusual amino acid replacements in most or all of these homeodomains provides additional evidence that the newly discovered homeobox genes are related to one another. The combined use of probes 121 and 125, which corre- spond to the overlapping hexapeptides shown at the top of Fig. 7, should detect only those homeobox genes that encode the amino acid sequence Gln-Val-Lys-Ile-Trp-Phe-Gin-Asn 4s residues 44—51 of the third a-helix of the homeodomain, vhich is part of the putative nucleotide sequence recognition site of homeobox proteins. Only zen-/, zen-2, lab, and cad, in addition to the homeobox genes described in this report would be expected to give positive autoradiographic signals with both 121 and 125 probes. It is uncertain whether msh, Dil, and Abd-B genes would give positive signals with 121 and 125 probes because both Dil and Abd-B genes contain introns oe TART AITGATCOTAAACTAGTGCCTAGTCCCTTAACGAGTTATAAC TATTATAGTIA -150 <a “TRY RYN ETCTACTATATICTCSATATPGSCTATCTPICAGAGY GAT AAC AGC -34 Asp Asn Ser 72 An GTG ACE TCC TCG CGT TCC GAG CTG CGA ABA AAC AGS ATC AGT -43 (. Val Thr Ser Ser Arg Ser Glu Ueu Arg Lys Asn Ser lle Ser -1? In RAC AGC AAT CCG GGG AGC AAC AGT GGT TCC ACC AAG CCC C sly Asn Ser Asn Pro Gly Ser Asn Ser Gly Ser Thr Lys Pro > CGA AAG CCT CGC GTG CTC TTT TCC CAG GCA CAG STC ys Ary Lys Pro Arg Val Leu Phe Ser Sln Ala Gin Val G GAG TGT CGC TTT CGA CTC AAA AAG TAT CTG ACG GGT u Glu Cys Arg Phe Arg Leu Lys Lys Tyr Leu The Gly - Hindill G CGC GAG ATA ATC GCG CAA AAG CTT AAC CTG TCG GCC ACC Glu tle Ile Ala Gln Lys Leu Asn Leu Ser Ala Thr . ATT TGG TTC CAG AAT CGG CGC TAC AAA TCG AAA CGT Tle Trp Phe Gln Ase Arg Arg Tyr Lys Ser Lys Arg         GAC TGC GAG GGC ATC GCC AAG CAT CTG AAG TTG AAG spl Ile Asp Cys Glu Gly Ile Ala Lys His Leu Lys Leu Lys GAG CCC CTG GAC TCG CCC ACT TCT CTG ecc ceG CCG ATT CCC AAC 267 slu Pro Leu Asp Ser Pro Thr Ser Leu Pro Pro Pro Ile Pro Asn a9 SAC GTG ATG TGG CCC CCA ACC ATG CAG CAA TCG CAG CAG CAG CAG 312 His Val Met Trp Pro Pro Thr Met Gin Gin Ser Gln Gln Gin Gin 104 “AG CAT CAT GCA CAS CAG CAA CAG ATG CAG CAC ATG TAG TGGACAT 358   sin His His Ala Gin Gln Gln Gin Met Gin Kis Met *** 116 oe SACAGGACORACCATTCGAGTCTCTAATTTATIGCAGT TT CAAGAAGAATACATGTT al? SROTCOTAAGCAAACGCTCATAGTTCTAGTTCTTTSTTT 456 Fic. 5. Nucleotide sequence and deduced amino acid sequence of the homeobox region of the NK-4 gene. The homeobox is enclosed ina box. A CAX repeat is underlined, which encodes repetitive Gln residues. Arrowhead represents a potential splice acceptor site.   Proc. Natl. Acad. Sci. USA 86 (1989) 719             ANSE et Jadesce ae eS wBasdak | BAITAZICA TT CA LE TJAGCA SUM ITIACOE attactfara AGAEL ES NK-3 qu NK-1 NK-4 (93E3-5) (93E1-3)   Fic. 6. (A) Jn situ hybridization of genomic DNA probes for NK-1 (first panel), NK-4 (second panel), NK-3 (third panel), NK-1 and NK-3 (fourth panel), and NK-2 (fifth panel) to Drosophila polytene chromosomes. A-F and vertical markers represent chro- mosomal band subdivisions in the 93 A-F region of the right arm of the third chromosome. The DNA probes hybridize to the following locations: NK-1, 93E3-5; NK-4, 93E1-3; NK-3, 93E1-3; NK-1 and NK-3, 93E1-5; NK-2, 1C1-5. Arrowheads indicate labeled chromo- somal bands. (B) The approximate locations of the NK-3, NK-4, and NK-1 genes are indicated on Bridges’ revised map of chromosomal bands (24). of unknown sequence between codons for the 44th and 45th homeobox amino acid residues, and it is not known whether the msh gene contains an intron at this site. Probe 125 hybridizes to the NK-1 gene because the 3’-terminal nucle- otide sequences of both exon 1] and intron 2 are CAG. Both zen-1 and zen-2 genomic DNA were detected and cloned with probes 121 and 125 in addition to NK-1, NK-2, NK-3, and NK-4, but Jab and cad genes were not detected. The Drosophila genome has been screened many times with DNA fragments containing homeobox sequences as probes; however, NK-1, NK-2, NK-3, and NK-4 homeo- domains may not have been detected because their overall homology to other Drosophila homeodomains is relatively low. The use of oligodeoxynucleotide probes that correspond to other amino acid sequences in homeobox proteins should provide a means of detecting additional sets of homeobox genes that have some structural features in common. 1. Gehring, W. J. (1987) Science 236, 1245-1252. 2. Niisslein-Volhard, C., Frohnhéfer, H. G. & Lehmann, R. (1987) Science 238, 1675-1681. Scott, M. P. & Carrol, B. C. (1987) Cell 51, 689-698. 4. Herr, W., Sturm, R. A., Clerc, R. G., Corcoran, L. M., Baltimore, D.. Sharp, P. A., Ingraham. H. A.. Rosenfeld, M. G.,. Finney, M., Ruvkun, G. & Horvitz, H. R. (1988) Genes Dev. 2, 1513-1516. Doe, C. Q. & Scott, M. P. (1988) Trends Neurosci. 11, 101-106. 6. Doe, C.Q., Hiromi, Y.. Gehring, W. J. & Goodman, C. S. (1988) Science 239, 170-175. 7. Blochlinger. K.. Bodmer, R., Jack. J., Jan. L. Y. & Jan, Y. N. Nature (London) 333. 629-635. Ww ” 7720     Biochemistry: Kim and Nirenberg Proc. Natl. Acad. Sci. USA 86 (1989) Li } I 2} { 3 } 29 EN ENR QVRIWF = #125 PERCENT HOMOLOGY #126 ELEKEF KIWFON #121 Nil Nkc3 NK WK-2 1 10 21 28 38 42 52 61 NK-1  PRRARTAFT YEQLVSLENKFK TTRYLS VCERLNLALSL SLT RT ONR RTKWKKQNP 100 54 44 49 NK-3 KKRSRAAFS BAQVFELERRFA OQRYLS GPERSEMAKSL RLT ETQVKIWFQNR RYKTKRKQ] 54 100 59 66 NK-4 KRKPRVLFS QAQVLELECRFR LKKYLT GAEREILAQKL NLS ATQVKIWFQNR RYKSKRGDI 44 $8 100 59 NK-2 KRKRRVLFT RAQTYELERRFR QORYLS APEREALASLI RLT PTQVKIWFQNH RYKTKRAQN 49 66 59 100 msh §NRKPRTPFT TQQLLSLERRFR EKQYLS ZARRAEVSSSL RLT ETQVKIWFQNR RAKAKRLOE 57 54 84 84 © (29] Dll  MRKPRTIYS SLOLQQLNRAFQ RTQYLA LPERAELAASL GLT QTQVKIWFQNR RSKYKKMMK 53 51 48 48 [22] lab NNSGRTNFT NRQLTELEKEFA FNRYLT RARRIEIANTL OLN ETQVKIWFONR RMKQKKRVK 51 48 48 43 © [19, 20] zan-1 LKRSRTAFT SVQLVELENEFK SNMYLY RTRRIEIAQRL SLC ERQVKIRFQNR RMKFKKDIQ 87 5i 44 39 [15} zan-2 SKRSRTAFS SLQLIKLERRFH LNKYLA RTRRIBISORL ALT ERQVKIWFQNR RMKLKKSTN 49 54 48 41 [15] bed PRRTRTTFT S8QIARLEQHYL OGRYLT APRLADLSAKL ALG TAQVKIWFKNR RRRHKIOSD 46 41 39 43 ~~ [26} Dfd = PKRORTAYT RHQILELEKEWH YNRYLT RRRRIEIAHTL VLS ERQIKIWFQNR RMKWKKDNK 53 48 44 36 [32] Scr TKRORTSYT RYQTLELEKEFH FNRYLT RRRRIELAHAL CLT ERQIKIWFQNR RMKWKKEHK 49 48 44 41 =~« [33] ftz | SKRTROTYT RYQTLELEKEFH FNRYIT RRRRIDIANAL SLS ERQIKIWFQNR RMKSKKDRT 44 43 44 38 {30} Antp RKRGROTYT RYQTLELEKEFH FNRYLT RRRRIELABAL CLT ERQIKIWFQNR RMKWKKENK 49 46 43 41 [34,35] Ubx - RRRGROTYT RYQTLELRREWH TNHYLT RRRRIEMABAL CLT ERQIKIWFQNR RMKLKKEIQ 48 46 43 41 [36,37] abd-A RRRGROTYT RYQTLELEKRFA FNHYLT RARRIEIAHAL CLT ERQIRIWFQNR RMKLKKELR 49 46 44 41 [38] Abd-B VRKKRKPYS KFQTLEUEKEFL FNAYVS KOQKRWELARNI, QLT ERQVKIWFONR RMKNKKNSQ 46 46 44 46 = {21) en EKRPRTAFS SEQLARLKREFN ENRYLT ERRRQQLSSEL GLN RAQIKIWFQNK RAKIKKSTG 46 43 38 34 = [16] inv  DKRPRTAFS GIQLARLKHEYN ENRYLT EKRRQQLSGEL GLN EAQIKIWFQNK RAKLKKSSG 49 46 39 39 [393 BSH4 ORRSRTTFT AKQLEALERAFS RTOYPD VYTREELAQTT ALT EARIQVWESNR RARLRKHSG 44 41 933 34 = [40] BSH9  ORRSRITFS NDQIDALERIFA RTOYPD VYTREELAQST GLT EARVQVWFSNR RARLRKQLN 48 44 38 38 [40] prd ORRCRTTFS ASQLDELERAFE RTOYPD IYTREELAQRT NLT EARIQVWFSNR RARLRKQHT 44 34 30 30 [25] ro QRRORTTFS TROTLRLEVEFH RNEYIS RSRRFELAEKTL RLT KTQIKIWWQNR RAKDKRIEK 51 51 44 46 [8,9] cad  KDKYRVVYT DFQRLELEKEYC TSRYIT IRRKSELAQTL SLS ERQVKIWFQNR RAKERTSNK 43 41 44 39 [42] H2.0 RSWSRAVFS NLORKGLEIOWQ QQKYIT KPDRRELAARL NLT DAQVKVWFQNR RMKWRHTRE 39 46 41 «939 [42] eve = VRRYRTAFT RDQLGRLEKEFY KENYVS RPRRCELAAQL NLP ESTIKVAFQNR RMKDKRQORI 41 36 34 33 [43,44] cut  SKKQRVLFS EEQKEALRLAFA LDPYPN VGTIEFLANEL GLA TRTITNWFENE RMRLKCQVP 31 28 33 31 [7] 00 @ 00 e eo o e ° oo oO e Oo COoOo@eeCceO @ 0 OO Fic.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ezfk.xq6y.hv6f", "00000000-0000-0000-3C67-5E4FD23D0F32", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to Francis Crick", "101584910X70", "101584582X278", "1962", "15 January 1962", "In this letter, Nirenberg praises Crick for his work on the code and informs Crick of his recent research findings.  A brief reference to the American press suggests that there is a popular conception that Nirenberg's work offers widespread applications and enormous potential.", "Letters (correspondence)", "Genetics", "Public Reactions to the Genetic Code, 1961-1968", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "January 15, 1962 Dear Dr. Crick: David Davies has made all of the arrangements for your talk here in February and we are all looking forward to seeing you.  Your paper on the General Nature of the Genetic Code is beautiful.  Your findings with this system certainly agree well with our results.  We have recently found that the code is partially degenerate, at least with respect to leucine, for both poly UC and poly UG stimulate the incorporation of leucine into protein.  Also, we were able to show that nonsense exists for a number of polynucleotides, including poly A, do not code for any amino acid.  We revised our manuscript which is in press in Biochemical and Biophysical Research Communications to include the results on degeneracy.  Also included are the nucleotide compositions of coding units corresponding to two additional amino acids. We tested the ability of many preparations of poly C to stimulate proline incorporation.  About six preparations gave a 5- to 10-fold stimulation; three preparations gave a 75- to 100- fold stimulation, and four or five preparations did not stimulate proline incorporation.  We have also found that poly CU stimulates proline incorporation effectively, more so than poly C. When we analyzed the base ratios of our polynucleotides, we could not find traces of uridylic acid in poly C.  Since we now know that the code is partially degenerate, it seems likely that both poly CU and poly C will code for proline and that this is another example of degeneracy.  Probably an E. coli coding unit corresponding to proline would contain both U and C. I haven't seen the English newspapers but the American press has been saying that this type of work may result in (1) the cure of cancer and allied diseases (2) the cause of cancer and the end of mankind, and (3) a better knowledge of the molecular structure of God.  Well, it's all in a day's work. Sincerely yours, Marshall W. Nirenberg", "Nirenberg, Marshall W.", null, null, null, "Crick, Francis, 1916-2004", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2y3c-98gp-r226", "00000000-0000-0000-FDA1-FD0EC00BBBF8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francis Crick to Marshall W. Nirenberg", "101584910X71", null, "1962", "21 September 1962", null, "Letters (correspondence)", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Francis Crick.", "Copyright may apply", null, null, "21st September, 1962. Dear Marshall, Enclosed you will find the review I mentioned. If there is anything I've quoted that I shouldn't have done, or any other alteration you might like, let me know and I'll alter it in proof. I hope you are happy with the way the Scientific American article turned out. I look forward to reading your own article in due course. Nothing new here for the moment. Yours ever, Francis F. H. C. Crick", "Crick, Francis, 1916-2004", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wf7q.a7b6_whda", "00000000-0000-0000-8C66-0E0A8C83C988", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francis Crick to Marshall W. Nirenberg", "101584910X72", null, "1962", "4 January 1962", "In this letter from the Cavendish laboratory, Crick shares his ideas on the coding process with Nirenberg, offers to give a talk at NIH as requested, and insists that Nirenberg's discoveries revealed in the Proceedings of the National Academy of Sciences were the \"real\" breakthroughs in genetic investigation.", "Letters (correspondence)", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "2", "pages", "Text", "English", "Reproduced with permission of Francis Crick.", "Copyright may apply", null, null, "4th January, 1962. Dear Dr. Nirenberg, It was very kind of you to send me your two papers, and I look forward very keenly to your next one.  I liked the first paper very much, but I felt that perhaps Ochoa had rushed you into publishing the second one in rather a hurry.  We hear that Leon and Maxine are helping you pre- pare the polymers.  I hope that this is true, and that all is going well.  It must be maddening to have the whole world jumping into one's problem.  I must confess that we are doing a little on it ourselves, in collaboration with Marianne.  Our results are broadly the same as everyone else's, except that we get incorporation with poly UC having a measured U/C ratio of about 0.24.  This incorporates a lot of proline, among other things.  In our hands poly C doesn't appear to work, but we think this is because it is double-stranded.  However, we haven't pursued this.  I should be surprised if CCC is not proline, as you claimed in your PNAS footnote. I will certainly give a talk at NIH, but I think it had better be on the genetic work.  We can talk about the bio-chemistry privately. I have to be in Washington for the Biophysics meeting, and expect to be around till at least the evening of 20th February.  Perhaps you could collate your invitation with David Davies, who has suggested I give a general evening lecture.  Let me know what you fix up between you. The English papers have made rather a fuss about our Nature paper, which was published on Saturday, but as far as I have been able I have stressed that it is your discovery which was the real break-through. Looking forward greatly to seeing you in February. Yours sincerely, F. H. C. Crick", "Crick, Francis, 1916-2004", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-y7rr_7igg.djmf", "00000000-0000-0000-00A4-2CA48A566EEC", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Bernardo A. Houssay, Instituto de Biologia y Medicina Experimental to Marshall W. Nirenberg", "101584910X73", null, "1962", "6 December 1962", "This letter from Bernardo A. Houssay thanks Marshall Nirenberg for sending him his curriculum vitae and reprints of his work on the genetic code.", "Letters (correspondence)", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "December 6th, 1962 Dear Dr. Nirenberg: Many thanks for your curriculum vitae and the brief resume of your work concerning the genetic code and reprints. I was born on April 10th like you. With best regards, I am, Sincerely yours, Bernardo A. Houssay", "Instituto de Biologia y Medicina Experimental ; Houssay, Bernardo A. (Bernardo Alberto), 1887-1971", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bmr3-ci5j.mwza", "00000000-0000-0000-A7D0-79CA4B39188F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to Wendell M. Stanley", "101584910X74", null, "1962", "6 March 1962", "Nirenberg respectfully declines the offer for a position at Berkeley and decides to remain at NIH due to his primary interest in research and concerns with devoting part of his time to teaching.  Additionally, more space, help, and a promotion from NIH lead him to make this decision.", "Letters (correspondence)", "National Institutes of Health (U.S.)", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "March 6, 1962 Dear Dr. Stanley: I enjoyed meeting your son last week. We had a long talk about messenger and ribosomal RNA and so forth.  I was sorry to learn that you had broken your hip and I certainly hope that it is mending properly and not giving too much trouble.  I also decided not to go to Phoenix because of the pressure of work. I must apologize for having taken these last few months to come to a decision.  Needless to say I have very seriously considered the position at Berkley from many angles.  It was difficult to make a decision because the various possibilities were almost equally good.  However, I believe it would be best for me to remain at NIH.  The major reason for this choice is that I am primarily interested in research and felt hesitant about devoting part of my time to teaching.  I wanted to leave NIH because adequate space and technical help were not available to me.  However, I. learned today that some more space, help and a promotion will be available to me at NIH and these factors were the deciding ones.  The position here in many respects is equivalent to the one in your department. I very much appreciate your interest and willingness to take me into your department.  I am glad, at least, that I had the opportunity of meeting you and I look forward to seeing you again soon. Respectfully yours, Marshall W. Nirenberg", "Nirenberg, Marshall W.", null, null, null, "Stanley, Wendell M. (Wendell Meredith), 1904-1971", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ncmj.vzf2.scm7", "00000000-0000-0000-C58D-767519F9D17F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francois Jacob, Institut Pasteur (France) to Marshall W. Nirenberg", "101584910X75", null, "1962", "20 March 1962", "This letter from Jacob discusses the possibility of Nirenberg coming to the Institut Pasteur and their anticipated meeting at Royaumont in the summer of 1962.", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of Francois Jacob.", "Copyright may apply", null, null, "20 mars 1962 Dear Dr. Nirenberg, Thank you for your letter of March 12. I understand very much how difficult it must be for you at this time to establish plans so far ahead, but we need to know definitely who will be coming in the laboratory. I hope very much you will be able to come sometime here and I keep you some laboratory space for the year 1964-1965 as you suggest. We shall probably meet in the Royaumont meeting this summer. I hope it will be possible at this time to make more precise plans. With best regards, Yours, Francois Jacob", "Institut Pasteur (Paris, France) ; Jacob, Francois, 1920-2013", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7jay.vs4d.4wfi", "00000000-0000-0000-ED50-A57437C8BC75", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Mrs. Jonan W. Williams to Marshall W. Nirenberg", "101584910X76", null, "1962", "16 March 1962", "In this handwritten letter, a woman from Texas responds to an article in Look magazine by asking Nirenberg for information regarding his research as it applies to treatment for nerve damage caused by polio.  Williams offers Nirenberg prayers and hope for success in his work.", "Letters (correspondence)", "Poliomyelitis", "Public Reactions to the Genetic Code, 1961-1968", "2", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "206 S. Mill Winnsboro, Texas March 16, 1962 Dr. Marshall W. Nirenberg National Institutes of Health Washington, D.C. Dear Dr. Nirenberg, In a recent issue of \"Look Magazine\" I read of your work with nucleic acids and especially with the molecule called DNA. The article was fascinating -- especially to me, since I am a polio victim. I wonder if your research in any way indicates a help for the nerve damage caused by [END PAGE ONE] [BEGIN PAGE TWO] such diseases as polio. Any information you might be able to give me would be greatly appreciated. I wish that I could be of some assistance to your research, and I do offer my prayers for your success in all your work. Sincerely, Mrs. Jonan Williams", "Williams, Jonan W., Mrs.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-q3pf~n44c_3isy", "00000000-0000-0000-2FFC-FB7913C8AF59", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from John E. Bruno to Marshall W. Nirenberg and J. Heinrich Matthaei", "101584910X77", null, "1962", "23 March 1962", "In this letter, a man from Wisconsin responds to an article in Science Digest describing Marshall Nirenberg's work in genetics.  Bruno asks Nirenberg and Matthaei if their work might explain the cause of albinism and recommends an experiment for the researchers.", "Letters (correspondence)", "Albinism,Pigmentation", "Public Reactions to the Genetic Code, 1961-1968", "2", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "John E. Bruno 1404-54th St. Kenosha, WI March. 23-'62 Drs. Nirenberg and Matthaei. Bethesda Md. Dear Drs. In my April issue of Science Digest an article was written on the wonderfull [sic] work use are doing on genetics and Metabolic Diseases. Now the reason I write is to inquire of a metabolistic [sic] problem I have, Albinoism. In the article it states of the work uses are doing on herditary [sic] Materials DNA and RNA. In your experiments have you found the cause of Albinoism? Have uses done any experiments with Albino mice? Is there any way of inducing pigment formations? My name is John E. Bruno. I am of Italian extraction. I have 20/200 vision. I have ben [sic] to a number of Oculist [sic] and their diagnosis is of the same pattern a lack of pigment in the retina. My O.D. has not ben [sic] able to give me any anatomical improvement. Is there a chemical that can be induced [sic] in the blood stream to stain the retina? Over Please [END PAGE ONE] [BEGIN PAGE TWO] Drs. I have an experiment which would be very interesting to work with! Understanding that the retina is a continuation of the brain and being a very complex mechanism to work with! My experiment deals with employing charged particles of chemicals that would be exceptable [sic] to tissue. Using a screen and a plate. The screen is charged and the solid metal plate being of opposite polarity from that of the screen Placing the chemicals in front of the screen the chemicals particles become charged and are drawn to the plate. The retina being between the chemical and plate becomes impergnated [sic]. [diagram] Hoping to receive an answer to this letter I remain Yours Truly John E. Bruno 1404-54th St. Kenosha, Wisconsin", "Bruno, John E.", null, null, null, "Matthaei, Heinrich ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dtcx-g5n8.p4c5", "00000000-0000-0000-87CE-C26D7FC17E1B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Recorded observations of nature in Florida", "101584910X78", "101584910X125", "1944", "[1 June-13 August 1944]", "These handwritten notes by Nirenberg list various flora and fauna and include some brief descriptions.", "Diaries", null, "Biographical Information", "13", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Rw Oe a ll) yg Co 4    ‘ me 5 | ‘ se K i : v LAN de eee eee mae me ewe TN eee Bee Lee : sf : ‘ ~. pe aS hae cori Acer     Sy oe : A. hp ii ip ‘ Kf es ae", "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2jaj.yi6f~bbw9", "00000000-0000-0000-5074-BC102E2170C8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Lewis J. Brown to Marshall W. Nirenberg", "101584910X79", null, "1962", "10 February 1962", "This letter from Nirenberg's former roommate attests to the acclaim granted Nirenberg from articles like the one published in Chemical Engineering magazine that caught Brown's eye.  Brown predicts Nirenberg will soon assume the chair of an outstanding biochemistry department.", "Letters (correspondence)", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "2", "pages", "Text", "English", "Reproduced with permission of Lewis J. Brown.", "Copyright may apply", null, null, "Feb. 10, 1962 Dear Marshall, My father saw this article about you in Chem and Eng. Magazine in regard to protein metabolism My old roommate has made his mark in the world and we're all proud of you. I visualize a rapid climb to the top of the biochemical heap within a few short months or years. Perhaps at this moment you are in the process of packing and moving to some large university to assume the chair of an outstanding department. How have you been? I would like to hear that you are married but have my doubts what about this point? Victor Blom recently wrote that Connie and June are living in Tennessee somewhere, associated with Vanderbilt. I guess that sort of leaves you the lone Wolverine remaining in D.C. [END PAGE ONE] [BEGIN PAGE TWO] My program is moving along really. I'm now doing some time in neuroanatomy teaching the freshman students the basic element -- fancy that, can you? Soon I shall return to the wards and assume the chief resident position I'm hoping to be out of here within 2 years. Where I shall finally settle remains a mystery -- perhaps we will return to the midwest. The world's fair is being held in Seattle this year, as you know. What are the chances for your coming out here to go to the fair and see us in Portland -- you could use a nice vacation trip -- Think about this, I'm serious We would appreciate a letter from you about your present status and what the future will bring -- it looks very bright from the way I look at it Lew New address: 6730 S.W. Vermont Court Portland 19, Oregon", "Brown, Lewis J.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cy59_h92h_i99q", "00000000-0000-0000-84A5-E5AAC2431FC1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Xandra Breakefield to Marshall W. Nirenberg", "101584910X80", "101584910X81,101584910X87", "1962", "19 February 1962", "In this letter from a sophomore biology major at Wilson College, Breakefield asks Nirenberg for the opportunity to observe his work in the NIH lab.", "Letters (correspondence)", "Mentors", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Xandra Breakefield.", "Copyright may apply", null, null, "February 19, 1962 Dear Dr. Nirenberg; I am a sophomore at Wilson College and a biology major.  I am writing to you because I read an article about your research concerning RNA in the newspaper and also because you are employed at N. I. H. and I am very interested in working there someday. I realize I have a long way to go before I am sufficiently educated to be worthwhile in research, but I would appreciate the opportunity to find out a little about how it works.  In other words, I wondered if I could be allowed to observe how you and, or your colleagues work.  Over Spring Vacation, March 16-26, I will be in the Washington area and would be available anytime to come out to N. I. H.   I realize you must be very busy but perhaps someone you know would be willing to put up with a few questions. I can't tell you how much I would appreciate the opportunity.  Please, answer this letter even if just to say, \"No! \". Thank you for your consideration. Sincerely, Xandra Breakefield", "Breakefield, Xandra O.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3rmc~mse4-m872", "00000000-0000-0000-9193-84AEA3E142D8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to Xandra Breakefield", "101584910X81", "101584910X80,101584910X87", "1962", "12 March 1962", "Nirenberg welcomes Breakefield's visit to the lab to discuss working in the field.", "Letters (correspondence)", "Mentors", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "March 12, 1962 Dear Miss Breakefield: Thank you very much for your letter. I think your interest and enthusiasm in this field of research is wonderful and I would be happy to have you visit the laboratory so that we could talk for a little while during your spring vacation. I hope that in a short visit you can get at least a partial idea of how we work and what we think about. Call me at Oliver 6-4000, extension 2881, during your spring vacation to make sure that I will be here. Sincerely yours, Marshall W. Nirenberg", "Nirenberg, Marshall W.", null, null, null, "Breakefield, Xandra O.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-926d~g5ub.54rp", "00000000-0000-0000-B904-EC10B152166A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to Kiyoshi Kurahashi", "101584910X82", null, "1962", "16 January 1962", "This letter to Kurahashi at the Institute for Protein Research at Osaka University reveals the pressing research issues in early 1962--determining the character of RNA coding units, working out coding ratios, resolving issues of degeneracy and specificity, and obtaining evidence of the universality of the code.  Nirenberg laments, \"There is so much to do that the work seems to crawl along at a snail's pace.\"  Personal matters are also included.", "Letters (correspondence)", "Sequence Analysis, RNA", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "2", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "January 16, 1962 Dear Kiyoshi: Thank you very much for your letter.  It was wonderful to hear from you. Enclosed are reprints and two preprints of our recent work; one of which is in press in Proceedings of the National Academy of Sciences, the other in Biochemical and Biophysical Research Communications.  I am in the midst of writing a few more papers and I will send manuscripts when they are finished. The work is pretty exciting at this stage.  Currently we are trying to determine characteristics of RNA coding units, such as the coding ratio, degeneracy, specificity of coding units, the effect of secondary structure upon the activity of coding units, etc.  However, there is so much to do that the work seems to crawl along at a snail's pace.  Leon Heppel and Maxine have been doing some work with us and this has been a great help.  One other thing that we are trying to do is to obtain an estimate of the universality of the code.  It will be months though before we have really clean data concerning this question. Gordon flew back from Paris last week for a few days and it was a lot of fun to see and talk to him.  Both he and Millicent are enjoying Paris very much and he is very excited about the work that he is doing there.  It was wonderful hearing about the gossip from Paris.  Actually, Bruce and Giovanna came back from Paris several weeks ago and we heard quite a bit about current research going on at Pasteur from them.  They both had a good time in Cambridge and in Paris. Mike Yarmolinsky is engaged to a Finnish girl - an artist whom he met this past summer when he was vacationing in Europe.  They expect to be married this summer. Let me know how things are going and give my best regards to your mother. Sincerely yours, Marshall W. Nirenberg", "Nirenberg, Marshall W.", null, null, null, "Kurahashi, Kiyoshi", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6hs7-2eg3.qknz", "00000000-0000-0000-0D67-98BCAD836247", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Postcard from Zhores Medvedev to Marshall W. Nirenberg", "101584910X83", null, "1962", "3 March 1962", "Medvedev thanks Nirenberg for the reprints and preprints of his work on protein synthesis and the coding problem.  Medvedev is hopeful that the work in genetics will finally end the legacy of Lysenkoism.", "Postcards, Letters (correspondence)", "Amino Acid Sequence", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Zhores Medvedev.", "Copyright may apply", null, null, "March 3 1962          from Zh. A. Medvedev Dear Dr Nirenberg: Many thanks for your kind letter of January 26 and for the sending to me reprints and pre-print of your remarkable works in protein synthesis which I received yesterday and have read with great admiration. Your works together with Ochoa and Crick's publications on coding problem made a great impressions here and this connected with some reasons First of all -- we have here rather influencial [sic] group of biologists and geneticists who for the very long time criticized and rejected the coding problem as non-scientific and idealistic (morganism-mendelism). You probably know about long and great discussion on heredity which was started by Lysenko and which seemed to be permanent. The new experimental direct attack in coding problem -- is a good basis for the settlement of this hard discussion. Therefore I see to this achievement not only as the great discovery of natural sciences, but as to the important step which can make influence to the situation in genetics here and to improve this situation. Of course, I should be happy to receive information about the new results of this interesting work with best wishes and many thanks Sincerely yours Medvedev", "Medvedev, Zhores A., 1925-2018", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-guud_9yuu~dpgw", "00000000-0000-0000-B461-A69320A99F1B", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Telegram from Francis Crick to Marshall W. Nirenberg", "101584910X84", null, "1962", "27 August 1962", null, "Telegrams", "Publishing", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Francis Crick.", "Copyright may apply", null, null, "Marshall Nirenberg Natl Inst of Health Very distressed to hear from Scientific American that your article is not ready STOP Most reluctant for mine to be published without yours STOP Suggest you phone Scientific American to clarify situation STOP Let me know result Francis", "Crick, Francis, 1916-2004", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rui3-4sx9~69y7", "00000000-0000-0000-3686-4D8E885C1672", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Marshall W. Nirenberg to G. Schramm", "101584910X85", null, "1961", "22 November 1961", "In this letter to Dr. G. Schramm of the Max Planck Institute, Nirenberg requests information regarding the use of German-produced synthetic deoxypolynucleotides in his enzymatic investigations.  Enclosing reprints of his previous articles, Nirenberg suggests that their collaboration could yield quick results in the study of protein synthesis crucial to deciphering the genetic code.", "Letters (correspondence)", "Polynucleotides,RNA, Messenger", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "November 22, 1961 Dear Dr. Schramm: I read your synthetic procedure for obtaining deoxypolynucleotides with great interest.  I very much would like to get or make 2 to 5 mg of deoxypoly A, deoxypoly T, deoxypoly C, deoxypoly G, and also mixed polymers.  I am interested primarily in cell-free protein synthesis from an enzymatic point of view.  Recently we found that polyribo U served as a synthetic messenger RNA which directed the synthesis of polyphenylalanine in a cell-free system.  I am enclosing reprints of this work which describe these experiments in detail.  I would like to use the deoxypolynucleotides in this work for I feel certain that some will work in our enzymatic system.  I would be glad to collaborate with you, if you would like to, on aspects of our work dealing with these polymers.  Certainly they would have a great deal of potential in our cell-free system.  Such a collaborative effort could yield important results in a short time. I would be glad to either synthesize the polymers if you would send me the detailed protocol, or to receive the polymers from you. Looking forward to hearing from you, I remain, Sincerely yours, Marshall W. Nirenberg", "Nirenberg, Marshall W.", null, null, null, "Max-Planck-Institut fur Virusforschung ; Schramm, G.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rprq.ugr5_mb5v", "00000000-0000-0000-94C8-8B4BF5B32511", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Evelyn Berk to Marshall W. Nirenberg", "101584910X86", null, "1961", "24 October 1961", "In this letter from Maryland eleventh-grader Berk, she asks Nirenberg for additional information that can be used to demonstrate the role of DNA experimentally.", "Letters (correspondence)", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "4113 Kennedy Street Hyattsville, Maryland October 24, 1961 National Institutes of Health Bethesda, Maryland Dear Dr. Nirenberg, I am an 11th grade student interested in DNA. I have already done a Feulgen Squash and I would appreciate and information or other experiments you could send me. My address: Miss Evelyn Berk 4113 Kennedy St. Hyattsville, Md. Thank you. Sincerely yours, Evelyn Berk", "Berk, Evelyn", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yhmb_zwsm-4gee", "00000000-0000-0000-E000-E522F3A19190", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Xandra Breakefield to Marshall W. Nirenberg", "101584910X87", "101584910X80,101584910X81", "1962", "25 March 1962", "Breakefield thanks Marshall Nirenberg for her experience observing his work in the NIH laboratory and the \"real impact [it has] on the individual.\"  She also extends her admiration for the \"sense of personal achievement that you seem to glow with.\"  She hopes that she will also be able to use her talent \"in a way that will profit me and others.\"", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "2", "pages", "Text", "English", "Reproduced with permission of Xandra Breakefield.", "Copyright may apply", null, null, "March 25, '62 Dear Dr. Nirenberg, I want to thank you for really showing me what research is to the individual engaged in it. I can't tell you how impressed I was with the sense of personal achievement that you seem to glow with. And this is what I wanted to know, not facts and figures that my lack of education would render unmeaningful [sic], but the real impact on the individual. I needed some kind of proof that all this memory that I'm filling full of facts is going to be used eventually in a way that will profit me and others. You showed me what I was hoping was there. Now I just [END PAGE ONE] [BEGIN PAGE TWO] have to sit down and start studying. There's something so challenging about a profession that you must prepare for. Thank you again. You'll probably be hearing from me when I need advice again. Sincerely, Xandra Breakefield", "Breakefield, Xandra O.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-gn5y.esq6-bzy4", "00000000-0000-0000-6953-641286F64D0F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from H. J. Muller to Marshall W. Nirenberg", "101584910X88", null, "1962", "1 February 1962", "This letter from Indiana University thanks Marshall Nirenberg for the recent symposium on RNA coding held at Bloomington and extends an open invitation for a return.", "Letters (correspondence)", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Feb. 1, 1962 Dear Dr. Nirenberg: Let me express the thanks and appreciation of the Committee that arranged the recent symposium on RNA coding for your kindness in having come here and for the truly remarkable contribution that you made to it. It was inspiring to the older and to the younger hearers alike to follow the course of the marvelous break-through that you described to us. We hope this will by no means be you last visit to Bloomington.  You have made many permanent friends here and we shall be watching your future work with the highest hopes and expectations. With kind personal regards from all of the Committee, Yours cordially, H. J. Muller", "Muller, H. J.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e6z2_ndum~w2ba", "00000000-0000-0000-BA2B-E7ED9F8ACB2D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Program of the \"Symposium on RNA Coding\"", "101584910X89", "101584910X90", "1962", "23 January 1962", "This symposium was held at Indiana University in Bloomington, Indiana on January 23, 1962.  In this copy of the program featuring Khorana, Nirenberg, Mahler, and Benzer as guest speakers, Nirenberg sketches some possible combinations of nucleotides for RNA codons.", "Programs (documents)", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "2", "pages", "Text", "English", "Reproduced with permission of Indiana University.", "Copyright may apply", null, null, "Symposium On RNA Coding Indiana University January 23, 1962 Program Prof. H. J. Muller in the Chair 9:30 A.M.  Dr. Gobind Khorana, Enzyme Institute, University of Wisconsin \"Chemical synthesis and sequential analysis of polynucleotides\" 10:30 A.M.  Dr. Marshall Nirenberg, National Institutes of Health \"Characteristics of RNA Coding Units\" 11:30 A.M.  Prof. Severo Ochoa, New York University School of Medicine \"Synthetic polynucleotides and the genetic code\" 12:30 P.M. * * * Lunch * * * * Prof. H. R. Mahler in the Chair 2:30 P.M.  Prof. Fritz Lipmann, The Rockefeller Institute \"Notions on the mechanism of code translation into peptide sequence\" 3:30 P.M.  Prof. Seymour Benzer, Purdue University \"Genetic fine structure and the code\" 5:00 P.M.  Informal Reception - University Club Lounge, Union Building [Handwritten columns of genetic code notes]", "Indiana University", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xwz4_4ps5-ncy8", "00000000-0000-0000-85E6-5F6DA0380B75", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Announcement of \"A Symposium on the RNA Code\"", "101584910X90", "101584910X89", "1962", "[Before 23 January 1962]", "This symposium was held at Indiana University in Bloomington, Indiana on January 23, 1962.  This announcement for a symposium devoted exclusively to the topic of the RNA code suggests that the race to uncover the code was both widespread and at least partly cooperative.  Speakers included S. Benzer, F. Lipmann, M. Nirenberg, and S. Ochoa.", "Announcements", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Indiana University.", "Copyright may apply", null, null, "Indiana University Bloomington, Indiana A symposium on the RNA code On January 23, 1962, the speakers listed below will discuss current work bearing upon this important problem. Sessions will be held in Jordan Hall on the Bloomington Campus, beginning at 9:30 A.M. C.D.T. and lasting throughout the day. Interested scientists are invited to attend. Reservations for overnight accommodations at the Union Building will be made by Mrs. Margaret Zurbrigg, Department of Zoology, 220 Jordan Hall, Indiana University. Kindly call this meeting to the attention of colleagues or students who might be interested. Speakers Prof. Seymour Benzer, Dept. of Biological Sciences, Purdue Univ. Prof. Fritz Lipmann, The Rockefeller Institute Dr. Marshall Nirenberg, National Institute of Health Prof. Severo Ochoa, Dept. of Biochemistry, New York University College of Medicine", "Indiana University", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hewc_3sah-m6tb", "00000000-0000-0000-A36D-7AF7494356B0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francis Crick to Marshall W. Nirenberg", "101584910X91", "101584582X281", "1962", "5 December 1962", "Crick congratulates Nirenberg on winning the Nobel Prize and uncovering more triplets.", "Letters (correspondence)", "Nobel Prize", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Francis Crick.", "Copyright may apply", null, null, "5th December, 1962. Dear Marshall, Very many thanks for your congratulations on the Nobel prize and for your most interesting preprint. I had one from Severo at about the same time, and was delighted to find that both of you are uncovering more triplets. We are all intrigued by your reference to \"quantative work . . . shows that some codewords are triplets\" and look forward to seeing the data. I wish I could get to the bottom of the degeneracy - it's clearly not random but what it is baffles me. Did you ever write that article for Scientific American? Yours ever, Francis F.H.C. Crick", "Crick, Francis, 1916-2004", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-swmk~x5wf.uvni", "00000000-0000-0000-9875-5A4B030AFEA0", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Roger Harris to Marshall W. Nirenberg", "101584910X92", null, "1963", "14 May 1963", "This letter requests Nirenberg's autograph as part of a \"collection of autographs of prominent persons.\"  The author also requests that Nirenberg reply with either a photo or a response to what he thinks his greatest contribution has been.", "Letters (correspondence)", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "May 14, 1963 My dear Dr. Nirenberg I should like to compile a collection of autographs of prominent persons. I hope you do not mind my imposing upon you and will permit me to include you to include you in my collection as I sincerely believe that you, through your inspiring efforts and outstanding contributions, are endearing yourself to many people. If you would be so kind as to autograph the enclosed card, I shall be very grateful. A stamped, return envelope is included for your convenience. If you have a photograph available that you could autograph, it would please me immensely. I would be interested in knowing, Dr. Nirenberg, what you consider your greatest satisfaction thus far during your career. With cordial best wishes, Sir, for continued success and much happiness, Respectfully yours, Roger Harris", "Harris, Roger", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cd8j_g6sf~jdxv", "00000000-0000-0000-7F06-FA46928F8C9A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Albert Baird Hastings to Marshall W. Nirenberg", "101584910X93", null, "1962", "[1962?]", "This letter congratulates Nirenberg for his work and honors while thanking him for the \"sparkling and impressive\" presentation made before the Public Health Service Council.", "Letters (correspondence)", "United States Public Health Service", "Public Reactions to the Genetic Code, 1961-1968", "2", "pages", "Text", "English", "Reproduced with permission of Albert Baird Hastings.", "Copyright may apply", null, null, "June 24 Dear Dr. Nirenberg: I congratulate you on your work and on the honor that has been bestowed upon you for it. But I am writing you especially because of the sparkling and impressive presentation you made to our Council on Saturday. Though we did not know the details of the subject, you gave us a sense of intuitive understanding. It was a fine job. I envy you the life and times you have ahead of you -- and I am glad that you are having this phase of it in the Public Health Service. As you probably know the University of Michigan is my [END PAGE ONE] [BEGIN PAGE TWO] Alma Mater -- and Hal Christiansen is one of my Ph.D's. I may therefore the pride in you as a sort of scientific grandson. You may not know, [ . . . ], that I entered the Public Health Service in 1917 (Assistant Sanitary Chemist, attached to the Hygiene Laboratory, the forerunner of N.I.H..). I stayed in it until 1921, during which I got my Ph.D. As a result I feel a great debt to the P.H.S. -- and work for it whenever they ask me. So far, I'm on my fourth Council, and have been in on the establishment of the fellowship and Grant programs, Tahm [?], Shock, Anform [?] and many others, I count as my \"boys\". With great faith in your future -- Most sincerely, Baird Hastings", "Hastings, Albert Baird, 1895-1987 ; Scripps Clinic and Research Foundation", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-x45r~nvk3.545m", "00000000-0000-0000-D1C3-04885D88D1F8", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from James F. Hogg to Marshall W. Nirenberg", "101584910X94", null, "1962", "29 January 1962", "Marshall Nirenberg's former advisor remarks on Nirenberg's dramatic change in status in the wake of his 1961 work on synthetic RNA.", "Letters (correspondence)", null, "Biographical Information", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "29 January 1962 Dear Marsh, In view of the very extensive recent publicity, we are considering putting a sign on our house, as follows \"Painted by Marshall V. Nirenberg\". AD 1953 Would you please send us a letter of authentication? We could then perhaps obtain a tax exemption as an historical site! Certainly your status has changed locally since last September when Christensen lectured me about your conduct and said I should not write any more letters for you. Perhaps sometime you will tell me what did go on last summer for I did get amazing stories about it all. Today, after an unpardonably long delay, Lucy and I were able to get together on a wedding gift for you and Perola and it should be in the mail tomorrow. Since we had no better address, it is being sent to you at NIH. Perhaps you should keep an eye out for it and let us know if nothing appears within the next 10 days. I regret that we did not receive the announcement in England (it was not forwarded) so that we could have obtained something unique but we hope that the present item will be compatible with your new household arrangements. Will you have any opportunities to visit Ann Arbor in the next few months. I would be only too glad to make arrangements if you would give me a schedule. With best regards, Jim Hogg", "Hogg, James F.", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-guqk~x23e.7yri", "00000000-0000-0000-6581-B446880C0A4A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francis Crick to Marshall W. Nirenberg", "101584910X95", "101584582X279", "1962", "29 January 1962", "In this letter, Crick shares the problems his team has encountered with evidence of degeneracy, and extends the hope of discussing the issue with Nirenberg in the future.", "Letters (correspondence)", "Poly U", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Text", "English", "Reproduced with permission of Francis Crick.", "Copyright may apply", null, null, "29th January, 1962. Dear Dr. Nirenberg, Thank you for your revised paper.  We, too, thought we had evidence for degeneracy, as in our hands both poly (U,C) and Poly (U,A) incorporate leucine, and the result is not due to an impurity in the isotope.  However, the argument obviously depends upon poly U not incorporating leucine, so we retested this.  To our surprise and annoyance we found that our poly U does stimulate the incorporation of some leucine.  The amount varies but on one occasion it was as high as 25% of the phenyalalamine incorporated. Naturally we believe that the result with poly U is an artefact, but we have not been able to track it down.  Until we have done this we don't feel we can trust the evidence from the other polymers. As far as we can see the result is not due to impurities in our poly U.  Apart from the fact that the polymers have been analyzed by Marianne we do not find the incorporation of other amino acids which we should expect if other bases were present in our poly U. I hope by the time we meet we shall have found the cause of the trouble. Looking forward to discussing all this with you. Yours sincerely, F. H. C. Crick", "Crick, Francis, 1916-2004", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-aga5-d8nt_2d8s", "00000000-0000-0000-26DF-C36985D2EF21", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "An Intermediate in the Biosynthesis of Polyphenylalanine Directed by Synthetic Template RNA", "101584910X96", null, "1962", "January 1962", "Building on earlier poly-U investigations, this article suggests that the genetic code may not be universal, offering that it may differ from species to species because variant sRNA base-pairings may substitute one amino acid for another during protein synthesis.", "Articles", "Phenylalanine,Poly U,RNA,Enzyme Activation", "Synthetic RNA and the Poly-U Experiments, 1959-1962", "6", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Reprinted from the Proceedings of the NaTronaL ACADEMY OF SCIENCES Vol. 48, No. 1, pp. 104-109. January, 1962. AN INTERMEDIATE IN THE BIOSYNTHESIS OF POLYPHENYLALANINE DIRECTED BY SYNTHETIC TEMPLATE RNA By Marsuatu W,. Nirenserc, J. Heinrich MarrHar,* AND OLIVER W. JONES NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by Richard B. Roberts, November 24, 1961 We have recently found that, simple, synthetically prepared polyribonucleotides such as polyuridylict acid and polycytidylic acid function as RNA templates in a cell-free protein-synthesizing system prepared from £. coli. 2 In this system, poly U contains the information for the synthesis of polyphenylalanine; therefore, the code for phenylalanine is one or more uridylic acid residues. Poly U was much more effective in increasing the rate of cell-free protein synthesis than naturally occurring informational RNA,? possibly because the synthesis of a protein containing only one amino acid was faster than the synthesis of a protein containing 20 amino acids. We are using this model system currently to study the enzymatic sequence of protein synthesis. Although sRNA and amino acid—activating enzymes have been studied exten- sively, there is controversy concerning their relationship to protein synthesis.*—¢ Vou. 48, 1962 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES 105 One purpose of this investigation was to determine whether sRNA is an intermediate in the synthesis of polyphenylalanine directed by a synthetic RNA template. Our results demonstrate that the incorporation of phenylalanine into sRNA and its subsequent transfer from sRNA are steps in the synthesis of polyphenylalanine. Methods.—-Dialyzed extracts of E. coli containing ribosomes and 100,000 x g supernatant solu- tions were obtained as described perviously.2. These extracts correspond to the previously described 8-30 fractions.2. Ribosomes were sedimented by centrifuging 8-30 extracts at 105,000 x gfor 2 hrat 3°. The supernatant solution was aspirated and will be referred to hereafter as 100,000 X g supernatant solution. The ribosomes were resuspended in 0.01 Jf Tris (hydroxy- methyl)aminomethane, pH 7.8, 0.01 17 magnesium acetate, 0.06 Af KCI, and were centrifuged again at 105,000 x g for 2 hr. The supernatant solution was decanted and was discarded. The ribosomes were washed two more times in the same manner by resuspension and centrifugation. Both 100,000 x g supernatant solutions and washed ribosomes were stored in small aliquots under liquid nitrogen. Thawed preparations were not refrozen and reused. sRNA was purified from #. colt 100,000 X g supernatant solutions by phenol extraction. sRNA was charged with C'-phenylalanine enzymatically, and the C!-aminoacylsRNA was purified by the method described by von Khrenstein and Lipmann.‘ The specific radioactivity of the C-aminoacyl-sRNA varied from 800 to 36,000 counts/min/ng sRNA. The optical density of img of sSRNA in H2O at 260 my was assumed to be 24.4 The RNAase-digested aminoacylsRNA described in Table 2 was prepared by incubating Cl4- phenylalanine-sRNA with L yg crystalline RNAase (Worthington Biochemical Corporation) per ml at 37° for 1 hr. The RNAase was removed by 5 consecutive phenol extractions using equal volumes of phenol. The aqueous phase was then dialyzed overnight against 1,000 volumes of HO. The alkali-degraded C'-phenylalanine-sRNA described in Table 2 was prepared by incubating aminoacyl-sRNA in 0.3 47 KOH at 35° for 18 hr. The solution was then neutralized and was dialyzed against 1,000 volumes of H.O. Also, C'-phenylalanine-sRNA was incubated in 0.4 M glycine buffers, pH 11.0 at 37° for Lhr. The solution was then dialyzed against 1,000 volumes of H.0. Radioactive protein precipitates were washed and counted as before? by a modification of the method of Siekevitz.?’ Protein concentrations were determined by a micro modification of the method of Lowry. All assays reported in this paper were performed in duplicate. Materials —U-C''-L-phenylalanine was obtained from the Nuclear-Chicago Company and had a specific radioactivity of 5-10 mC/mmole. The purified transfer enzyme (after DEAE column chromatography, purified about 10-fold’) and some C1phenylalanine-sRNA used were the gener- ous gift of Daniel Nathans and Fritz Lipmann. Polynucleotide phosphorylase was used to synthesize poly U. The molecular weight of the poly U was between 30,000 and 50,000. Results-—Components of reaction mixtures are given in Table 1. The data pre- sented in Table 1, Experiment 1, demonstrate that C'-phenylalanine was incor- porated into protein only when poly U was added to the reaction mixture. When 2 ymoles of unlabeled, C'’-phenylalanine were added to a reaction mixture contain- ing 0.019 umoles of C!*-phenylalanine, the incorporation of C!phenylalanine into protein was markedly reduced as was expected, due to dilution of the tracer. C'-phenylalanine-sRNA was prepared by incubating sRNA with C'phenyl- alanine and nonpurified phenylalanine activating enzyme present in 100,000 X g supernatant solutions. These extracts catalyzed a phenylalanine-dependent exchange of labeled pyrophosphate into ATP. Other amino acid-activating en- zymes, stimulating a similar exchange, were also present. In Experiment 2, the tracer present in reaction mixtures was C!~phenylalanine- sRNA in place of free C'-phenylalanine. ATP, CTP, and UTP were omitted. In the absence of poly U, little C'phenylalanine was incoporated into protein. However, in the presence of poly U, approximately 60 per cent of the C'4-phenyl- 106 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES Proc. N. A.S. TABLE 1 Errscr or C12-PHENYLALANINE UPON C!PHENYLALANINE INCORPORATION INTO PROTEIN Experiment No. C1t-tracer Addition Counts/min 1 C'_L-Phenylalanine None 1,430 — Polyuridylie acid 68 + 2 »moles C!*-L-phenylalanine 93 — ATP, PEP, and PEP kinase 62 None, zero time AQ 2 44L-Phenylalanine-sRNA None 683 — Polyuridylic acid 9 + 2 ymoles C*-L-phenylalanine 603 — GTP, PEP, and PEP kinase 10 None, zero time 6 The reaction mixtures for Experiment 1 contained the following in wmoles/ml: 100 Tris (hydroxymethyl) - aminomethane, pH 7.8; 10 magnesium acetate; 50 KCl; 6 mercaptoethanol; 1 ATP; 5 phosphoenolpyruvate, K salt; 2 wg phosphoenolpyruvate kinase, crystalline; 0.03 each of GTP, CTP, and UTP; 10 ug polyuridylic acid: 0.019 wmoles of Cl4-L-phenylalanine, ~75,000 counts/min; and 0.46 and 1.04 mg of ribosomal and 100,000 x g supernatant solution protein, respectively, The reaction mixtures for Experiment 2 contained the following in zmoles/ml; 100 Tris (hydroxymethyl)- aminomethane, pH 7.8; 10 magnesium acetate; 50 KCl; 6 mercaptoethanol; 0.3 GTP; 5 phosphoenolpyruvate, K salt; 2 ng phosphoenolpyruvate kinase, crystalline; 10 wg polynridylic acid; 0.45 mg C1-phenylalanine-sRNA, ~1100 counts/min; and 0.46 and 1,04 mg of ribosomal and 100,000 X g supernatant solution protein, respectively, Total volume was 0.5 ml. Samples were incubated at 35° for 10 min, were deproteinized with 10 per cent tri- chloroacetic acid. alanine was transferred from sRNA to protein. The transfer required GTP and a GTP-generating system; however, with this dialyzed but unpurified system, ATP and an ATP-generating system was 70 per cent as effective as GTP in facilitating transfer. Addition of 2 wmoles of unlabeled C!*-L-phenylalanine did not decrease appreciably the transfer of C'-phenylalanine from sRNA to ribosomal protein, in contrast to the data of Experiment 1. These results demonstrate that C'-phenyl- alanine can be transferred undiluted from sR.NA to protein in the presence of a large pool of unlabeled, free phenylalanine and that poly U directs the transfer. Tn Figure i, the quantity of phenylalanine transferred from sRNA into ribosomal   i rs rs es es     tPOLY U COUNTS /MINUTE x 3   MINUTES Fic. 1.-Stimulation of C-pnenylalanine transfer from sRNA to protein by polyuridylic acid ©.5 ug polyuridylic acid added; a without polyuridylic acid. The components of the reaction mixtures are given in Table 1, Experiment 2. 0.17 mg of C'™phenylalanine-sRNA. 400 counts/ min, 0.46 mg ribosomal protein and 1.04 mg 100,000 X g supernatant fraction protein were added to each reaction mixture. protein is plotted against time in minutes. In the absence of poly U, little C! phenylalanine was incorporated. In the presence of poly U, C!-phenylalanine was transferred rapidly from sRNA to protein. Almost all of the C'phenylalanine was transferred during the first: five minutes of incubation. Some control experiments are presented in Table 2 demonstrating that C!+- Vou. 48, 1962 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES 107 TABLE 2 AminoacyL, RNA Controu EXPERIMENTS Additions Counts/min C1+-phenylalanine-sRNA 704 C}*phenylalanine-sRNA treated with RN Aase* 2 14_phenylalanine-sRNA treated with 0.3 @ KOHt 9 C'-phenylalanine incubated at pH 11f 18 None, Zero time 4 The composition of the reaction mixtures is presented in Table i, Experiment 2. 1.04 mg protein in 100,000 X g supernatant solution, 0.46 mg ribosomal protein, and 0.45 mg Cu-phenylalanine sRNA containing ~1100 counts/min (before digestion) were added to each reaction mixture. *sRNA preparations were deproteinized by phenol extraction after RNAase treat- ment as specified in the Methods section. { Alkali treatment of C'*-phenylalanine-sRNA and incubation at pH 11 are described in the Metheds section. phenylalanine-sRNA has properties similar to those reported for aminoacyl-RNA. Treatment of C'*-phenylalanine-sRNA with RNAase or with 0.3 VN KOH destroyed its activity. Aminoacyl-sRNA is hydrolyzed to free amino acids and sRNA at pH 10.° Incubation of C'-phenyl-alanine-sRNA at pH 11 resulted in the hydrol- ysis of phenylalanine from sRNA as expected. In addition, our preparations of C'-phenyl-alanine-sRNA had a sedimentation value of 4.6 as determined by sucrose density-gradient centrifugation.\" The data of Table 3 demonstrate that both 100,000 X g supernatant fractions TABLE 3 REQUIREMENT FoR 100,000 < g SUPERNATANT SOLUTION AND RrposoMEs Additions Counts/min None 341 — 100,000 X g supernatant solution 14 — Ribosomes 5 None, zero time 7 The components of the reaction mixtures are presented in Table 1, Ex- periment 2. 0.46 and 1.04 mg protein were present in the ribosome and 105,000 X_g supernatant fractions, respectively. 1.0 meg of C!&phenyl- alanine-sRNA, ~800 counts/min, were added to each sample. and ribosomes were required for transfer of C'*-phenylalanine from sRNA to protein. Since the transfer enzyme is found in 100,000 X g supernatant solutions, it seemed likely that the requirement for this fraction could be replaced by purified transfer enzyme. The data of Table 4, Experiment 1, demonstrate that transfer enzyme TABLE 4 REPLACEMENT OF 100,000 X g SupERNATANT FRACTION wiTH TRANSFER ENZYME Experiment No. Addition Counts/min 1 None 404 ~— Transfer enzyme 10 — Polyuridylic acid 11 None, zero time 6 2 + GTP, + PEP, + PEP Kinase 566 ~ GTP, + PEP, + PEP Kinase 17 — GTP, + ATP, + PEP, + PEP Kinase 68 + GTP, + ATP, + PEP, + PEP Kinase 271 + GTP, — PEP, — PEP Kinase 17 + GTP, + ATP, — PEP, — PEP Kinase 13 + GTP, + PEP, + PEP Kinase, Zero Time 3 Components of the reaction mixtures are presented in Table 1, Experiment 2. 100,000 X g super- natant solution was omitted. 2.0 «moles of ATP/ml of reaction mixture were present where specified. 0.46 mg of ribosomal protein were present and 0.095 mg transfer enzyme protein were present except where specified. 0.30 mg C14phenylalanine-sRNA, ~800 counts/min, were added to each sample in Experiment 1 and 0.05 mg, ~1300 counts/min, were added to each sample in Experiment 2. In Ex- periment 2, for simplicity, the presence or absence of GTP, ATP, PEP, and PEP kinase are noted. 108 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES Pnroc. N. A.S. could replace 100,000 X g supernatant solution. The data of Experiment 2 show that GTP was necessary for the transfer, and that, in this purified system, ATP could not replace GTP effectively. The data of Tables 1, 3, and 4 show that C'- phenylalanine transfer from sRNA to protein required ribosomes, transfer enzyme, poly U, and GTP and a GTP-generating system. Discussion.—The data presented in this communication demonstrate that amino- acyl-sRNA is an intermediate in phenylalanine incoporation into protein mediated by poly U. Ina previous communication, we showed that the protein synthesized had unusual characteristics similar to authentic polyphenylalanine.? The initial steps in polyphenylalanine synthesis appear to be: phenylalanine- activating enzyme   L-phenylalanine + ATP AMP-phenylalanine + P-P. (1) phenylalanine- activating enzyme     AMP-phenylalanine + sRNA phenylalanine-sRNA + AMP. (2)   Poly U ribosomes transfer enzyme GTP > 2 9 Phenylalanine-sRNA — —» > polyphenylalanine + sRNA. (3) The detailed mechanisms of the steps involved in reaction (3) are under investiga- tion. It should be noted that our data do not preclude the possibility of alternative routes of synthesis of polyphenylalanine. When poly A and poly U are mixed, doubly- and triply-stranded RNA is formed (U-A and U-U-A).!2 13) We have shown previously that poly U in the doubly- and triply-stranded state was completely inactive as template RNA.2 Further experiments have corroborated and extended these findings and will be published in a separate communication. These data strongly suggest that the portion of the RNA molecule which functions as a template for protein synthesis is single-stranded. Simple predictions may be made concerning the primary and secondary structures of the hypothetical “template-recognition portion” of phenylalanine-sRNA. Since a sequence of one or more uridylic acid residues in poly U is the code for phenylalanine in this system, it is probable that phenylalanine-sRNA contains a complementary sequence of one or more adenylic acid residues which base-pair with the template. It is also probable that the portion of sRNA recognizing the template is single-stranded. The genetic code may not be universal ; lt may differ from species to species. Since sRNA may be a cofactor which functions as an “adaptor” carrying an amino acid to its proper place on template RNA, a variant sRNA base-pairing with a different code letter of template RNA would substitute one amino acid for another during protein synthesis.‘ Although E. coli sRNA can be used for the cell-free synthesis of rabbit hemoglobin,*: 4 it is possible that in species other than £. cold, poly U may be either meaningless or may serve as a template for a different amino acid.t Changes in the code could occur at different stages in the translation of information from DNA to the finished protein, for example, at the level of the DNA or RNA templates, at the level of sRNA, or at the level of amino acid—activating Vou. 48, 1962 BIOCHEMISTRY: NIRENBERG, MATTHAEI, AND JONES 109 enzymes. We arc using the poly U system to determine whether the code for phenylalanine is the same in different species. Summary. —Phenylalanine-soluble RNA was shown to be an intermediate in the cell-free synthesis of polyphenylalanine directed by a synthetic template RNA, polyuridylic acid. We wish to thank Mrs. Linda Greenhouse for her excellent help in performing some of the analyses. * NATO postdoctgral fellow. } The following abbreviations are used: Polyuridylic acid, poly U; polycytidylie acid, poly C; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; sRNA, soluble ribonucleic acid; RN Aase, ribonuclease; ATP, adenosine triphosphate; UTP, uridine triphosphate; CTP, cytidine triphos- phate; and GTP, guanosine triphosphate; PEP, phosphoenolpyruvate; and PEP kinase, phos- phoenolpyruvate kinase. ¢ That this may be the case is suggested by preliminary experiments performed in collaboration with Harry Gelboin showing that poly U does not stimulate incorporation of C!phenylalanine in a rat liver amino acid—incorporating system. Similar results have been obtained by S. Ochoa (personal communication). Nirenberg, M. W., and J. H. Matthaei, V International Congress of Biochemistry, Moscow, August 1961. 2 Nirenberg, M. W., and J. H. Matthaei, these ProcEepines, 47, 1588 (1961). 5 Beljanski, M., Biochim. et Biophys. Acta, 41, 104 (1960). ‘ Khrenstein, G. von, and F. Lipmann, these Procerpinas, 47, 941 (1961). 5 Aronson, A. I, E. T. Bolton, R. J. Britten, D. B. Cowie, J. D. Ducrksen, B. J. MeCarthy, Ix. MeQuillen, and R. B. Roberts, Carnegie Institute of Washington, Fearbook (1959-1960), p. 229. § Nisman, B., and H. Fukuhara, C. &. Acad. Sci. (Paris), 248, 2036 (1959). 7 Siekevitz, P., J. Biol. Chem., 195, 549 (1952). § Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, zbid., 193, 265 (1951). ° Nathans, D., and F. Lipmann, these Procenpinas, 47, 497 (1961). 1 Hecht, L. I, M. LL. Stephenson, and P. C. Zamecnik, ibid., 45, 505 (1959). 1 We thank Robert Martin for performing these determinations for us. 2 Rich, A., and D. R. Davies, J. Am. Chem. Soc., 78, 3548 (1956). 13 Felsenfeld, G., and Rich, A., Biochim. et Biophys. Acta, 26, 457 (1957). 1 Benzer, 8., and B. Weisblum, these ProceEpinGs, 47, 1149 (1961).", "Nirenberg, Marshall W. ; Jones, Oliver W., Jr ; Matthaei, Heinrich", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3ikc~y9pg~fm6w", "00000000-0000-0000-F365-9F3D91EE4699", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "The Effect of Secondary Structure on the Template Activity of Polyribonucleotides", "101584910X97", null, "1963", "March 1963", "One of the key discoveries in early code translation efforts is discussed in this article--that single-stranded RNA, but not double or triple stranded RNA, serves as a template for protein synthesis.  This article discusses the possible functional significance of RNA's secondary structure in the coding mechanism.", "Articles", "Nucleotides,RNA, Messenger ; Amino Acids", "Translating the Code of Life and the Nobel Prize, 1962-1968", "8", "pages", "Text", "English", "Reproduced with permission of Marshall W. Nirenberg.", "Copyright may apply", null, null, "Reprinted from the Proceepines or THE NATIONAL ACADEMY OF SCIENCES Vol. 49, No. 3, pp. 392-399. March, 1963. THE EFFECT OF SECONDARY STRUCTURE ON THE TEMPLATE ACTIVITY OF POLYRIBONUCLEOTIDES By Maxine F. Sincer, OLIVER W. Jones, AND MARSHALL W. NIRENBERG NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES AND NATIONAL HEART INSTITUTE, BETHESDA, MARYLAND Communicated by Robert J, Huebner, January 16, 1963 Current experiments on the synthesis of polynucleotides and proteins seem to us to emphasize the functional importance of polynucleotide secondary structure. Thus, for example, the highly ordered double-helical structure of DNA! readily leads to ideas concerning DNA replication.2:? Although our notions concerning the Vou. 49, 1963 BIOCHEMISTRY: SINGER ET Al. 393 secondary structure of the various types of RNA are much less precise, it does appear that RNA molecules are primarily single stranded and contain varying degrees of helical content.4:® Recently, investigators from two different labora- tories have proposed similar helical structures for transfer-RNA® 7 and both groups have discussed the functional significance of the suggested configurations. It is therefore of interest to consider the secondary structure of template RNA. Pre- vious reports from this laboratory’: * described the template RNA dependent in- corporation of amino acids into proteins in a stable cell-free system from LE. colt. Using this system and randomly mixed polyribonucleotides composed of various combinations of the four common ribonucleotides, nucleotide codewords corre- sponding to almost all of the protein amino acids have been determined.!—\" Observations made with this system suggested that the secondary structure of poly- mers influenced template efficiency. For example, the ability of poly U to direct polyphenylalanine synthesis is lost when poly A-poly U double or triple helices are formed.* The present report describes certain physical properties of a series of poly UG prep- arations as well as the efficiency of these polymers in directing amino acid incor- poration in the cell-free system. The data indicate that secondary structure in a polyribonucleotide limits its efficiency as a template. his finding is discussed and a specific functional role for polynucleotide secondary structure in the coding mechanism is proposed. A preliminary account of some of these data has been published. Materials and Methods —The procedures for the synthesis of polymers, determination of base ratios in the polymers, and measurement of amino acid incorporation have been described pre- viously.®, +4 The UG polymers are isolated by a modified procedure!’ designed to concentrate the longer chain length polymer and eliminate some of the shorter chain material. At the end of the polymerization the reaction mixture is deproteinized by the method of Sevag.7 The aqueous solution of polymer is made 2 Af in KCl by addition of an appropriate amount of solid KCl. Polymer is precipitated by the addition of 0.2 volume of cold absolute alcohol, and collected by centrifugation. The precipitate is dissolved in a small amount of water. In several cases the polymers did not dissolve readily in water unless a small amount of EDTA (final concentration about 5 mM) was added. Precipitation with KC] and alcohol is repeated two more times. The final precipitate is washed successively with 80%, 95%, and 100% alcohol and finally with ether and dried over paraflin. Polymers were subsequently dissolved and dialyzed against, distilled water before use. The phosphorolysis of the polymers by polynucleotide phosphorylase was determined by meas- uring the formation of P-labeled nucleoside diphosphate in the presence of P;32. The procedure referred to as Assay A by Singer and Guss!8 was used: the pertinent polymer was substituted for the poly A of that method. For the phosphorolysis studies, Micrococcus lysodetkticus polynucleo- tide phosphorylase, purified approximately 250-fold (Fraction VIII‘) was used. Measurements of the temperature dependence of the spectra of polymers (‘‘melting curves’) were carried out either in a Cary recording, model 14M, or Beckman, model DU, spectropho- tometer. The Cary instrument was equipped with a thermostatted cell holder and the temperature was measured inside the scaled quartz cuvette by means of a hypodermic needle type thermistor. The Beckman instrument was equipped with thermospacers and the temperature was estimated in a water-filled, unsealed, blank cuvette. All solutions were gassed with helium just before filling the cuvettes and the cuvettes were sealed with a coating of General Electrie Company RTV-60, silicone rubber compound.” The chain lengths of polymers were determined by measuring the ratio of total organic phos- phate to phosphate removable by £. cold alkaline phosphatase (Worthington Biochemical Cor- poration). The procedure outlined by Heppel and co-workers?! was followed. Sedimentation velocity studies were performed at 56,100 rpm and 20°C using the Spinco 394 BIOCHEMISTRY: SINGER ET AL. Proc. N. A.S. Model E Ultracentrifuge equipped with UV absorption optics. The solutions contained ap- proximately 0.03 mg of polymer per ml of 0.05 M cacodylate and were 0.1 M in NaCl. For measurements at neutral pH the cacodylate served as a buffer at pH 7.2; for alkaline measure- ments the solutions were made 0.01 M in KOH (pH 12). Tracings were obtained from photo- graphic images of the cell using the Joyce-Loeble Recording Microdensitometer. The sedimenta- tion coefficient was calculated®? from the rate of change of position of the 50% point of the bound- ary. Optical rotation was measured on a Rudolph Polarimeter using the mercury line at 365 my and a 1 decimeter light path. Results —Dependence of amino acid incorporation on the base ratio in poly UG: Table 1 shows the incorporation of phenylalanine, valine, leucine, and tryptophan into protein, measured with a series of poly UG preparations of increasing G con- tent. The relative amounts of the four amino acids incorporated with any partic- ular polymer are clearly related to the proportion of U and G. The data show that efficient incorporation of tryptophan requires a greater proportion of G in the polymer than is necessary for incorporation of valine or leucine. These data, there- fore, confirm the earlier assignment of codewords UUG, UUG, and UGG to valine, leucine, and tryptophan, respectively.!!: 12. 14 It is evident that the efficiency of a polymer as template RNA (Table 1) for any of the amino acids shown decreases sharply when the U/G ratio becomes less than 1. For example, the incorporation of phenylalanine with the polymer of U/G ratio 6.7/1 is about 60-fold greater than that obtained with the polymer of U/G ratio, 0.58/1. Although the decreased ability of the latter polymer to direct phenylala- nine incorporation is expected, the lack of activity in coding for leucine, valine, and tryptophan is surprising. In an attempt to understand this observation various properties of the polymers were investigated and the results of these studies are presented below. Chain length of polymers: Fach of the polymers shown in Table 1 had a chain length in excess of 300 nucleotide units but probably not greater than 500 units. Because of the large amounts of polymer required for the chain length determina- tion, more accurate measurements were not made. Sedimentation characteristics of polymers: The scdimentation coefficients of the polymers described in Table 1 are given in Table 2. At pH 7.2 the S» of the poly- TABLE 1 STIMULATION OF C4 Amino Acip INCORPORATION BY PoLty UG Polymer base ratio U/G)* 6.7/1 3.3/1 1.6/1 0.58/1 Nucleoside diphosphate Control minus ratio (U/G) 8/1 5/1 3/1 1/1 polynucleotide Incorporation above Controlt uumoles C4 amino acid Phenylalanine 901 1708 1067 15 45 Valine 287 1086 1050 61 13 Leucine 267 834 856 35 64 Tryptophan 38 210 276 10 60 * Base ratio of polymer determined as previously described.14 Ratio of UDP to GDP used in polymer synthesis. Figures represent incorporation of C'4 amino acid above the basal incorporation obtained in the absence of added polymer. Basal incorporation is given in the last column. Reaction mixtures (0.5 ml) contained 0.1 M Tris buffer, pH 7.8, 0.01 M magnesium acetate; 0.05 M KCl; 6 X 10-4 M B-mercaptoethanol; 1 X 10-3 M ATP; 5 X 1071 M potassium phosphoenolpyruvate; 10 yg crystal- line phosphoenolpyruvate kinase (CalBiochem); 2 % 10-4 M of each of 19 L-amino acids lacking the C14 amino acid; 0.8 X 10-¢ M C14 amino acid; 5 wg of polynucleotide; and preincubated, dialyzed S-30 £. cold extract.9 Each assay was performed in duplicate. The specific radioactivities of the amino acids varied 2-6 millicuries per millimole. All reaction mixtures were incubated at 37° for 90 min. These incorporation data therefore represent total incorporation of C'4-amino acids into protein rather than rate of incorporation. VoL. 49, 1963 BIOCHEMISTRY: SINGER ET AL. 395   TABLE 2 SEDIMENTATION COEFFICIENTS OF Poty UG PReparaTIoNs Polymer base ratio Soo* (U/G) pH 7.2 pH 12 6.7/1 5.9 5.2 3.3/1 3.1 2.3 1.6/1 5.7 3.3 0.58/1 11.0 2.1 * Inspection of the densitometer tracings indicated relatively little or no breakdown of the polymers during sedimentation at pH 12. Nonsedimenting material amounted to ap- proximately 5%, at pH_7 and 12, with all the polymers except the last (U/G, 0.58/1), when it was about 20% at pH 12. Furthermore, in each case the sedimenting material was more homogeneous at pH 12 than at pH 7 and this was most striking with the last polymer (U/G, 0,58/1). mer containing 70 per cent G is considerably higher than the S.o values of the others. However, the decrease in S2) noted for all polymers at pH 12 is much greater for this high G containing polymer. Phosphorolysis of polymers by polynucleotide phosphorylase: The susceptibility of the poly UG preparations to phosphorolytic cleavage by M. lysodeikticus poly- nucleotide phosphorylase was studied and the results are presented in Table 3. TABLE 3 PxospyoRroiysis oF Poty UG sy PoLynucLEoTipE PHOSPHORYLASE Base Ratio Phosphorolysis Rate Phosphorolysis Ratet (U/G) (mumoles/15 min.) Rate with poly A poly U* Lee 6.4 6.7/1 22.6 3.2 3.3/1 25.9 3.7 1.6/1 11.6 1.7 0.58/1 1.1 0.16 * Experiment carried out at a separate time. t vad numbers in the last column represent the rate of phosphorolysis of the given polymer relative to that of poly A. The incubation mixtures contained approximately one zmole of polymer phosphate per ml (see section on Afa- tertals and Methods for details). The rate of phosphorolysis (estimated with a measurement at a single time when the reaction rate is known to be linear with homopolymers) depends on the relative con- tent of uracil and guanine. In particular, when the polymer has more guanine than uracil the rate falls off very sharply. This effect is not the result of the de- creasing uracil content since the members of an analogous series of poly UC prep- arations were all phosphorolyzed at similar rates. Thus, when the ratio of uracil to cytosine was 4/1, 3.3/1, 1.7/1, and 0.6/1, the rates of phosphorolysis relative to poly A were 3.3, 4.0, 3.7, and 2.2, respectively. The slow phosphorolysis of poly UG containing more guanine than uracil appears to result from the high guanine content. Effect of temperature on the ultraviolet absorption spectra of poly UG preparations: Thomas** and Doty and co-workers* * have discussed the use of hypochromicity as an indicator of secondary structure in polynucleotides. We have determined so-called “melting curves” for polymers described in Table 1. Polymers having ratios of uracil to guanine greater than 2/1 showed essentially no change in absorp- tion between 200 and 300 my between room temperature and 85°. With poly UG having a uracil to guanine ratio of 1.6 to 1, we observed a slow increase in the absorption at 250, 260, 270, and 280 my upon increasing the temperature from 22° to 82°; however, the total increase at 270 mu was only about 5 per cent (Fig. 1). 396 BIOCHEMISTRY: SINGER ET AL. Proc. N. A. 3.   ny & J AbSo7q Ot t/AbSy70 at ty 5 I     OO F-                 | I | { i 30 40 50 60 70 ~=80 90 TEMPERATURE (°C)   Fic. 1.—Changes in ultraviolet absorption of poly UG as a function of temperature. The ordinate is the ratio between the absorption at 270 mu at the indicated temperature and the absorption at 270 my at the first temperature measured. —@—, poly UG, U/G ratio equal to 0.58/1, in 0.1 M@ KCl, pH 7.8; —O— poly UG, U/G ratio equal to 0.58/1, in 0.01 M potassium phosphate, pH 7.0; —A—, poly UG, U/G ratio equal to 1.6/1 in 0.01 M potassium phosphate, pH 7.0. Under the same conditions (0.01 M phosphate buffer) poly UG having a uracil to guanine ratio of 0.58 to 1 gave a total increase in absorption at 270 my of 12 per cent on raising the temperature from 30° to 85° (Fig. 1). The data are given for 270 my since the hypochromicity of G containing structures appears to be optimal at that wavelength.2* Figure 1 shows two curves for poly UG (U/G, 0.58/1); one was obtained in 0.1 M KCl, pH 7.8, the other in 0.01 M phosphate buffer, pH 7.0. The curves are similar although the start in the increase in absorption is somewhat delayed in 0.01 M phosphate buffer, pH 7.0. This observation is consistent with the finding™ that the “T,,” of d-pGpGpGpG is lower in 0.2 M NaCl than in phos- phate buffer alone. The experiment in 0.1 47 KCl was carried out in the Cary in- strument and a temperature dependent shift in the wavelength of maximum ab- sorption from 255.5 mp to 253 my was also observed. This shift took place at about 90°. Optical rotation of poly UG preparations: As an independent measure of poly- nucleotide helical content‘ the optical rotations of several polymers shown in Table 1 were measured. Determinations were carried out at room temperature in 0.01 M potassium phosphate buffer, pH 7.0, using the mercury line at 365 my. The specific rotations of the polymers having U/G ratios of 6.7/1, 3.3/1, and 0.58/1, were +99, +74, and +399, respectively. Discussion.—Amino acid incorporation into protein: The relative amounts of phenylalanine, valine, leucine, and tryptophan directed into protein by the poly UG preparations (Table 1) is clearly related to the base ratio of the polymer and Vou. 49, 1963 BIOCHEMISTRY: SINGER ET AL. 397 these data provide strong support for earlier codeword assignments.!!: }2) 4 How- ever, several properties of the poly UG preparations used indicate that meaningful comparisons between theoretical frequencies of doublets or triplets and relative amino acid incorporations cannot be made. One such factor is the secondary struc- ture of the polymers and is discussed in detail below. Another factor is the pos- sibility that the polymers may be nonrandom. Since G is incorporated into poly- mer preferentially (Table 1) the ratio of UDP to GDP changes during polymer syn- thesis. Thus, the base ratio determined by analysis could represent the average base ratio rather than that of any particular polynucleotide region or molecule. Preferential incorporation of G into polymers has been noted previously,!*. and similar observations have been made by Bretscher and Grunberg-Manago,* using A. agilzs polynucleotide phosphorylase. The mechanism of this concentra- tion phenomenon is currently under investigation.”” Effect of secondary structure on the template activity of messenger RNA: Several lines of evidence presented above indicate that copolymers of U and G contain a high degree of secondary structure when the relative content of G is high, and this interpretation is consistent with recent reports on the secondary structure of poly G itself.°+ % 5~*° (1) Although the four UG preparations described have approxi- mately the same chain lengths the sedimentation coefficient of poly UG (0.58/1) at neutral pH is markedly higher than that of the others. This increase in § value may result from greater aggregation (due to hydrogen bonding) with increasing G content for, at pH 12, where the secondary structure of poly G collapses,” % all the polymers have similar sedimentation characteristics. (2) The sharp drop in phosphorolysis rate observed on going from a poly UG with a U/G ratio of 1.6/1 to one with a U/G ratio of 0.58/1 can also be explained by an increase in secondary structure. Ochoa*' and Grunberg-Manago® have shown that polyribonucleotides having ordered secondary structures are phosphorolyzed much less rapidly than polymers existing as random coils. Poly G, for example, is completely resistant to phosphorolysis by polynucleotide phosphorylase.!*. #2 (3) Only polymers contain- ing relatively large amounts of G (U/G, 1.6/1, and 0.58/1) showed any increase in ultraviolet absorption at raised temperatures, and the increase is greater the higher the G content. (4) The relatively high optical rotation of poly UG (U/G, 0.58/1) also indicates ordered secondary structure.! Thus, the first three polynucleotides listed in Table 1 contain only moderate amounts of secondary structure and direct amino acid into protein with high effi- ciency. The last polymer, which contains almost 70 per cent G, exhibits a great deal of secondary structure and a markedly decreased ability to serve as a template for protein synthesis. This correlation between template efficiency and secondary structure is consistent with our earlier observation that the ability of poly U to direct polyphenylalanine synthesis is lost when poly A-poly U helices are formed.® furthermore, we have found! that the extent of inhibition of polyphenylalanine synthesis by oligoadenylic acid preparations increases with the length of the oligo- nucleotide chain and can be correlated with the stability of oligoadenylic-poly U helices. Thus, single strandedness and lack of extensive intramolecular hydrogen bonding appear to be requisite for messenger RNA activity in this zn vitro system. Results obtained with natural RNA preparations are in accord with this conclusion. For 398 BIOCHEMISTRY: SINGER ET AL. Proc. N. A. 5. example, TMV-RNA directs protein synthesis with high efficiency® *4 in this sys- tem, compared to the efficiency of other RNA preparations. Melting curves for TMV-RNA* 5 indicate that a larger per cent of its secondary structure is destroyed at 37° than is destroyed with ribosomal or transfer RNA preparations. From an experimental point of view, caution should be used in handling template RNA to be used in in vtiro systems. In the course of experiments on the melting of polymers we noted that the change in absorption with temperature is not always readily reversible. Thus the storage (for example, freezing and thawing) of poly- mer solutions may cause significant changes in secondary structure and therefore in template efficiency. At present three factors influencing template activity are known—the size of the polymer chain,’ ®: *. 35 the nucleotide sequence, and the secondary structure. Previously we showed that poly U fractions of relatively high molecular weight are more active as templates than smaller molecules!* and more recent data indicate that high template activity corresponds to average chain lengths of 100 or greater.**. 3 The effeet of nucleotide sequence on template efficiency is difficult to assess. Recent data show that the code is very degenerate but it is not known whether all nucleotide sequences will code. Although nonsense sequences may exist, thus far none have been definitively demonstrated. As indicated in the pres- ent report, the secondary structure of a polynucleotide must also be considered in any evaluation of its ability to serve as template RNA in the in vtro system. The molecular basis for the genetic information specifying the beginning or end of a protein is unknown. Previously we suggested that nonsense nucleotide sequences might function in this manner. The results of the present study suggest an al- ternative explanation. Thus, a limited region of messenger RNA containing a high degree of secondary structure as a result of intramolecular hydrogen bonding (for ex- ample, G-G interaction) might also specify the beginning or end of a protein. Areas of marked secondary structure, which may be likened to knots in a rope, might separate the messenger molecules containing information for the synthesis of more than one protein into functional units. Mechanisms enabling certain pro- teins to be synthesized in close geographic proximity may be advantageous as, for example, in the synthesis of proteins containing different subunits** ® or for the syn- thesis of coordinately repressed enzymes. In a more general sense, we expect that future studies of the three distinct RNA fractions, transfer RNA, ribosomal RNA, and messenger RNA, will indicate functional significance for the specific conforma- tional aspects of their structures. With regard to messenger RNA, we would sug- gest that both the nucleotide sequence and secondary structure are relevant to the decipherment of the genetic code. Summary.—aA series of copolymers containing varying amounts of uracil and guanine have been studied as templates in a cell-free system for protein synthesis. Polymers containing relatively large proportions of guanine have low template ac- tivity. Evidence indicating that the guanine-rich polymers contain a high degree of ordered secondary structure is presented. It is suggested that the existence of such secondary structure accounts for the poor template activity of these polymers. The possible functional significance of RNA secondary structure in the coding mechanism is discussed. Vo. 49, 1963 BIOCHEMISTRY: SINGER ET AL. 399 It is a pleasure to thank Linda Greenhouse and Helen Maleady for valuable technical assist- ance. We would also like to thank Dr. Samuel Luborsky for his experimental and theoretical as- sistance with the sedimentation studies. * The following abbreviations are used: Poly U, polyuridylic acid; poly A, polyadenylie acid; poly C, polycytidylic acid; poly G, polyguanylic acid; poly UC, copolymer of uridylie and eytidylic acids; poly UG, copolymer of uridylic and guanylic acids; G, guanyliec acid; U, uridylic acid; A, adenylic acid; C, cytidylic acid; TMV, tobacco mosaic virus. All other abbreviations conform to those acceptable to the Journal of Biological Chemistry. 1 Watson, J. D., and F. H. C. Crick, Nature, 171, 737 (1953). ? Watson, J. D., and F. H. C. Crick, Nature, 171, 964 (1953). 5 Josse, J., A. D. Kaiser, and A. Kornberg, J. Biol. Chem., 236, 864 (1961). * Doty, P., H. Boedtker, J. R. Fresco, R. Haselkorn, and M. Litt, these Proceeprines, 45, 482 (1959). 5 Doty, P., H. Boedtker, J. R. Fresco, B. D. Hall, and R. Haselkorn, Ann. N. Y. Acad. Sci., 81, 693 (1959). § Spencer, M., W. Fuller, M. H. F. Wilkins, and G. L. Brown, Nature, 194, 1014 (1962). 7™McCully, K. §., and G. L. Cantoni, J. Mol. Biol., 5, 497 (1962). 8 Matthaei, J. H., and M. W. Nirenberg, these Procerpinas, 47, 1580 (1961). 9 Nirenberg, M. W., and J. H. Matthaei, these Procerprnas, 47, 1588 (1961). 0 Nirenberg, M. W., J. H. Matthaei, O. W. Jones, R. G. Martin, and S. H. Barondes, Fed. Proc. Symp. 1962, in press. 1 Speyer, J. F., P. Lengyel, C. Basilio, and S. Ochoa, these PRocrEDINGS, 48, 63 (1962). 1 Lengyel, P., J. F. Speyer, C. Basilio, and S. Ochoa, these ProcBEDINGS, 48, 282 (1962). 13 Speyer, J. F., P. Lengyel, C. Basilio, and 8. Ochoa, these ProcrEpines, 48, 441 (1962). 14 Matthaei, J. H., O. W. Jones, R. G. Martin, and M. W. Nirenberg, these PRocEEDINGs, 48, 666 (1962). 8 Jones, O. W., and M. W. Nirenberg, these Procerprinas, 48, 2115 (1962). 16 Heppel, L. A., unpublished procedure. \"Sevag, M. G., D. B. Lackman, and J. Smolens, J. Biol. Chem., 124, 425 (1938). 18 Singer, M. F., and J. K. Guss, J. Biol. Chem., 237, 182 (1962). 19 Singer, M. F., and B. M. O’Brien, J. Biol. Chem., 238, 328 (1963). »” Felsenfeld, G., and G. Sandeen, J. Mol. Biol., 5, 587 (1962). *\\\\ Heppel, L. A., D. R. Harkness, and R. J. Hilmoe, J. Biol. Chem., 237, 841 (1962). 22 The Svedberg and Kai O. Pederson, The Ultracentrifuge (Oxford: Clarendon Press), 1940. 23 Thomas, R., Biochim. et Biophys. Acta, 14, 231 (1954). 4 Gellert, M., M. N. Lipsett, and D. R. Davies, these ProcrEpines, 48, 2013 (1962). > Ralph, R. K., W. J. Connors, and H. G. Khorana, J. Am. Chem. Soc., 84, 2265 (1962). *6 Bretscher, M. S., and M. Grunberg-Manago, Nature, 195, 283 (1962). 27 Brenneman, F. N., and M. F. Singer, unpublished observations. 78 Fresco, J. R., personal communication. 29 Fresco, J. R., and Der-Fen Su, J. Biel. Chem., 237, PC3305 (1962). * Lipsett, M. N., and L. A. Heppel, J. Am. Chem. Soc., 85, 118 (1963). 31 Ochoa, 8., Arch. Biochem. Biophys., 69, 119 (1957). 32 Grunberg-Manago, M., J. Mol. Biol., 1, 240 (1959). 33 Heppel, L. A., H. A. Sober, O. W. Jones, and M. W. Nirenberg, unpublished data. #4 Tsugita, A., H. Fraenkel-Conrat, M. W. Nirenberg, and J. H. Matthaei, these ProcEEDINGS, 48, 846 (1962). 3 Martin, R. G., and B. N. Ames, these Proceeprnas, 48, 2171 (1962).  Ttano, H. A., S.J. Singer, and E. A. Robinson, in Ciba Foundation Symposium on Biochemistry of Human Genetics, ed. G. E. W. Wolstenholme and C. M. O’Connor (1959), p. 96. 7 Ttano, H. A., and E. A. Robinson, these ProceEpinas, 46, 1492 (1960).", "Singer, Maxine ; Nirenberg, Marshall W. ; Jones, Oliver W., Jr", null, "Proceedings of the National Academy of Sciences of the United States of America", "National Academy of Sciences (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9hqq-jqs5_uc4a", "00000000-0000-0000-14BC-04F32F36462F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Arthur J. Walker to Marshall W. Nirenberg and J. Heinrich Matthaei", "101584910X98", "101584910X99,101584910X100", "1962", "3 May 1962", "In this letter, Arthur Walker quotes a Long Island newspaper, stating, \"It is apparent that a considerable number of biochemical geneticists are looking ahead to the time when man can plan for himself, draft and carry on programs to improve the actual human being as to intelligence, physique and resistance to disease.\"  Walker suggests that any attempt to improve man's character should be pursued.  He requests Nirenberg's and Matthaei's reaction.", "Letters (correspondence)", "Genetic Code,Genetic Engineering", "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, "c/o Eric J. Wells, Esq. 64 Browning Avenue Boscombe - Bournemouth Hants. - England May 3, 1962 Doctor M. W. Nirenberg Doctor J. H. Matthaei National Institute cf Health Bethevce, Faemilend Dear Doctors; There has been some fairly recent publicity here about biochemical genetic research, and I note that your names were mentioned with those of Cambridge scientists in this respect. It seems to me that all engaged in this work are making an equally important contribution, and I am taking the liberty of submitting a copy of a letter that I wrote to Cambridge, together with reply. Some time earlier when at home on Long Island, New York, I read from a Long Island newspaper: \" It is apparent that a consid- erable number of biochemical geneticists are looking ahead to the time when man can plan for himself, draft and carry on programs to improve the actual human being as to intelligence, physique, and resistance to disease.\" Personally, I feel that every possible way to improve mans’ character needs urgent exploration and attention right now, if humanity is to survive. I am wondering what, perhaps, your reactions may be. Sincerely yours, Ctr <i Ladi beer Arthur J. Walker", "Walker, Arthur J.", null, null, null, "Matthaei, Heinrich ; Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rpud~83k6.xfhs", "00000000-0000-0000-A9B4-C28D022C244E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Letter from Francis Crick to Arthur J. Walker", "101584910X99", "101584910X100,101584910X98", "1962", "2 February 1962", "Crick thanks Walker for his letter and expresses both wishfulness that his work \"could be used to make people feel better and better\" but also doubt that \"there is a simple effect of environment on character\" as Walker suggests.  Crick anticipates insight in this area will come from study of the higher nervous system more than it will come from biochemical genetics.", "Letters (correspondence)", "Environment", "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Text", "English", "Reproduced with permission of Francis Crick.", "Copyright may apply", null, null, "2nd February, 1962. Dear Mr. Walker, Thank you for your letter. I wish that our work could be used to make people feel better and act better, but it will be many years before we are likely to be able to do this. I very much doubt if there a simple effect of environment on character, apart from the well-known effects of a hot climate. In any case I think that research on the higher nervous system is more likely to lead to scientific insight into man's behaviour than our work will. Thanking you for your good wishes. Yours sincerely, F. H. C. Crick", "Crick, Francis, 1916-2004", null, null, null, "Walker, Arthur J.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
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, [ "row-hsp3_jh9x-r2cb", "00000000-0000-0000-25B5-8797CFD5E70D", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg", "101584910X368", null, "2002", "2002", null, "Photographic prints, Portraits", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Public Domain", "Public domain", null, null, null, "National Institutes of Health (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bue4-chaq.nde3", "00000000-0000-0000-ED4F-E0EA94E938BF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg reading data in lab", "101584910X369", null, "1975", "[September or October 1975]", "This unpublished photograph was intended for publication in National Geographic Magazine.", "Photographic prints", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of John Neubauer.,copyright 1975 John Neubauer", "Copyright may apply", null, null, null, "Neubauer, John", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rbpi-6wqb.hqjw", "00000000-0000-0000-4B26-B0D4B206C97A", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "John Minna", "101584910X37", null, "1990", "[1990s?]", "Minna collaborated with Nirenberg on his neuroblastoma research throughout the 1970s.  As a leading therapeutic oncology researcher, Minna has since argued that understanding the functions of mutated genes, the pathways disrupted, and the expression \"signatures\" will provide a foundation for a translational (bench to bedside) research approach to human cancer and also help us understand fundamental aspects of cell regulation.", "Photographic prints", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yin2~2j6y_e2qv", "00000000-0000-0000-1E49-D04FD33C861E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg with test tubes", "101584910X370", null, "1990", "[1990s]", null, "Photographic prints", null, "From Neuroblastoma to Homeobox Genes, 1976-1992", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of Richard T. Nowitz.", "Copyright may apply", null, null, null, "Nowitz, Richard", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9qe5~e328.ubys", "00000000-0000-0000-8AA4-893576BCC313", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg in home office", "101584910X38", null, "1960", "[1960s?]", "This photograph was taken during the dramatic period of rapid code translation.", "Photographic prints", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cpbr.pjmh~u9k9", "00000000-0000-0000-F3F2-CCC2F08A0D40", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg, Norma Heaton, and others in lab", "101584910X39", null, "1960", "[1960s?]", "The two other individuals are F. Anderson and Fritz Rotman.  Heaton, Anderson, and Rotman were post-doctoral lab technicians and researchers who remained with Nirenberg's team at the National Heart, Lung, and Blood Institute at NIH for years.", "Photographic prints", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ia9a-6dfa.auay", "00000000-0000-0000-CE2B-9A045062164E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg with Kellogg, Sidney Pestka, and others", "101584910X40", null, "1960", "[1960s?]", "Photograph taken in an NIH office.", "Photographic prints", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5sg4_une6~8mh5", "00000000-0000-0000-D46A-1971ED8DF557", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg", "101584910X41", "101584910X6", "1968", "[ca.1968]", "Official portrait taken during the year of his celebrated Nobel Prize in 1968.", "Photographic prints, Portraits", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, "Guenveur, Carl", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-f6kr-6y7w_kend", "00000000-0000-0000-9171-432C9EACEC05", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg", "101584910X42", null, "1968", "[ca. 1968]", "One of many professional photographs of Marshall Nirenberg from the Nobel Prize era.", "Photographic prints, Portraits", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-im2t.hr5z~v7y4", "00000000-0000-0000-5543-F5F5C313D060", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg in his study at home", "101584910X43", null, "1968", "[ca. 1968]", "Private photograph of Nirenberg at home during the Nobel Prize era.", "Photographic prints", null, "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-iu3p~2n86.fqmh", "00000000-0000-0000-3716-DD383AA088F2", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg", "101584910X44", null, "1970", "[1970s?]", "Candid shot of Nirenberg from his neurobiological research period.", "Photographic prints", null, "Neuroblastoma Research, 1967-1976", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3bub_p2sb~f2in", "00000000-0000-0000-82A3-770FCF27E71F", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg with molecular models", "101584910X45", null, "1962", "[ca. 1962]", "Photograph taken during the period of the genetic code translation effort.", "Photographic prints", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hc7z_gqfj~nau9", "00000000-0000-0000-00B8-A784907D48A1", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg performing experiment", "101584910X46", null, "1962", "[ca. 1962]", "Photograph of experiment likely related to protein synthesis and genetic code translation.", "Photographic prints", null, "Transition to Neurobiology, 1965-1969", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the National Institutes of Health (U.S.).", "Copyright may apply", null, null, null, "MacVicar, N.,National Institutes of Health (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j463_r6sf-d4mf", "00000000-0000-0000-DD64-ABAF3C3F7E88", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg and Heinrich Matthaei", "101584910X47", null, "1961", "[ca. 1961]", "Heinrich Matthaei was Marshall Nirenberg's first postdoctoral researcher.  Their collaboration led to the now famous protein synthesis poly-U experiments and the first clue to the genetic code.", "Photographic prints", null, "Synthetic RNA and the Poly-U Experiments, 1959-1962", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the National Institutes of Health (U.S.).", "Copyright may apply", null, null, null, "MacVicar, N.,National Institutes of Health (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-iw5q.nn89~hyz8", "00000000-0000-0000-6905-8FD6167421C9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "UUU Are Great Marshall", "101584910X470", null, "1968", "[1968]", null, "Photographic prints", null, "Public Reactions to the Genetic Code, 1961-1968", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Marshall W. Nirenberg.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6eey_qgew~7s3y", "00000000-0000-0000-C44F-0AB54DBAA316", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Genetic code chart", "101584910X471", "101584910X475", "1965", "18 January 1965", "By 1966, Nirenberg announced that he had deciphered the 64 RNA codons for all 20 amino acids.  This chart from 1965 lists the correspondence between these codons and amino acids.. NOTE: Image obtained by scanning a negative of the genetic code chart.", "Charts (graphic documents), Negatives (photographic)", "Genetic Code", null, "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5rpe-m7ui-k3jm", "00000000-0000-0000-10C3-F75733EFB5B9", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Second and last summary of the genetic code", "101584910X472", "101584910X475", "1965", "[Late 1965 or early 1966]", "By 1966, Nirenberg announced that he had deciphered the 64 RNA codons for all 20 amino acids.  This chart lists the correspondence between these codons and amino acids.. This image was obtained by scanning a color copy of the genetic code chart.", "Charts (graphic documents), Laboratory notes", "Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-92gq_i6mj.rruu", "00000000-0000-0000-0F2D-0F95F2D86D22", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg and his lab staff", "101584910X473", null, "2000", "[After 2000]", null, "Photographic prints", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", null, "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-f2ju_qwbr_amux", "00000000-0000-0000-33AC-992724E53383", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "First summary of the genetic code", "101584910X475", "101584910X472", "1965", "18 January 1965", "By 1966, Nirenberg announced that he had deciphered the 64 RNA codons for all 20 amino acids. This chart from 1965 lists the correspondence between these codons and amino acids.", "Charts (graphic documents), Laboratory notes", "Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kq7e.6m4t-wtep", "00000000-0000-0000-0724-F1069A701A37", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Template used to compile data for the genetic code", "101584910X477", null, "1964", "[1964]", null, "Charts (graphic documents), Laboratory notes", "Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kbm2_czr4_uh6w", "00000000-0000-0000-A666-316EE88A2F9E", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Compilation of data contributing to the genetic code", "101584910X478", null, "1965", "[1965]", null, "Charts (graphic documents), Laboratory notes", "Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Heaton, Norma" ]
, [ "row-zvrp~428k_jwij", "00000000-0000-0000-14CB-2854920042CA", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Large working copy of genetic code chart (first half)", "101584910X479", null, "1965", "[1965]", null, "Charts (graphic documents), Laboratory notes", "Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Heaton, Norma" ]
, [ "row-g98c_uucb-uy8j", "00000000-0000-0000-1E96-E8191DE38924", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Marshall Nirenberg", "101584910X48", null, "1960", "[1960s]", "Portrait of Nirenberg before his rapid rise to scientific fame.", "Photographic prints, Portraits", "National Institutes of Health (U.S.)", "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the National Institutes of Health (U.S.).", "Copyright may apply", null, null, null, "National Institutes of Health (U.S.)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5xjr~7py7-2hwz", "00000000-0000-0000-1EB7-74F8058FA431", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Large working copy of genetic code chart (second half)", "101584910X480", null, "1965", "[1965]", null, "Charts (graphic documents), Laboratory notes", "Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Heaton, Norma" ]
, [ "row-7wm6-2dit~dqr4", "00000000-0000-0000-282F-D936258307EF", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Compilation of data contributing to the genetic code chart", "101584910X481", null, "1965", "[1965]", null, "Charts (graphic documents), Laboratory notes", "Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Heaton, Norma" ]
, [ "row-uav2_c8ui_cf5j", "00000000-0000-0000-9CF0-B9BE0373C955", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Data contributing to \"large working copy of code\" chart", "101584910X482", null, "1965", "1965", null, "Charts (graphic documents), Laboratory notes", "Genetic Code", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Heaton, Norma" ]
, [ "row-yik4_6qrx-46mg", "00000000-0000-0000-3D9E-ACB7852659B5", 0, 1750962675, null, 1750962675, null, "{ }", "The Marshall W. Nirenberg Papers", "101584910", "Experiments related to the genetic code examining longer polynucleotides", "101584910X483", null, "1965", "[1965]", null, "Charts (graphic documents), Laboratory notes", "Genetic Code,Polynucleotides", "Translating the Code of Life and the Nobel Prize, 1962-1968", "1", "pages", "Still Image", "English", "Public Domain", "Public domain", null, null, null, "Nirenberg, Marshall W.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Levenson, Darlene" ]
, [ "row-ge2i.5t4t_ip2j", "00000000-0000-0000-D3D6-813057179B6D", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Lee Frankel", "9918573882206676X98", null, "1919", "01 December 1919", "Dublin wrote to Frankel regarding tentative plans to do a large-scale survey of U.S. urban health departments.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "December 1, 1919.  Dr. Frankel, Third Vice President:     In reply to the attached memorandum, I would say that ne teabulaticn was made three years ago of the material received from the Superintendents. This was because the replies were not in form for tabulation but were rather descriptive end very leese in statement.          I would now suggest that «a similer letter /f go out, this time with a definite questionnaire attached, covering the fields of public health and sanitation for each , district sresa and that the Superintendents be required to / : complete this form at regular intervals, say, every six-merrthas” ~~ In this way, we would get a very useful fund of information concerning the local problems of public health throughout the country. .In many cases we could help local heslth officers with their difficulties. The information received would also have considerable business value to us.  / f -              I am attaching a form which should be sent out at the beginning of 1920, to be completed before the Convention date.  wm F7i- 7 //A of  if f (ff o 4] £6 A yy Se  ff wy a  a1 /3 3 Statistician.", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Frankel, Lee Kaufer, 1867-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hf2q-hf9w.3amc", "00000000-0000-0000-AB78-6260E701CF12", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Lee Frankel", "9918573882206676X99", null, "1919", "22 December 1919", null, "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "December 22, 1919     Dr, Frankel, Third Vice President,  In reply to your memorandum of December Sth, I strongly recommend that the schedule prepared by us be kept in preference te another and shorter questionnaire as you suggest. Our schedule was prepared with great care and covers only the essential facts of health administration, I am afraid that our Superintendents would not be able alone te fill out satisfactorily any questionnaire which would have any real value, My suggestion is to keep the questionnaire, to send it to Superintendents with a request that they get in touch with the health authorities and, working together, have the form completed,  ; This precedure would have the advantage that the technical part, te which you evidently objected, would be filled out by those who would be competent te do so, that the Superin- tendents would be interested in the procedure and would get in touch with the health officers, many of whom have not had an occasion to do so, Finally, such cooperation with the health officers would make the answer to the important last question very valuable,  If we succeed in putting this across, we will have the most complete infcrmation, with regard te public health, existing in the United States, and an important contribution could then be made on the present state of American public health administration and what it most needs, We are in an excellent position to carry out such an investigation and I cannot urge you too strongly to do it,  GSROESKRHS OR MR SRR RRR ero RS  «2 Statistician", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Frankel, Lee Kaufer, 1867-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-neme_rzbs_iiss", "00000000-0000-0000-13C3-12311F90CFAE", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Lee Frankel to Louis Dublin", "9918573882206676X100", null, "1920", "21 January 1920", "Frankel shared his thoughts regarding a survey of U.S. urban health department administration.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, ": Jan. 21st, 1920 DR. DUBLIN:  I have again carefully gone over your memorandum  of December 22nd and the schedule thereto attached, I am more than ever convinced that our Superintendents are not able to have this schedule filled out. They surely are not in a position to do  it themselves and I question very much whether they can obtain co- operetion of health officers,  It would seem to me that if this infomation is : desirable we had better communicate with health officers directly, Judging from the results which I obtained two years ago in the completion of the questionairye which I sent out at thet time, health officers are very responsive and we can count upon help from most of them. With proper follow-up letters we ought to be able to have practically all of them send us the desired data,  Will you rearrange the schedule so that it may be sent with a letter to health officials.", "Frankel, Lee K. (Lee Kaufer), 1867-1931", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tsf2-bkny~7nkx", "00000000-0000-0000-8E49-78D13DF0F2BB", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Lee Frankel", "9918573882206676X101", null, "1920", "24 April 1920", null, "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "ee :     Qe  f april 24, 1920  Dr. Frankel, Third Vice President,     In re: Survey of health service in American cities  : In accordance with the suggestion..contained inyour memorandum of January 21, we have revised our questionnaire so  that it may be sent to health officers rather than to our superin- tendents. As we now conceive it, our plan is to collect informa- tion from the two hundred and fifty larger American cities, where there is an attempt at health administration along the usual lines. Our form, if completed, will obtain for us a cross section of health service as now practiced in our larger cities. We, attempt- ed to cover all the more important activities. We have also asked the health officer to state his more urgent needs and his plans  for future expansions and also what the Company can do to cooperate with him,  The questionnaire is submitted for your criticism. I suggest alse that it be sent to a few of the more critical saniterians of the country, such as, Winslow, Chapin, Armstrong, McLaughlin, and Drake, to get their reaction to it. We want to know whether they think health officers will complete such a form. Armstrong was very favorable to the idea when I discussed it with him the other day, saying that the time was very opportune. The American Medical Association and The Red Cross, The National Tuberculosis, sre think- ing along these lines. If our questionnaire is success ul, we can give all these associations basic date for their own programmes.  He also urged the necessity of putting this through at the earliest possible date.  o1", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Frankel, Lee Kaufer, 1867-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xb55_mgek_npr9", "00000000-0000-0000-B9D8-624B4E2AEE7A", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Lee Frankel", "9918573882206676X102", null, "1920", "17 May 1920", null, "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "May 17, 1920  Dr. Frankel, Third Vice Presidents     ‘In re? Survey of Public Health Work in American Cities  You and I have long realized the utter lack of trustworthy information on the kind and amount of public health work that is being carried on in American cities. With the hope of obtaining this information, not only for our own purposes in the Company, but as a contribution to the public health movement, we planned to send out a questionnaire to health officers of cities which, when collected and tabulated, would give the necessary facts. We would thus have a cross-section of health service as now practiced in the lerger cities of the United States and Canada = about 250 of them with 25,000 population or over. We would also learn the more urgent needs of the health officers, their plans for future expense 08 and,  also, what the Company could do to cooperate with thems  With your general guidance, such a questionnaire was drawn up and submitted to five leading sanitarians, including Prof. CaHy As Winslow  of Yale, Dr. Donald B. Armstrong of Framingham, Dr. C. V. Chapin of  Providence, Dr. Wade H. Frost of Johns Hopkins and the Public Health Service, - and Dr. W. S. Rankin, President, American Pabits Health Association. There  is unanimous, agreement on the.part.of these. ve..mer is investigation is most timely. Everyone is enthusiastic over the “possibilities of the inquiry. But, ‘there is considerable doubt expressed as to our Ability to gather this information by the questionnaire method. Winslow is especially — skeptical. In order to make the answers worthwhile, the questions have to.  be specific and, because of the wide scope of the inquiry, the questionnaire must be lengthy and involve a great deal of labor on the part of the health officers. The result would be that only a small percentage of the forms would be sent ine        There is virtual agreement among our saeecepuedonte that the best procedure would be to have the actual inquiry work done in each state by a trained person working under the auspices and with the authority of the State — Board of Health. Local health officers would cooperate with a representative of the State Department where they might refuse us information. It is, theres  |) fore, suggested that the Company ask the Section on _on Public Health Administra-=  / tion of the American Public Health Association to 5 appoint. a special committee     to take charge of the work for us, the funds required for the actual canvassing to be furnished by the Company. I agree with 1 Winslow that $5,000           i would sée the work through in a satisfactory manner. i 5  z -soacuuead that the Executive be asked to appropriate $5, 000 for this purpose,  LV}.  : SASRSSOSASO DAG ASHE SED “2 2 3 : Statistician  —— 4 a", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Frankel, Lee Kaufer, 1867-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tsad.besb.ivwp", "00000000-0000-0000-D157-8993DCF081A8", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Lee Frankel to Haley Fiske", "9918573882206676X103", null, "1920", "20 May 1920", "Frankel wrote to Fiske, then president of the Metropolitan Life Insurance Company, asking him to appropriate funds to support a survey of U.S. urban health department administration.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "MR. HALEY FISKE, PRESIDENT.  2 oe The attached memorandum fron . Dr. Dublin was prepared by him at my  request. We have had this matter under consideration for some time. At first we had the hope that we might — obtain the desired information through Superintendents, but we soon realized  - that this was not feasible. The Questionnaire (herewith attached) is lengthy and should be accurately filled out if the data is to be of value.  Work of this kind should really be done by the United States Public Health Service. They have never attempted however, and there is no like- lihood, so far as I know, of their under- - taking it in the near future. The | suggestion of Professor Winslow that a  special Committee of The American Public  Health Association should be appointed to collect the information is a very good one. Such a Committee would volunteer its services end the fund © asked forwould be used to pay compilers of data in the respective communities.  The infomation when finally collected and tabulated would be of value to the Company, both from an Agency and from a health conser- vation standpoint. I approve of the recommendation that the Company make an appropriation to finance the work. If you agree you will pro- bably wish to refer the matter to the Meeting of the Welfare Committee  on May 25th.     Welfare Division, May 20th, 1920.", "Frankel, Lee K. (Lee Kaufer), 1867-1931", null, null, null, "Fiske, Haley, 1852-1929", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hk3g~t3jw~sx96", "00000000-0000-0000-1AF1-83775F09ABEC", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from W. S. Rankin to Lee Frankel", "9918573882206676X104", null, "1920", "04 June 1920", "Rankin, then president of the American Public Health Association, wrote to tell Frankel that the APHA had selected committee members to oversee the survey of U.S. urban health department administration that Frankel's company, Metropolitan Life Insurance, had agreed to fund.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "* EXECUTIVE COMMITTEE W.S. Rankin, M. D., Raleigh, N. C., President A. W. Hedrich, Boston, Massachusetts, Secretary G. H. Sumner, M. D., Des Moines, Iowa, Treasurer Peter H. Bryce, M. D., Ottawa, Ont. Chas. J. Hastings, M. D., Toronto, Ont. W. A. Evans, M. D., Chicago, Ill. A. J. McLaughlin, M. D., Washington, D. C. Lee K. Frankel, Ph. D., New York City M. P. Ravenel, M. D., Columbia, Mo.  VICE-PRESIDENTS  A. J. Douglas, M. D., Winnipeg, Man. S. L. Jepson, M. D., Charleston, W. Va. W. H. Robin, M. D., New Orleans, La.  BOARD OF DIRECTORS 1919-1920 C. J. McGurren, M. D., Devil’s Lake, N. D. John B. Anderson, M. D., Seattle, Wash. W. iH. Kellogg, M.D., Sacramento, Cal. B. L. Arms, M. D., Jacksonville, Florida P. M. Hall, M. D., Minneapolis, Minn. L. A. Galaretta, M. D., Havana, Cuba S. J. Crumbine, M. D., Topeka, Kan.  E. R. Kelley, M. D., Boston, Mass.  M. S. Fraser, M. D., Winnipeg, Man. M. M. Seymour, M. D., Regina, Sask.     A. J. McLaughlin, M. D., Washington, D. C. G. H. Sumner, M. D., Des Moines, Iowa Oscar Dowling, M. D., New Orleans, La.  J. T. Black, M. D., New London, Conn. F. L. Hoffman, LL. D., Newark, N. J.        THE AMERICAN ‘PUBLIC HEALTH ASSOCIA TION     W. S. RANKIN, M. D., PRESIDENT  STATE HEALTH OFFICER, RALEIGH,N.C.  June 4, 1920  Dr. Lee K. Frankel, Metropolitan Life Ins. Co., 1 Madison Avenue,  New York City.  Dear Dr. Frankel  ° e     Industrial Hygiene, J. W. Schereschewsky, M. D., Wash., D.C.     Haven Emerson, M. D., New York City M. P. Ravenel, M. D., Columbia, Mo. J. A. Kappelman, M. D., Canton, O. J. D. Robertson, M. D., Chicago, Ill. Henry F. Vaughan, D. P. H., Detroit, Mich. PAST PRESIDENTS ON DIRECTORATE Stephen Smith, M. D., New York City Henry B. Baker, M. D., Holland, Mich. Frederick Montizambert, M. D., Ottawa, Ont. Samuel Durgin, M. D., Millbrook, Massachusetts Eduardo Liceaga, M. D., Mexico City, Mex. Peter H. Bryce, M. D., Ottawa, Ontario Richard H. Lewis, M. D., Raleigh, N. C. Gardner T. Swarts, M. D., Providence, R. I. Charles O. Probst, M. D., Columbus, Ohio Robert M. Simpson, M. D., Winnipeg, Man. John N. Hurty, M. D., Indianapolis, Ind. Rudolph Hering, D. Sc., Montclair, N. J. William C. Woodward, M. D., Boston, Mass. William T. Sedgwick, D. Sc., Cambridge, Mass. John F. Anderson, M. D., New Brunswick, N. J. W. A. Evans, M. D., Chicago, Illinois Chas. J. Hastings, M. D., Toronto, Ont. Lee K. Frankel, Ph. D., New York City  SECTION REPRESENTATIVES ©. . Laboratory, G. W. McCoy, M. D., Washington, D.C. Vital Statistics, Louis I. Dublin, Ph. D., New York City Public Health Admin., T. D. Tuttle, M. D., Topeka, Kan. Sociological, Louis I. Harris, M. D., New York City Sanitary Engineering, E. D. Rich, Lansing, Michigan  Food and Drugs, Prof. James O. Jordan, Boston, Mass.           I wish to acknowledge with very deep appreciation  your letter of June end,  stating that the Metropalitan Life  Insurance Company is prepared to finance a study of the activ- ities of various health departments throughout the country  to the extent of a maximum of. $5,900, desire of your company that this study be made under the au- spices of the American Public Health Association, and throubh  @ special committee to be appointed by the President.  and that it is  the  In an-  ticipation of this action by your company I have been author- ized by the Executive Committee to appoint such a committee as you suggest in the event that the appropriation for the  aforesaid purpose be made.  I am notifying the Secretary of the Association of the appointment of the following committee:  Prof. C.-E. A. Winslow, School of Medicine, Yale University,  New Haven, Conn.  Dr. Chas. V. Chapin, Superintendent of Health, Providence, R. I,  Dr. Louis I, Dublin, Statistician, Metropolitan Life Ins. Co., New York City  Dr: W. 8. Frost,  School of Hygiene & Public Health, Johns Hopkins,  .-Baltimore, Md.  with the request that he notify this committee of their appoint- ment,  As President of the A. P. H. A. I wish to express the official thanks of the Association for the isterest 6f  lt _  Dr. Frankel - 2  the Metropolitan Life Insurance Company in this important study, and for their generous appropriation in making the study possible.  Very truly yours,  YESS rrnhen.  op President.  Copy ir. Hedrich.", "Rankin, Watson S. (Watson Smith), 1879-1970", null, null, null, "Frankel, Lee Kaufer, 1867-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-a7k7-6are_js4p", "00000000-0000-0000-3ABC-0B751284249F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Haley Fiske", "9918573882206676X105", null, "1921", "19 November 1921", "Dublin commented on the initial results of the public health department survey report funded by the Metropolitan Life Insurance Company, and noted other ways the collected data could be used.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "¢ ‘= i  ( enw > 4 : November 19 : 1921  President.  You may wish to have a bound copy of our report.  The practical uses to which this repert can be put are unlimited. I+ was well received on Thursdey at the meeting. But, the Committee and the Association will follow up the report in. many ways. A number of health officers have already asked us to get behind them in their local efforts to remedy the situations in their cities. They have waited for the report and now know in what respects their city is deficient as compared with the best practice in other cities. They can use these facts in connection with their appeals for appropriations, etc. The fuller report which will probably run into three or four hundred pages, is well on the way, and we shall finish during the next four or five months. The staff at New Haven and at Johns Hopkins will be put to work on it without any expense to us, and we will, in that way, work out  very definite standards as to what the most effective health depert-|  ment should be like and what it should cost for each group of items.  You are right in your statement that we cannot get any- where without well paid full-time health officers. We are placing great emphasis on that, but the difficulty is that there are not enough well-trained health officers to go around. The schools of Johns Hopkins, New Haven, and, more recently, the one established by the Rockefeller Foundation at Harvard are trying to do this on a large scale.  On every hand, I heard praise for the Company and grati- tude to you personally for having made this Committee's work pos- gible through the generous contribution last year.  S©SGOSCSCOCGCEGCOSOHSCBS  #2 Statistician", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Fiske, Haley, 1852-1929", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yxdh-jejh-mtcg", "00000000-0000-0000-A3FB-BF5A91857774", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Lee Frankel to Haley Fiske", "9918573882206676X106", null, "1922", "18 February 1922", "Frankel wrote to Fiske suggesting that data from a recent public health department survey funded by Metropolitan Life could be used for a campaign to educate communities about the need for permanent, well-funded public health departments.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "THE PRESIDENT: . =  I spoke to you some time ago about my plan to use the data which has been prepared by the Committee on Municipal Health Department Practice of the American Public Health Association, as the basis of an intensive campaign to be conducted by the Company in various cities in co-operation with the respective health officers to im- prove-the status of health departments. This campaign, as i stated to you, is to educate the community generally through Chambers of Commerce and other civic bodies regarding the es~ sential needs, financial and otherwise, of health departments in the hope that the city officials will see the necessity of making adequate appropriations to the health department So as to place it on a foundation which will enable it to do the very best type of worke  The preparation of the material obtained by the Committee was, as you will recall, financed by the Company, an = appropriation of #5000 having been made for this purpose. The complete report is now in preparation, detailing the facts’ pub=  lished in the preliminary report, copy of which is attached herewith. : : ;  For our purpose, however, it is necessary to re- arrange this data. It will require a man thoroughly acquainted with health administration to do this shecessfully. Dre Dublin and I have discussed it and are of the impression that the best . man available for this purpose is Drs Haven Emerson, formerly | Health Commissioner of New York City. I attach herewith a memo« randum from Dre Dublin. He has spoken to Emerson and we believe- his services can be obtained for $2500. The work will require from three to six months.  Will you approve of Drs Emerson's employment on this basis?         February 18, 1922. , Third Vice-President.  : al : 7 | = cern | 2 - 2 =  t } i Se : - q : ee d 5  (4 ;", "Frankel, Lee K. (Lee Kaufer), 1867-1931", null, null, null, "Fiske, Haley, 1852-1929", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-net5~vi65-xmxa", "00000000-0000-0000-E54B-5B8AE82BACA7", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Wade H. Frost", "9918573882206676X107", null, "1922", "24 February 1922", "Dublin wrote to Frost regarding ongoing work on summaries of individual city health department statuses, from the survey conducted in 1920, noting that Metropolitan Life Insurance Company would agree to fund the work.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "an E4 - | ts et caus      Em = Vf rhewe- 4, 4 PS WL a BO 7 eee wes 4 ne”  February  24, 1922 : £4 : sro £ me *  Dr. Sade H. Frost,  Johns Hopkins University, 310-312 West Monument Street, Baltimore, Maryland.  My dear Dr. Frost:  You will recall that it was a part of the plan of the Committee to prepere a third section of the report which would consist of & series of summaries covering the important points in the practice of each one of the 83 cities studied. Because of the lack of funds and the inability of the individual members to further this project, no progress on this pert of the - report hes been made. :  Hore recently, Dr. Frankel has raised the question whether this was not after all, the most important part of the Committee's work. He ‘i. especially interested from the point of view of the Company in utilizing in a practical way the findings of the Committee on conditions in the individuel cities. The Company would look with approval and help te support an active propaganda in those cities where it would be possible to do so to make goed — the deficiencies disclosed by the report.  i think the Committee could obtain financial support from the Company for the completion of this third section of the report. In fact, it would be possible to employ Dr. Haven Emerson to complete this work during the next three months. The Company is ready to finance this work, provided the findings were freely made available to it in order to carry out the proe motion work in the individual cities in which i¢ may be interested. Will you be good enough to let me know at your earliest convenience whether you | favor such an arrangement between the Committee and the Company.  Very truly yours,  <a. : 7 Secretary", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Frost, W. H. (Wade Hampton), 1880-1938", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-t8ca.8kya_qtt7", "00000000-0000-0000-B119-4E4E5794DD5F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Charles V. Chapin to Louis Dublin", "9918573882206676X108", null, "1922", "01 April 1922", "Chapin, then superintendent of public health for Providence, Rhode Island, provided his comments on Haven Emerson's first summaries of survey data for individual cities, including his opinions on which data were indicative of the overall heatlh of urban populations.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "fi  Gepy  HEALTH DEPARTMENT OFFICE OF THE SUPERINTENDENT OF HEALTH CITY HALL, PROVIDENCE     April 1, 1922.  ear Dr. Dublin:  Your favor ef the 20th of March with enclosures came duly to hand.  I agree entirely as to the desirability of brevity in the gummary reports n the different cities. The reports which you gent me seem to figure out a little : bove your upper limit. It seems to me desirable to cut these down, if possible, and_ | peldeve that this could be dene without sacrificing anything of importance. IT ‘oubt if it is necessary to say much, if anything, about the general organization f the city, and the organization of the health department could be touched upon very  _ phefly.. Nothing should be emitted of Dr. Emerson's excellent handling of the ap-  “tntment of the health officer, civil service, control of employees, and pernicious  elitics. It seems to me that, with care, these summaries could be cut down 20% and  et contain every bit of the meat that is new in them. The style ia excellent, but I  @ not believe that anything would be lost by a Little condensation. A more con-  -msged style is, of course, a little more difficult, but 1 believe, gains rather than ses in force. | ose ,  The material covered in these reports seems to be emtirely adequate, and I hink that Dr. Emerson has been very happy in stressing the good and the bad. Tf do wt think he has been too harsh. Perhaps it might vewell, in some cases, to make a wre direct statement of the major recommendation. :  | The greatest care should be taken in making the expenses of the health de-  ‘artments comparable. Dy. Emerson seems to have done this in these reports, but I  vould think it desirable in the preface or in the introduction to the series, to  ypeeify what is included in “health services\". I ama little skeptical about the use of vitalstatistics as a measure of  \\\\ealth department efficiency. Probably the crude death rate in the cities under sur ey depends more upon the geographical location and upon the race, nationality, age —  matitution and economic condition of the population than it does upon the conscious wealth activities of the municipality. The typhoid death rate is, however, a very ood indication of a certain class of activity, but these activities are not usually 4 function of the health department. ‘Infant mortality we know depends largely upon genomic status, and it would take several times the effort and money to reduce the nfant mortality rate of Fall River and New Bedford te 64, if it could ever be done, than it hag te reduce it to 84 in Rochester. We know too little about the causes of  _ the decline in tuberculesis to use this death rate as an index.  Of course I have no business to butt in on Dr. Emerson's expressed opinions,  but I am glad to see that he speaks of the difficulties of local officials carrying  on V. D. work when cases are reportable to State authorities. I am very firmly of  the opinion that the local authorities should carry on all V. D. and all T. B. work  and that cases should therefore be reported to the local health officer. It is = very minor matter, of course, but I was sorry +o see that in the report on Scranton, it ig stated that telephonic reports of contagious disease should be confirmed by written repogts. I believe this is an unnecessary hardship on the physicians. I have not required written reports for a long time, and have found no ebjection to the method whatever. ae |  : “On the whole I believe these reports are very satisfactory indeed, and my only real suggestion is that they be condensed which I think can be done without sacrifice. ,  a Yours truly, mak og  (Signed) Charles V. Chapin (Mm : S ‘ )", "Chapin, Charles V. (Charles Value), 1856-1941", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p88m~kaat-xuut", "00000000-0000-0000-EE43-6D2D72E2EF26", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from C.-E. A. Winslow to Louis Dublin", "9918573882206676X109", null, "1922", "04 April 1922", "Winslow, of Yale University School of Medicine, gave Dublin comments on Haven Emerson's first summaries of survey data for individual city public health departments.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "@ great deal of interest. In general they seem to me   propriations,, but perhaps there may ve furthe  copy YALS UsIVaRSITY The School of Medi cine  New Haven, Cte, Dre Ine Ie mine 8 Metropolitan Life Ina. COvs  New York City.  Dear Dubl", "Winslow, C.-E. A. (Charles-Edward Amory), 1877-1957", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-49xi_6km6_bji4", "00000000-0000-0000-201D-45A3B3910742", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from A. W. Freeman to Louis Dublin", "9918573882206676X110", null, "1922", "05 April 1922", "Freeman, of Johns Hopkins School of Public Health, gave Dublin his comments on Haven Emerson's first summaries of survey data for individual city public health departments.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "op mre ree enemy aetna ott ee  1 Madison Avenue  | ly dear Doctor Du  gent ig@ that they are too long  (310-312 fest Mon ment Street , /  /  , Baltimore, Md. 2 = ola De 19 22,4. A  American Publie Health Association,         New York City. . bli n: : : : i have your letter of Mareh 30th, and have just     ‘finished going over the summary reports Which accompanied it.  The first criticiem 1 would make of the reports . g and that eighty cities abstracted in this manner will make tee great a bulk of material. |      in reading the reports over I have wondered whether  er not. it is advisable to comment either favorably or unfavorably  by  over the material you sent me and am  upon the situation as it exists in the individual city. 1 feel that  Doctor imerson's comments, while in the main sound and justified, are in @ sense a violation of our promise to keep individual and specific information confidential, The implied eriticiem of the  I am sure, cause an unfavorable reaction.  — While I agree whh you thet it is diffienlt to avoid a “dull uniformity\" of statement, we must remember that the function of the Committee is to study present practice in health administra» tion and not te give gratuitous advice to individusl eities. I  present health comuissioner of New York and similer stetements will,  hesitate to speak so unfavorably of vhat impresses me as really a very  valuable and delightful set of swumeries. Yor my own reading I should enjoy very much secing the complete set but for the purposes of the Comittee I think a more direat statement of faets in regard to each city is essential, o es ae  I am devoting practi cally all of my time now to working L you much interested in it. I foree see only one difficulty and that is that each subject offers such            inviting opportunities for study that it is going to be difficult to s  bring them whhin any reasonable compass, | very truly yours,  (Signed) A. Ww. Vreaman, MeDe,     sea", "Freeman, Allen W. (Allen Weir), 1881-1954", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2wvr.ytr5_ty5y", "00000000-0000-0000-E76D-595FA3F8D995", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from W. H. Frost to Louis Dublin", "9918573882206676X111", null, "1922", "05 April 1922", "Frost, of Johns Hopkins School of Public Health commented on Emerson's summaries of survey data for individual city public health departments.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "rs #% fet }       | = Copy THE HONS HOPKINS UNIVERSITY School of Hygiene and Public Health _ 3106312 West Monument Street  r oe  Department of Epidemiology Baltimore, Md. “April 5, 2922. we 1  Metropolitan Life Ingurance Company, ae a 1 Madison Avénue, . ! : ae 4 New York City. | | o           My dear Dr. Dublins : I have vour letter of March 30th enclesing copies of Dr. Emerson's pS summaries of activities in a nimber of cities, and Asking for an opinion upon Fe these reports. i have not as vet had an opportunity to do more than look over _  -~ ft few of the reports, just enough to get the general plan, and as I will not ’ havé an opportinitys to do more for several devs I can undertake at this time * only to give mv first impressions, subject to revision if*this seems necessary after I have gone over the reports somewhat mere carefully. —  My firet impression ie very definitelw that the summaries as drawn up are not suitable for the following reasons: ke     (1) Thev are too diffuse; with the result that the facts as to - oYganization and activities in any citv can be dug out onlw by reading several ( peges of description and disciesion. I think it would be much better to have a ‘the facta stated as succinctly as possible in a skeleton outline with headings — as nearly uniform as possible for all cities. i s \"  (2) L object to the inclusion of opinion along with the state- ment of facts; believe that the committee's report should confine itself, as far as individual tities are concerned, to giving facts. I cannot see how it  aS is possible for afiyone without a thorough personal knowledge of the actual s workings 6f a city organization to express reliable opinions as te their efficiency. Moreover,*even if opinions could be reliably formed I doubt that - ae it would be good policy to inelude them. =     n (3) There is too much revelation of details as to individuals, | @speciallv as to the health commissioners, I think it should be a cardinal | principle te avoid revealing matters of individual training, salaries; ete. | Although as to salaries, these are matters of public record and it mar be  | Penne to include them. | Ay er     it seeme to me onthe whele that it would be much better to substitute for this form summary an entirely different form, giving in brief 4 outline the bare facts as to organization and activities of each cite, requir~ * q ing 1 should judge not more than about one printed page for each citw, certainly mot more than 100 to 125 pages for the whole series. oS ge 4s I find that Dr. Freeman has not received a similar letter from | | vou, 1 have turned mine over to him, asking him te answer it independentiw. ee  With best regards,  -  Very sincerele yours,  ame Se ig err ns", "Frost, Wade Hampton, 1880-1938", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-478c_ie29~f7xn", "00000000-0000-0000-D258-E8138D4C10F5", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Lee Frankel", "9918573882206676X112", null, "1922", "26 September 1922", "Dublin reported to Frankel on the last meeting of the Committee on Municipal Public Health Practices, the printing of the committee's survey results, the establishment of permanent APHA committees, and probable future support from the Rockefeller Foundation, along with other matters.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "e € = = S “Le =  ‘September 26, 1922  Dr. Lee K. Frankel, Brown Palace Hotel,  Denver, Colo.  By dear Dre Frankel:  You will be interested, I think, in the results of the mesting of the Committee on Municipal Health Department Practice which was held here yesterday. We had full attendance at what will probably prove to be the Last @eeting. The report was accepted and ought to go to the government printer (U. 8. Public Health Service) within ten days, making a document of e i suppose, between 450 and 500 pages. It is not the bulk, however, that will make this report important. We ell felt that we had for the first time collected the important facts with reference to the activities of American health departments and had worked cut a standard practice which would guide the individual health officers to higher  achievements. The book will be very much used and we #11 feel thet it will be a  most useful contribution,  7 What I want to tell you is that at the meeting, there was unanimous sentiment to thank the Company most heartily for what it has done to make this contribution possible. I could, of course, do nothing in that matter. But, all  the other teabers voted a statement which will go into the first page of the Iutreduction, acknowledging the indebtedness of the Association to the Company.  Sut, more important ie the fact that at the last meeting of the Committee on Re- organization of the American Public Health Association, it was moved that the Association continue te carry on the activities of our Committee as one of ite : permanent and vital functions. In fact, Dr. Vaughen’s plan is to make this work the starting point for the new life of the Association. With thie in mind, they have approached Dr. Yincent of the Rockefeller Foundation, asking for an appro- priation with which to establish a bureau. They have in mind about $25,000 a year for a number of years, which would obtain the services of an outstandingly able men to head up the work. He would keep the files of the Association up to date on everything that is going on in the health departments of the country,  . €fewer inquiries and stimulate the process of raising the tone of the varicus  departments of the country; evidently, something very much akin to the plan you developed last year and tried out on Emerson. Possibly, you have telked with Others about the plan because everybody on the Organization Committee now talks your language and feele that that is something for the Association to de. I  _ personally said nothing because I knew this was your plan which you would 1 probably want to keep for ihe Company as one of its activities.  At the luncheon which we gave to the Committee, I reiterated your plans, emphasizing the point that you felt that we were in an extraordinarily favorable position to stimulate public health work through our Managers and staff, and through the support which any good plan would receive from our policyholders.    Dre Frankel = September 26, 1922 = 2.  Te which, the comment was made that it would be better possibly for all con- cerned if the Company, the American Public Health Association and the Rockefel- ler Foundation joined forces, each doing what it could toward the common goal. The impression was that Vincent would be more likely to participate if he could count on the Company's cooperation, either in terms of money or service. He suggested the name of Gunn as the logical person to head up this work. I feel that you should knew right away all these items because you may wish to get in touch with Winslow at once and let him know what the Company is preparing to do.  ; You will be interested also to know that the antegonism to the Company which found expression during the last few months in some resolutions of  Dr. Harrie has given place to a resolution of appreciation, which Dr. Harris will offer at the Cleveland meeting. He was shown how silly his suspicions were of any domination by the Company and confessed that he had made a mistake and ie making amend by offering this resolution thanking the Company for all that it has done during the last decade to strengthen the American Public Health Association. Nobody hears anything from Coon or from Goler these days. I thought you would be  particularly pleased to know the change of front on the part of Dr. Harris.  I need hardly tell you what a load is taken off my hands in the comple-  tioncof the report. We have been at it for two years now. It was easily the  most important thing we were doing and yet not being a Company activity, it had to be carried on in what little spare time we had after the day’s work was done. We have had splendid support from the best men in the health movement including, Winslow, Chapin, Emerson, Freeman, and a really most valuable contribution is now at hand, which I know will bring credit to the Company.  i hope this finds you and the other officers well, and I beg you to  remember me to the President and to all the others.  Very truly yours,  ~  <2 | §$tatistician.", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Frankel, Lee Kaufer, 1867-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7qh7.3nmr_s277", "00000000-0000-0000-D922-274B8B515E85", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Memorandum from Louis Dublin to Lee Frankel (in re: Support for Committee on Municipal Health Department Practice)", "9918573882206676X113", null, "1924", "13 September 1924", "Dublin reported to Frankel regarding funding for a permanent Committee on Municipal Public Health Practices within the APHA.", "Memorandums", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Dr. Frankel : eer 15 Second Vice President ee  IN RE       Department Practice so      I would suggest that you take up with the Executive the question of continued support by the Company for the work of the Committee on Municipal Health Department Practice of the American Public Health Association.  This Committee was organized by us in 1920. Professor Winslow of Yale University was asked to be Chairman and I was Secretary. We also had a group of leading sanitarians join with use The purpose of the Committee was to study the actual methods of health administration used in the eighty-three largest cities of the country, and to discover the best procedures and ultimately to replace deficient organizations with the most approved practice. You hoped, also, that the Company could actively participate in stimulating the improvement of the organization in each of the cities as the deficiencies were disclosed. As the result of three years’ work in which the United States Public Health Service and many volunteers participated, a report was issued by the Committee, being Bulletin 136 of the United States Public Health Service, a volume of 468 pages.  This report was favorably received and the work of the Committee was continued and extended. Dr. Rankin, State Health Officer of North Carolina, was invited to become Field Director in the fall of last year. As the result of his labors, a new set of surveys has been made by the United States Public Health Service and by the American Child Health Association. Possibly the most important work done by Dr. Rankin has been the preparation of a schedule by which the health work of any city could be rated and its relative position in the list of cities determined. This schedule has been approved by all the national health organizations and by a large number of municipal health department officers.  The work is growing rapidly and we all hope that it can be made the basis for much improvement in municipal health department practice. Everything will depend upon adequate fi- nancing of the worke    po  In this connection, the Milbank Fund has made an appropriation of $10,000 for the year 1924-1925. An additional $10,000 is very much needled to see the work of the Committee . through the next year and I strongly recommend that an appropria-~ tion of this amount be made by the Company.  The Company has, to date, appropriated $22,500 to the work of the Committee es follows:  May 25, 1920 $ 5,000 February 28, 1922 2,500 April 24, 1923 15,000  ' This fund has been almost entirely expended for the salaries of the  investigators and for the Director and his small office staff.  Much more than the Company's appropriation has been expended by the Federal Government in making available to the Committee a large number of Officers who made surveys without any cost to the Committee.  @@eseeeeeeeogeeeeee ee &  September 13, 1924 : Statistician", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Frankel, Lee Kaufer, 1867-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qdm3~8v84~3f4r", "00000000-0000-0000-837C-CEAB28C40C60", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Memorandum from Louis Dublin to Lee Frankel (in re: Support for Committee on Municipal Health Department Practice)", "9918573882206676X114", null, "1925", "23 July 1925", "Dublin summarized progress made by the Committee on Municipal Public Health Practices.", "Memorandums", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Met. ‘NtusGo? ., Rise  July 23, 1925  Dr. Frankel Second Yice President  in FG: ou Op: ey     The Company appropriated $10,000 for the support of the  Committes on Municipal Health Department Practice. A similar amount was appropriated by the Milbank Memorial Fund. We have to date spent $6,695 of this $20,000 appropriation. The work of the Committee this year has been under the following heads:  le The working up of the reports made by the agents of  the Public Health Service of one hundred cities of seventy thousend population and over. Eighteen chapters are being prepared on all of the important phases of municipal health department practice, and a new book will be issued befere the close of the year bringing all this information up to date.  2- The Committee has worked up the appraisal form until it is satisfactory to all concerned, that is, to the Committee, the American Child Health Association, and the various groups of health officers. This has required many meetings and very great labor and dis- cretion on the part of our Chairman. The appraisal form now makes pos- sible a fairly satisfactory rating of the 186 cities which have been sur- veyed, Such ratings and appraisals will make possible field work on a fairly large scale beginning this fall and throughout next year.  3. Our Field Director hes made a large number of contacts with Health Officers, Mayors, and other officials, preparing the ground for more active cooperation in the fall. The cities of Ghic, Massachu- setts, New York, Pennsylvania, Memphis, Kansas City, Missouri, Wilkes- barre, and a number of ethers have been visited, interviews had with the Health Officer, and public officials, and it ie. expected that out of this number a choice will be made for an intensive demonstration by our Field Director, which will result in a qrest improvement in the health  practices in those cities.  4. We have made preparations for the publication of a number of the Survey Graphic which will be a popular presentation of the principal findings of the Committee to date and will serve also as en - appeal to social workers and others who read the Survey to get in touch with our Committee for advice and guidance in matters of their local health work. The entire number of the Survey wil] be used for this pur- pose. . = : =    b. \\\\  ; More then the feregoing would have been accomplished, especially in the field of intensive demonstration, but for the change of our Field Director. As you know, Dr. Renkin resigned to become associated with the Duke interests in the health work of North Carolina, and we have for some months been without an active Director. We have now found in Dr. Walker an ideal person to take hold of this work. He will teke hold on the first of September. We plan also to continue Dr. Drake for field work among the Pacifie Coast States. The rest of the  year should see a number of well planned and suceessful demonstrations  along the very lines thet you have slways urged, to show that larger appropriations can be obtained and direct facilities established through the spirt of the public, the Metrepoliten agents, and the members of this Committes &  The programme of 1925 calls for a budget of §25,000, of which $5,000 will be appropriated by the American Publie Health Associa-  tion itself, and it is heped that the Company will continue its appropri-  ation of $10,000 and thai the Milbank will do likewise. ‘The division of this budget under bread headings is as follows:  Salaries $16,000 Travel end expenses 4,000 Rent & incidental)  office expenses) 3,000  . Sotel | = $25,000 Our work for next year should be almost entirely in the  field. Our representatives will be concentrated on a group of cities armed with the complete facts, with appraisals of the work done, and with  many friends lecally to assist in every way. The five years’ work of the  Committee has come now to a head. Everything seems to be ready to gather in the fruit of much Labor, and I am convinced that this is the strategie time to support this work and encourage it in every way. ‘There are no difficulties in sight for the complete working out of our programme.  SOCCHGeoeecee 666  #2 Statistician", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Frankel, Lee Kaufer, 1867-", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xt3y-2zgn~5qw3", "00000000-0000-0000-6007-B58EEF00F60D", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Carl E. Buck to Louis Dublin", "9918573882206676X115", null, "1931", "31 December 1931", "Buck, then Field Director for the Committee on Administrative Practice, wrote to Dublin regarding expected funding shortfalls for the committee's activities due to the economic depression, and possible strategies for coping with such difficulties.", "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "3", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "Met. ‘NtusGo? ., Rise  July 23, 1925  Dr. Frankel Second Yice President  in FG: ou Op: ey     The Company appropriated $10,000 for the support of the  Committes on Municipal Health Department Practice. A similar amount was appropriated by the Milbank Memorial Fund. We have to date spent $6,695 of this $20,000 appropriation. The work of the Committee this year has been under the following heads:  le The working up of the reports made by the agents of  the Public Health Service of one hundred cities of seventy thousend population and over. Eighteen chapters are being prepared on all of the important phases of municipal health department practice, and a new book will be issued befere the close of the year bringing all this information up to date.  2- The Committee has worked up the appraisal form until it is satisfactory to all concerned, that is, to the Committee, the American Child Health Association, and the various groups of health officers. This has required many meetings and very great labor and dis- cretion on the part of our Chairman. The appraisal form now makes pos- sible a fairly satisfactory rating of the 186 cities which have been sur- veyed, Such ratings and appraisals will make possible field work on a fairly large scale beginning this fall and throughout next year.  3. Our Field Director hes made a large number of contacts with Health Officers, Mayors, and other officials, preparing the ground for more active cooperation in the fall. The cities of Ghic, Massachu- setts, New York, Pennsylvania, Memphis, Kansas City, Missouri, Wilkes- barre, and a number of ethers have been visited, interviews had with the Health Officer, and public officials, and it ie. expected that out of this number a choice will be made for an intensive demonstration by our Field Director, which will result in a qrest improvement in the health  practices in those cities.  4. We have made preparations for the publication of a number of the Survey Graphic which will be a popular presentation of the principal findings of the Committee to date and will serve also as en - appeal to social workers and others who read the Survey to get in touch with our Committee for advice and guidance in matters of their local health work. The entire number of the Survey wil] be used for this pur- pose. . = : =    b. \\\\  ; More then the feregoing would have been accomplished, especially in the field of intensive demonstration, but for the change of our Field Director. As you know, Dr. Renkin resigned to become associated with the Duke interests in the health work of North Carolina, and we have for some months been without an active Director. We have now found in Dr. Walker an ideal person to take hold of this work. He will teke hold on the first of September. We plan also to continue Dr. Drake for field work among the Pacifie Coast States. The rest of the  year should see a number of well planned and suceessful demonstrations  along the very lines thet you have slways urged, to show that larger appropriations can be obtained and direct facilities established through the spirt of the public, the Metrepoliten agents, and the members of this Committes &  The programme of 1925 calls for a budget of §25,000, of which $5,000 will be appropriated by the American Publie Health Associa-  tion itself, and it is heped that the Company will continue its appropri-  ation of $10,000 and thai the Milbank will do likewise. ‘The division of this budget under bread headings is as follows:  Salaries $16,000 Travel end expenses 4,000 Rent & incidental)  office expenses) 3,000  . Sotel | = $25,000 Our work for next year should be almost entirely in the  field. Our representatives will be concentrated on a group of cities armed with the complete facts, with appraisals of the work done, and with  many friends lecally to assist in every way. The five years’ work of the  Committee has come now to a head. Everything seems to be ready to gather in the fruit of much Labor, and I am convinced that this is the strategie time to support this work and encourage it in every way. ‘There are no difficulties in sight for the complete working out of our programme.  SOCCHGeoeecee 666  #2 Statistician    a  OFFICERS LOUIS I, DUBLIN, PH.D. President  E. L. BISHOP, M.D.  Chairman of Executive Board  KENDELL EMERSON, M.D.  Acting Executive Secretary  - fr  COMMITTEE  ON  ADMINISTRATIVE ~-PRACTICE  owe  C.-E. A. WINSLOW, DR. P.H. Chairman  HAVEN EMERSON, M.D. Vice-Chairman  -L, BISHOP, M.D. CHn...88.V. CHAPIN, M.D. MICHAEL M. DAVIS, PH.D. ALLEN W. FREEMAN, M.D. JOSFPH W. MOUNTIN, M.D. , C. NELSON, R.N. GEORGE T. PALMER, DR. P.H. W. S. RANKIN, M.D. JOHN L. RICE, M.D.  R. H. RILEY, M.D. GEORGE C. RUHLAND, M.D. HENRY F, VAUGHAN, DR. P.H. W. F. WALKER, DR. P.H.  LOUIS I. DUBLIN, PH.D. ex officio  owe  Cc” ME. BUCK, DR. P.H. ‘jeld Director     a ceelll  THE AMERIGAN PUBLIG HEALTH ASSOCIATION.  450 SEVENTH AVENUE~ NEW YORK, N. Y.  December 23, 1931  Dr. Louis I. Dudlin, Metropolitan Life Insurance Company, 1 Madisen Avenue  New York, N. Y.  My dear Dr. Dublin:-  The Committee on Administrative Practice is at present confronted with a very serious financial problem which I believe should be called to your attention.  The present staff of the Committee totals six in- cluding myself, Dr., Wallace, Mrs. Fraas, Miss Tonnele and two stenographer-clerks. Salaries for this group total $23,530.00. These salaries together with travel and maintenance items have been allocated, in accordance with the following table, to the Health Conservation Contest and Field. To Field have been allocated all the activities of the staff, other than those of the Health Conservation Contest, as well as the activities including travel and maintenance of the various sub-committees.  Field H.C.Contest  Salaries $11,614 $11,916\" Travel 2,500 6,500. : Maintenance 1,000 3,000 fi / Awards = 1,084 / 7 7 Total $15,114 — $22,500 $37,614 : 7  Of this $37,614 total $22,500 is cared for through the Health Conservation Contest leaving $15,114 (including $11,614 for salaries and $3,500 for travel and maintenance) for which no specific funds have thus far been provided.  In addition to these amounts the C.A.P. is charged with 40.6 percent of the total general administrative costs of eC the A.P.H.A. as a whole which amounts to slightly over 7 $7,000.00. ;  + q apo  The American Journal of Public Health is the Official Publication of this Association This means then that the C.A.P. has anticipated expenditures of $22,114 for which there is no assured income. Under normal conditions paid surveys plus the overhead charges which may permissibly be charged to special grants would cover these costs. This year, however, with the economic depression so widespread there is small chance of any very  _ extensive surveys and there are no special grants. (The Commonwealth _  Fund did not grant the $20,500 which we had expected for the studies of the sub-committee on the Evaluation of Administrative Practices).  In the past, as you know, we have counted on income from surveys to cover not only direct and indirect maintenance charges but also the cost of travel, preparation of reports etc. of the various sub-committees which did not have special grants. This coming year these expenses cannot be met in this manner for it is doubtful if ae come from surveys during the year will exceed $3,000 or $4,000.  (We have no contract for surveys at present).  There seem to be three possible means of solving the problem:-  1. By procuring grants for special projects, the income from which would be sufficient to maintain the present staff and assist in carrying on the work of the various sub-committees.  2. By procuring funds for carrying on the specific work of the sub-committees such as, Record Forms, Rural Health, City Appraisal Forms etc.  3- To drop the personnel of the staff other than those necessary 7 for carrying on the work of the Health Conservation Contest.  Of the three suggested solutions the third seems the least desirable in view of the fact that this would mean a discontinuation of all sub-committee activities as well as all field and consultant service. If the sub-committees are te function in the future, as  they have in the past, it is imperative that funds be procured not mn. f only for travel but for office personnel since se much of the detail work such as the preparation of the revision of the Appraisal Forms pr  and the like must of necessity be carried on in the office.  it would be impossible without maintaining the office staff  to carry out the objectives and policies of the C.A.P. which are: —7< \"The preparation, study, standardization and presentation of scientific Jf Q public health procedures by the collection of information in regard to  current administrative health practice, the analysis of the material Cot obtained to derive standards of organization and achievement and the ye pl + translation of these standards into terms of concrete achievement  through an information bureau and field service placed at the dis-  position of all members of the Association\".  It is unquestionably true that the work of the various sub- committees is of great value to public health progress and it would seem a real disaster to have to discontinue their activities. As you know, the work on revising the Appraisal Forms fer City Health work    is to be taken up in 1932, and this, of course, cannot be done unless funds are available.  Likewise the work of the Committees on record forms and rural health work planned for 1932 will be a valuable contribution to public health administration and should be continued if possible.  I should very much appreciate from you suggestions with regard to special projects for which funds mught be procured or which of the activities of the various sub-committees efforts should be put forth to get financial aid. Would you also suggest an organization or organizations that should be approached.  Sincerely yours, Os  CARL E. BUCK, Dr. P.H. Field Director", "Buck, Carl E.", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-igpw.g3ru-bvnu", "00000000-0000-0000-0AFF-023C0B26DFFD", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Meeting Minutes of the Committee on Administrative Practice, APHA", "9918573882206676X116", null, "1926", "22 November 1926", "These minutes recorded the committee's discussions regarding its expanding activities.", "Minutes (administrative records)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "8", "pages", "Text", "English", null, "Copyright may apply", null, null, "Minutes of the Meeting of the COMMITTER ON ADMINISTRATIVE PRACTICE November 22, 1926  Office of the Field Director, New York City  Present  Dr. Haven Emerson, Vice Chairman Dr. Louis I. Dublin, Secretary  Dr, James Roberts  Dr. ¢, Hampson Jones Mr. Homer N, Calver Dr, Henry F, Vaughan Dr. George T. Palmer Dr, George C. Ruhland Dr, W. F, Draper  Dr. E, L, Bishop  Dr, Michael M, Davis Dr. George D, lummis  Dr. W. F. Walker, Field Director  He 2 oe aK oR a ok ok ok ak a ae ak ake 2k  The meeting was called to order at 10:00 A.M. and the Chairman at once called for a report from the Field Director,  Dr, Walker, Field Director, referred to his report presented at the An- nual Meeting of the American Public Health Association at Buffalo, copies of which were already in the hands of the members of the Committee, This con- tained a description of the work of the field service for the year ending October 1, 1926. The report presented at this time covered activities since the annual meeting, which were, briefly: A visit to the Texas cities as a follow-up of Dr, Drake's extensive work there; Work with the sub-Committee on Appraisal Form for Rural Health Work in Nashville, Tennessee; Attendance at a meeting of Ohio Health Officers and some follow-up work in Springfield and Preble County, Ohie, and Terre Haute, Indiana; Conferences with offi- cials in Toledo, Ohio, and Washington, D. C., relative to a survey and ap- praisal of these twe cities,  The Chairman in accepting this report called attention to the very val- uable service which is being rendered cities through the Committee on Admin- istrative Practice by surveys and apnraisals of city health departments. Hs- pecial mention was made of the assistance rendered Chicago and Boston in securing the reappointment ef their respective health officers and the de- veloping of very excellent heaith departments.  Dr, Walker brought before the Committee at this point the question of Dr. Drake's accepting the invitation from the Beard of Health of San Antonio, Texas, and other interested groups in that city, to direct a Health Exposi- tion for the City of San Antonio. This was a matter that had been discussed at a previous meeting, at which time it was the general opinion that it would             22 «  be against the policy of the Committee to foster or approve of Health Exposi- tions. The situation which existed in Texas at the time of the first request for Dr, Drake's services in connection with the Health Exposition in San An- tonio was reviewed to refresh the memory of the members on the question. Dr. Dublin asked Dr. Walker to give his opinion upon the situation, in the light of his recent visit to Texas. Dr, Walker said that there had been some im- provement and that he was quite convinced that the Health Exposition in San Antonio would be a means of arousing the city to its immediate needs in pub- lic health work and would serve primarily the purpose of putting over the results of the survey and appraisal made by Dr. Drake.  Mach discussion followed on the subject of health shows and health ex positions in general. Dr. Vaughan was interested in knowing the ultimate good which would result to the Committee from such a work, Mr. Calver spoke at some length on the subject, touching upon the matter of coordinating the work of the Association with that of the Committee and setting forth the ad- vantages that would result both to the Association in its attempt to estab lish a branch office in Texas, and to the Committee in participating in or directing the Health Exposition in San Antonio. The question was then : brought before the Committee of releasing the time of Dr, Drake through the Central Office of the American Public Health Association rather than have him represent the Committee in this work. After further discussion Dr. Emerson moved, seconded by Dr. Vaughan, that the Committee release Dr. Drake 's time through the Central Office of the Association for the work of putting on  the Health Exposition in San Antonio, if the Executive Board should so desire. Carried.. .  Dr. Ruhland, Chairman of the sub-Committee on Record Fomms was asked to give a report of the work of that Committee. In giving his report, Dr. Ruhland went into the history of the Committee on Record Forms. Five meet- ings were held by this Committee. Progress on the work of this committee had been slow owing to the variety of questions to be considered in evolving forms that could be recommended as standard, Dr. Ruhland presented for the approval of the Committee as a whole forms for commmicable disease, labora~ tery, mursing service and school physical examination. The Committee on  Record Forms was authorized to continue its work for the remaining health activities.  The question was raised as to whether or not these forms had been sub- mitted to persons familiar with mechanical tabulation in order to be abso- lutely sure of its being possible to use them in this manner. Dr. Ruhland assured the Committee that this particular point had been in mind in work- ing out these forms. Dr. Emerson urged that these forms be put out as a publication of the Association. Dr. Dublin pointed out the potential value of the forms in money, the possible financial support te the Associa-~ tion, and suggested turning the matter of printing over to the Association,  Discussion arose as to the means of distributing these forms to mem- bers of the Committee and Association for criticiem before final publication, Mr. Calver suggested that Dr. Ruhland's report be published in the American Journal of Public Health and a special appeal be made to the membership of the Association to submit criticisms of the forms before a certain date. Dr, Dublin called attention to the necessity of having the forms copyrighted. It was suggested that to avoid undermining the copyright of the forms that the idea be copyrighted. Dr. Dublin moved, and it was seconded, that the Association be requested by the Committee to copyright the idea. Carried,    eis  Dr. Vaughan moved, seconded by Dr, Palmer, that the report of the sub-Committee on Record Forms be published in the Journal, and then be distributed to the health officers, Carried, Dr. Emerson moved, seconded, that the report of the sub-Committee on Record Forms be received, Carried,  Dr. Vaughan felt that difficulties might arise in cities that had here- tofore been printing their own record forms, and that they would not like to feel forced to buy the forms from the Association. He suggested a solution in that cities paying a retaining fee to the Association be furnished plates from which to print the forms for their own use.  The Committee voted that the Report of the Committee on Record Forme should be signed by all members of that committee, and that forms covering other functions when ready be handled in the same manner, regarding distri- bution, as those presented at the present time.  Dr. Bishop, Chairman of the sub-Committee on Appraisal Form for Rural Health Work, was next to report, giving a resume of the work leading up to the development of the tentative form. The minutes of the meeting of this committee held in Nashville on November 4.5 were read by Dr. Bishop and the appraisal form as worked out at this meeting offered for the approval of the Committee for one year of exporimental use. Dr. Bishop said that there was complete agreement among the sub-committee members as to the principles underlying this tentative appraisal form for rural health work.  Dr, Palmer moved, seconded by Dr. Vaughan, that the report of the sub- Committee on, Appraisal Form for Rural Health Work be received, and that the Committees the form as submitted for publication, with the understand- ing that when published the form will carry a statement that it is intended for experimental purpose and has not yet been approved by the Committee on Administrative Practice, The sub-Committee on Appraisal Form for Rural Health Work was authorized to continue functioning for the purpose of re- vision after a year of experimental use of the form,  Dr, Emerson suggested that since the sub-Committee on Appraisal Form for Rural Health Work and the sub-Committee on Record Forms were composed of a number of persons not members of the Committee on Administrative Prac- tice, and in view of the valuable service rendered on these committees, a resolution of appreciation be sent to the members of these sub-Committees, with a copy of the resolution approving the publication of these tentative forms. This was put in the form of a motion, seconded and carried.  Dr, Emerson asked that the Chairman be authorized to appoint a commit- tes tO prepare an appraisal form for state and provincial health activities, It was brought to the attention of the Committee that such a committes had been appointed at the last meeting, but since Dr, Griswold, Chairman of that committee, was not re-appointed as a member of the Committee on Administra- | tive Practice, it would be necessary only to re-appoint the committee rather than authorize a new committee. Dr, Bishop spoke briefly of the work al- ready done in studying state health departments and suggested that it would be wise to defer action of a committee on appraisal form for state and pro- Vincial health activities until a report of this study is available. Dr, Emerson agreed, but urged that the committee be appointed now before someone else attempts the work, in order that the fundamental principles may be the same as those developed in the other appraisal forms, Dr. Draper voiced    = 4s  the same opinion as that of Dr. Bishop in postponing the activities of such  a committee until material gathered in the study of state health departments be made available. The entire Committee agreed to this. Dr. Vaughan moved, and it was seconded, that the Committee on Appraisal Form for State Health Work be re-appointed, Carried, Dr. Winslow appointed Dr. Draper chairman of this committee, with power to coopt members, subject to his approval,  Dr, Davis was asked to make a report of a preliminary study on the Re- lation between Health Departments and Hospitals. Dr, Tavis told of the questionnaires sent out jointly by the Committee on Dispensary Development and the office of the Field Director on this subject, the results of which brought out the need and advisability of pursuing the study more systemati- cally. To that end funds have been secured to carry on the study during the coming year. Dr. Davis said it was not intended that this should be Gn elaborate study; it would still be carried on by the questionnaire method, but in greater detail than the first questionnaire, The sum of $1,500 was considered by Dr. Davis to be sufficient to pay the expected expenses in this study. This amount has already been pledged,  In discussing this work Dr, Emerson recalled a similar suggestion made previously by Dr. Craster as a possible plan for expanding the work of the Committee and gathering information on supervision of hospitals through such a committee, It was moved and carried that a committee be appointed to study the Relation between Health Departments and Hospitals. Dr, Davis was appointed chairman of this committee with Dr. Ruhland and Dr. Emerson members,  The latest information regarding the Report of the 1923 Surveys as re= ported by Dr. Walker and other members present indicated that practically all the sections have been proof-read and returned to the printer and that the book may be expected for distribution at an early date,  Dr, Walker next presented the program of the Committee for 1927, the first subject for consideration being the \"Plan for Coéperation with the U.S. Chamber of Commerce\", Some time was spent in giving the details of how this contact with the Chamber of Commerce Originated and the specific items in the plan for cooperation requiring the approval of the Committee were the following: — 7  1. Tabulation and summarization of replies from local chambers of commerce, indicating items in which interest is expressed and type of service needed,  é. Issuing @ bulletin by the Chamber of Commerce of the United States setting forth these results,  3. To furnish blank copies of the Appraisal Form to those cities interested in analyzing their health activities, to give an  idea of the scope and type of appraisal and surveys made by the Committee,  4, Educational bulletins to be prepared for distribution through= out the year:  a. Community health and commimity prosperity  b. Place of public health in the tax dollar  CG, Essential items in the health Organization of the typical American City  Gd. How the Chamber of Commerce can assist in bring- ing about improved community health    ~5-  5. Special bulletins, possibly prepared and sponsored by other agencies on such items as: Periodic physical examination; Venereal Disease from the standpoint of the patient; and others,  6. To carry items in the Health Officers' News Letter on affilia- tion of the Committee on Administrative Practice with the United States Chamber of Commerce and the possibilities of local application.  7, The publication under joint editorship of a News Letter for public health comnittees of local chambers of commerce setting forth activities and results obtained by other chambers and the relation of the U.S. Chamber of Commerce and the Committee on Administrative Practice.  8. To encourage the members of the Committee on Administrative Practice to establish strong contact with their local chambers, possibly acting as chairman of the public health committee of same, and to build up an active affiliation between the chamber and the local health work, urging Committee on Administrative Practice members to advise the Field Director, so far as pos- sible, of visits to other cities sufficiently in advance so that contacts with both the health officer and the chamber of commerce secretary may be made, in order that the program of the Committee may be stimulated in advance.  Dr, Emerson in discussing the question pointed out that the primary interest of the chamber of commerce in the Committee was as a means of pre- senting the human assets of the community on the same basis as the fire pre- vention work. He also stated that this plan for cooperation would not al- ter the policy of approaching a city through the health officer,  Dr. Dublin in discussing the plan pointed owt to the Committee that this was an excellent opportunity for cooperation. The local chambers of commerce have no desire to do the technical work which this Committee is prepared to do but are willing to put every facility at the disposal of this Committee. Dr, Jones spoke of the danger in local chambers of commerce in undertaking this work unless it comes directly threugh the local health of- ficer. Dr, Walker said it was not the intention of this plan of coopera- tion to take the work out of the hands of the health officer, but it is 4 splendid opportunity to guide local chambers of commerce in their health programs. He cited work already accomplished in Springfield, Ohio, 4s an example of what might be done by a local chamber, and said that under such guidance and advice which can be given by the U.S. Chamber of Commerce through well selected local men this health committee of the local chamber can become a tremendous power in the community. Dr. Palmer suggested that each item in the plan for cooperation as presented by Dr. Walker should be considered and acted upon separately by the Committee.  Items 1 and 2 required no action by the Committee, Item 3 was the subject of considerable discussion and it was agreed that if in furnishing the Appraisal Forms to local chambers of commercs the following statement contained in the Introductory Statement be underlined in red and the health officer be advised by letter of such action the Committees saw no objection te this part of the plan for cooperation: \"The Committee believes that    A atm AP  - 6 =  eee  there should be no appraisal of the health work of a city on this plan except with the approval of the health officers concerned; and that in no event should there be any publication of the appraisement of the health work of a city ex cept with the consent of the health officer,\" Dr. Ruhland put this into the form of a motion, which was duly seconded and carried,  Item 4 was approved by the Committee. Dr, Winslow believed this point to be really a function for Mr, Calvex's office and it was moved and carried  that the Committee approve this project but that it be referred to the Execu-~ tive Board for carrying out.  Items 5 and 6, It was moved and carried that the person responsible for the present Health Officers? News Letter should de delegated to carry out these two items, of which the Committee as a whole approved,  In discussing Items 7 and &, the question of the original purpose of the Health Officers' News Letter was set forth and it was felt that in undertaking this plan for cooperation no departure would be made from the original purpose. Dr. Dublin pointed out in his discussion of Item & that the contacts to be made through this plan would not be essentially new contacts. It is the present policy of the field staff to make contact in cities with influential persons, such as managers of chambers of commerce and leading business men's clubs, It would simply be a question of furthering the work of the Committee in a community. Dr, Dublin moved that these two items be approved. Carried,  Dr. Ruhland moved, seconded by Dr. Bishop, that the Committee approve the plan for Cooperation with the U. 8. Chamber of Commerce, as outlined by Dr. Walker, subject to the action of the Executive Board, Carried,  The need for model ordinances as indicated by the numerous requests re- ceived in the office of the Committee and the Association was brought to the attention of the Committee by Dr. Walker. This was deemed a most important work which could be undertaken by the Committee during the coming year, Af- ter the discussion of the subject, Dr, Vaughan moved that a committee be ap- pointed by the Chairman to formate model health ordinances. Carried,  Dr, Winslow appointed the following committee: Dr, Vaughan, Chairman, Dr,  Emerson, Dr, Bishop, and gave the chairman permission to appoint other mem bers, subject to his approval ,  A letter was read from Prof, Hiscock requesting that the Committee give scme thought to the question of standardization of health department reports. This matter had been discussed at the annual meeting in Buffalo, Dr. Vaughan moved that the Chairman appoint &@ committee to develop a standard form for he@lth department reports. Carried. Dr, Jones was appointed chairman with  permission to coopt such other members for his committee as needed, with the approval of the Chairman.  Dr. Winslow presented for the consideration of the Committee the question of revising the Appraisal Form for City Health Work, Though the time set for such @ revision is 1929 it was believed that a committee should be appointed to makes @ study of the Appraisal Form during 1927, receive the suggested changes and criticisms offered and incorporate them in a tentative form for experimental use in 1928, then be prepared for the revision in 1929, Dr, Vaughan distributed to the members of the Committee charts showing the appli- cation of the Appraisal Form to the work ef the Detroit Health Department for & period of five years and gave a brief discussion of various items, pointing    aos  out specific items which he felt from this extensive study should be revised, Dr. Emerson presented criticisms of the laboratory section as set forth in a letter from Dr, Wadsworth of the New York State Health Department.  The question was raised relative to whether the Committee on Analysis of Public Health Procedure could not do this work of revising the Appraisal Form, It was pointed out that this committee was appointed to investigate the validity of the criteria as a true measure of health services and that though it should furnish material for the revision of the Appraisal Form a special committee to undertake this work was necessary, and should be ap- pointed, It was the opinion of the Committee that close cooperation should be established between this committee on revision and the chairmen of the various sections of the American Public Health Association, such as the nursing section, laboratory, child hygiene section, and others. Dr. Vaughan moved, seconded by Dr, Palmer, that a committee be appointed to study and prepare material for the revision of the Appraisal Form for City Health Work, Carried, The following were appointed on this committee: Dr. Palmer, Chair- man, Dr. Hoberts, Dr. Freeman, Dr. Iummis, Dr, Vaughan, Mr. Platt.  Owing to contimied illness, Dr, Freeman, Chairman of the Committee on Analysis of Public Health Procedure, was not present and there was no report  from this committee, The Committee voted to authorize the continuation of Dr, Freeman\\\\s committee,  Dr, Lummis introduced a blanket motion making Dr, Walker member ex- officio of all sub-committees. Carried. , :  Dr, Winslow called to the attention of the Committee a resolution passed by the Governing Council of the American Public Héalth Association requesting the Committes on Administrative Practice to consider undertaking with the American Medical Association a systematic study for better organi- zation of medical service, looking toward the checking of incipient disease. The scope of such & study was discussed, ideas and opinions expressed as to how far ths Committee should go in work of this kind, the ultimate good which would rasult in the field of public health and the medical profession, the educational value attached to such a study, and many other phases of the question, It was the feeling of the entire Committee that the initial step in undertaking such a study should be the appointment of a committee ~ to confer with representatives of the American Medical Association upon the feasibility of the plan, and that the committee thus appointed should report at the end of one year, Di. Vaughan put this into the form of a _ motion, seconded by Dr. Ruhland, Carried. Dr. Emerson was appointed  Chairman of this committee, with Dr. Draper, Dr, Rankin, Dr. Bishop and Dr. Davis members,  Dr, Dublin gave a report of the finances of the Committee. He gave a& comparison of the expenditures for the past ten months and the budget for ten months, which showed an unexpended balance for ten months of $1,046.74, The budget for 1926 amounted to $25,900. The budget for the year 1927 in the sum of $33,800 was presented for the consideration of the Committee. Dr, Dublin went over each item of the proposed budget and pointed out the necessity for the increases. He also called attention to the expected in- comé for 1927, based upon expected g ants from the Metropolitan Life Insur- ance Company and the Milbark Memorial Fund, $7,500 each, and the income which would result from the sale of consultant service, surveys and ap- praisals by the Field Staff, some of which are definite commitments.    Dr, Winslow went inte considerable detaii concerning the relation of the Com- mittee and the Milbank Memorial Fund, explaining the conditions upon which previous grants from the Fund had been made and also touched upon the fact that the Metropolitan pledge has been contingent upon the sun granted by the Milbank, Dr. Walker felt that it was quite possible that within a few years the Committee on Administrative Practice would be self-supporting,  After this report was received Dr. Emerson brought before the Committee the question of full-time, trained public health personnel, and Suggested that some credit should be allowed for this in the Arpraisal Form. The feel- ing of the Committee after discussing the matter briefly was that this should be referred to the committee appointed for the purpose of revising the Ap- praisal Form for their consideration, This was done,  Dr. Winslow called attention to the fact that the positions of Field Director and Associate Field Director are annual appointments and some action should be taken by the Committee regarding these appointments. Dr. Emerson moved, seconded by Dr, Dublin, that the Committee re-appoint the present Field Director, Dr, Walker, and Associate Field Director, Dr, Drake, at the salaries as listed in the budget for 1927, Carried,  Meeting adjourned,", "American Public Health Association. Committee on Administrative Practice", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4yv7.dpne-k3pf", "00000000-0000-0000-140B-11F90DBDC7C4", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Meeting Minutes of the Committee on Administrative Practice, APHA", "9918573882206676X117", null, "1933", "20 September 1933", "These minutes reflected the committee's ongoing monitoring and appraisal of public health departments, and efforts to support and expand them despite the constraints of the economic depression.", "Minutes (administrative records)", null, null, "17", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "MINUTES OF MEETING OF COMMITTEE ON ADMINISTRATIVE PRACTICE September 20, 1933     Present: Professor C.-E. A. Winslow, Chairman Dr. Haven Emerson Dr. Louis I. Dublin Dr. E. L. Bishop Dr. Henry F. Vaughan se Dr. George T. Palmer Dr. George C. Ruhland Dr. Robert H. Riley Dr. John L. Rice Miss Sophie C. Nelson Dr. Joseph W. Mountin Dr. W. F. Walker Dr. Leverett D. Bristol Dr. Kendall Emerson (ex-officio) Dr. Carl H. Buck, Field Director Alma W. Fraas, Secretary  Absent: Dr. Charles V. Chapin Dr. Michael M. Davis Dr. Watson &. Rankin  Guests for Luncheon:  Dr. John A. Ferrell Mr. John A. Kingsbury Mr. Homer Folks  Professor Winslow reviewed the statement on the work of the C.A.P. which he had prepared for inclusion in the report of the Ghairman of the Executive Board to the Governing Council. He read the following statement on Future Ac- tivities of the Committee:  \"Insofar as funds and personnel are available, the Com- mittee will continue to carry on the same activities during the coming year, together with such new projects as may sub- sequently be discussed and approved by the Committee.  \"The most important service of the Committee has been, and will continue to be, the voluntary work of its tireless and devoted sub-committees, and this will continue, ir- respective of special funds. We are confident that the Health Conservation Contest will be financed in its present more eco- nomical form and we hope that funds may be again available for the vital work of the Evaluation Committee.  \"The major problem before us at the moment is, of course, the damage that has been done to the health machinery of the    country by reduction in appropriations, sometimes necessary, more often the result of thoughtless panic and lack of intelli- gent planning. In many states and cities the work of a decade has been undone. Heakth organizations have been wrecked in certain instances and reduced below the minimum of safety in many more. Now that the economic crisis appears to have passed its gravest stage, it would appear that the Committee on Admin- istrative Practice should be in position to take a position of real leadership in the struggle to restore our health machinery to a position of effectiveness. Nor should we be satisfied with a mere return to the conditions of 1950. We are living through & unique period of purposeful social and economic planning and ene of the leading features of such a period should be a program providing adequate health protection for the whole American people.  \"The Committee on Administrative Practice has already begun to collect material indicating the seriousness of recent reduce tions in health service. If authorized by the Governing Council - and particularly if special funds can be obtained for the purpose - this Committee should be in position to cooperate very effectively with the United States Public Health Service and other official and voluntary agencies in a positive and constructive program for the restoration and upbuilding of the health defenses of the nation.\"  In view of the importance of the problem, Professor Winslow said that he had taken the liberty of inviting Dr. Ferrell, Mr. Kingsbury and Mr. Folks to luncheon at which time the matter could be discussed in considerable detail.  It was voted that a letter from the Committee be sent to Dr. Chapin. The Committee next took up reports of sub-committees: Record Forms  This committee, of which Dr. Ruhland is chairman, has had no meeting this year. It still has before it the development of forms for sanitation. When these are ready the committee can proceed with the preparation of the booke— let which will contain the philosophy of records and Sample forms.  Professor Winslow asked the committee's opinion with reference to the stimulation of the sale of record forms. It was the consensus of opinion that the function of the Record Forms Committee is to prepare a booklet on the need for and use of records with samples giving the minimum essential information, rather than to stimulate the sale of forms prepared by the Committee. It was felt, however, that state health departments might encourage the use of forms developed by the committee in county or rural health districts.  Rural Health Work  Dr. Bishop read the following report on the work of his committee;    \"The report of the study of rural health work is being bound and will be ready for distribution before the first of the month. The complicated nature of the material and the necessity of hav- ing the manuscript in final form and proofs read by a number of people has been primarily responsible for the delay in publica- tion. However, the use of a large part of the material in the Rural Appraisal Form means that the survey has not lain dormant during this period. The retardation of rural health work due to the depression makes the findings and conclusions of the report and recommendations equally good at the present time.  \"The special sub-group of the committee on rural health work which has been at work on rural record forms has been carry- ing on throughout the year, giving consideration to the general scope of record systems and the items that should be incorporated on specific case records. The committee has felt the necessity of proceeding cautiously in this matter in order not to develop a too elaborate program. A feeling has been expressed by some organizations that records are complicated and too time consuming. It is, however, the feeling of the members of the records com-~ mittee from experience in a number of small as well as large units, that well planned and carefully used records do, in fact, increase the effectiveness of the organization and can be provided for with state cooperation at a reasonable figure. For example, our exper-~ lence in Tennessee with a state system of records indicates that the ordinary four-piece unit can have satisfactory record forms provided for from $35 to $40 per year. The committee is planning the publication of a record manual which will indicate the minimum essentials of satisfactory records. Though the committee does not feel that records identical in form and arrangement must be adopt- ed in all places, it seeks to encourage the recording of certain items and the acceptance of standard definitions of terms.\"  Professor Winslow said that the Rural Study was one of the most valu- able contributions to public health by the Committee. Reference was made to the splendid work of Dr. Freeman on the report. It was voted that a letter be sent to Dr. Freeman expressing the gratitude of the Committee for his very excellent work.  In discussing record forms for rural health work, Dr. Emerson point- ed out that there was bound to be duplication if two booklets on record forms are issued, one for urban and the other for rural health work. He felt that for the purposes of teaching and for sale it would be advantageous to issue one book. Since the principles are essentially the same, it might be possible to do this. Other members pointed out certain difficulties in carrying out this idea.  It was moved by Dr. Emerson, seconded by Dr. Bishop, that the C.Aa.P. recommend that the Committee on Record Forms and the Committee on Rural Health Work consider the possibility of a unified publication, insofar as it seems . practicable. CARRIED. ,    Vital Statistics  Dr. Dublin stated that he had been advised that all the Vital Statistics Section Committees allocated to this sub-committee would make re- ports at the Annual Meeting in Indianapolis. Copies of these reports are not yet available.  Organized Care of the Sick  In the absence of Dr. Davis, Chairman, Dr. Walker reviewed the work of the committee during the past year. The tentative draft of the hospital survey schedule and appraisal form is now ready for review by Dr. Davis's com- mittee, which will meet for that purpose the latter part of October or early in November.  Dr. Emerson said he had many misgivings concerning the C.A.P- enter- ing the field of evaluation of hospital work.  Dr. Walker said that from discussion of this form with the representa- tives of the American Hospital Association it was his feeling that they would welcome the work of the committee.  Dr. Bristol told of the favorable reaction of the committee in Montclair, New Jersey, to the report which Dr. Buck presented after studying the hospitals in Montclair, using the tentative survey schedule developed by Dr. Davis's committee.  It was suggested that a representative of the nursing profession be added to this committee.  Manual of Public Health Administration  Dr. Vaughan stated that because of lack of funds nothing has as yet been done toward the development of a manual. His committee had served as a Clearing house on questions of administration referred from the central office. He felt that the committee might well disband.  The committee opposed this suggestion. Annual City Health Department Reports  Dr. Rice reported that his committee had prepared a form setting up basic information which the committee thought might be secured from cities and published early in the year. The form was sent to the central office, but nothing further had been done about it.  Dr. Buck stated that this form was considered in connection with the work of the sub-Committee on Current Practices of Health Departments and that it was felt that the information called for on the form might be tabulated from the Health Conservation Contest material.  It. was suggested that further discussion of the work of Dr. Rice's committee be deferred until after the report of the Committee on Current Prac- tices of Health Departments.    ms  Public Health Nursing  Miss Nelson said that she had no forml report to present for this Committee. She reported that the Evaluation Study of Public Health Nursing is about ready for publication. She called attention to the wide distribu- tion of the statement on \"Objectives\" issued by the N.O.P.H.N.  Health Conservation Contest  Dr. Dublin presented a very gratifying report on the Contest. There are 141 cities already enrolled in the fifth Contest. The state health depart- ments are cooperating in providing field service which had to be discontinued from the central office because of curtailed income for the Contest.  Professor Winslow in commenting upon Dr. Dublin's report spoke of the credit due Dr. Buck for his efficient management of this project. Dr. Buck was unwilling to take full credit for the Contest but told the Committee that due credit and recognition should be given Miss Tonnele, of the office staff, for her untiring efforts and loyal work in connection with the Contest.  Dr. Emerson brought up the question of what should be done with the group of cities that have won first prize in the Contest for several years. The matter was discussed at some length and then referred to Dr. Dublin's com- mittee for further consideration. Dr. Dublin asked that committee members send to him any specific suggestions which they may care to make on the subject.  Medical Participation In the absence of Dr. Rankin, Dr. Vaughan read the following report:  \"The purpose of the committee is to bring about a larger, more effective, and more harmonious cooperative relationship between members of the medical profession engaged in private practice and health officials.  \"In discussing the possible service that this sub- committee might render both health departments and the medical profession, there was universal agreement by the sub-Committee that:  (1) Organized medicine has a legitimate interest in the field of prevention as well as in the field of cure, which should be respected, en- couraged, and promoted by health departments.  (2) To ignore or disregard the interest of organ- ized medicine in the prevention of disease would ultimately lead to:  (a) the employment, at great and unneces-~- sary public expense, of excessive , public health personnel, physicians, nurses, dentists, etc.;    (bd) the assumption by public officials of a large service for which physicians are educated and prepared in every way to render and for which they are entitled to reasonable compensation, and  (c) a spirit of conflict instead of one of cooperation in a problem of mutual inter- est between the public, on the one hand, and the profession on the other.  \"While the committee has itself been inactive because of lack of funds, one member of the committee, Dr. Henry F. Vaughan, has done a great deal of splendid work in bringing about harmonious working relationships between general prac- titioners of medicine and health authorities.  \"The sub-Committee asks that it be continued and it is to be hoped that funds may be procured for carrying on an active program. \"  Some discussion arose as to whether or not the publicity for the principles set forth in this report would be psychologically wise at the pres- ent moment. It was voted to lay the report on the table until another meeting at which Dr. Rankin can be present.  Evaluation of Administrative Practices Dr. Emerson reported as follows:  \"The prenatal study, under the direction of Professor Winslow, has been completed and copies of the report sub- mitted to the committee.  \"The laboratory diphtheria studies carried on by Dr. William H. Park have been completed. The epidemiologic diphtheria study, under the supervision of Dr. Edward S. Godfrey, Jr., was discontinued in May. The study was not completed but the funds were exhausted and no further sup- port could be secured at the present time. However,  Dr. Godfrey is continuing certain phases of the study without financial assistance from the committee.  \"The scarlet fever study, under the guidance of Dr. J. BE. Gordon, has been continued this year and funds are available to carry this work on until the fall, at which time it is hoped that additional financial assist- ance will be secured so that this project may be carried on for at least another year.\"  The committee expressed the hope that it might be possible to get the prenatal study published and permission was granted Professor Winslow to arrange’ for publication, if possible.    PO  Dr. Walker pointed out that as yet no summary of the evaluation  studies has been published. It was sugges tee prepare for publication a report stati study and conclusions.  Current Practices of Health Departments  Dr. Mountin reviewed the activit  ted that the chairman of the commit-~ ng the problems studied, method of  ies of his committee since its ap-  pointment. Because of lack of funds the work of the committee this year has  been confined to summarizing the data from schedules. It is to be hoped that for th collected from states, cities and counties  (1) Personnel (classified by typ (2) Expenditures (by functions -  In discussing this report Dr. Ri the Contest would be published giving the officers were interested in knowing what w rather than for a group of cities.  Dr. Buck stated that according t National Chamber of Commerce the informati  the Health Conservation Contest @ coming year information may be on:  e, full-time, part-time) if possible)  ce asked if the data summarized from name of the city. He felt that health as the practice in a given city,  Oo our present agreement with the on from cities is considered confiden-  tial and may not be released except in summarized form. However, he thought  it would be possible to secure the consent it could be published giving the name of t  Further discussion brought out t the United States Public Health Service an work of this committee. It was voted to a Provincial Health Authorities to appoint a committee, to work with the sub-Committee partments. It was further suggested that purpose of developing a plan of work.  Professor Winslow asked Dr. Kend when it meets.  ‘Relation of Social Worker to Public Health  Dr. Walker presented the followi  \"Something more than a year appointed to consider the relati public health work. As all appo respondence and no previous disc purpose of the committee had tak confusion in the minds of those purpose. It was finally agreed dividual consultation, that the sideration to the use of social program and to discuss with the  of health officers concerned so that he individual cities.  he need for the close cooperation of d state health departments in the  sk the president of the State and committee, or designate an existing on Current Practices of Health De-  a joint meeting be held for the  all Emerson to sit in with this group  ng report:  ago, the committee was  on of social workers to intments were made by cor- ussion of the function and en place, there was some appointed as to its exact  by the members, through ine function was to give con- workers in the public health American Association of    Hospital Social Workers, as an organized representative of this profession, what they believed to be sound relation-~ Ships and how they might be furthered and what would be gained by collaboration of the two organizations.  \"As is well known to many of us, Dr. Bigelow and other state and local administrators have made excellent use of socially trained individuals in the promotion of the public health program both in the fields of tuberculosis and venereal disease and chronic disease service.  \"In accordance with this program, the American Asso- clation of Hospital Social Workers was approached early this year and the project discussed with Miss Helen Beckly, the executive secretary. The questions which might finally come before such a joint committee were as follows:  1. What is there in the field of public health which requires the skill and training of a social worker?  2. Should this be a medical social worker, or is it more the field of a generalist?  3. If it requires special training, what ought to be included in that training?  4. What administrative set-up should there be: should this person be a part of the department of public welfare or under the health department?  \"Miss Beckley indicated the interest of the Associa- tion in discussing these questions and at her suggestion Miss Elizabeth Gardiner, the president, has appointed Miss Marguerite Spiers of Oakland, Cal., Mrs. Charles W. Webb, of Cleveland, Ohio, and Miss Hlizabeth P. Rice, of Boston, as members representing the A.A.H.S.W. to discuss the quese tions with us.  \"No meeting has been held of the Committee on this subject as it was believed that at the present time the urgency of the question did not justify the expenditure of funds.  \"At the suggestion of the committee, Dr. Buck has sent questionnaires to certain state health departments and to the 50 largest cities to inquire concerning the use of so- cial workers in the health departments. The returns so far show that three state health departments and 16 cities have. indicated that they have social workers on their staffs.  It is the plan of the committee to seek a meeting of rep-  resentatives from some of these states and cities at least at Indianapolis with representatives of the A.A.H.S.W. for the purpose of discussing this material further and seeing  to what extent such a joint committee may be helpful or ad« visable.\"    Pre,  Appraisal of Industrial Health Service  As copies of the report of this committee were available at the meete  ing, Dr. Bristol made the following summary of activities:  \"The method of procedure agreed upon was that so far aS practicable each committee member would place in the hands of a company manager or supervisor who might be will- ing to cooperate in the trial of this Form: (a) a copy of the original reprint on the subject, and (») a mimeographed copy of the preliminary and tentative Survey Schedule which is nothing more nor less than a questionnaire, with the re- quest that this be filled out and returned with personal comments or criticisms as to its value and feasibility to the committee member, the latter would then have the Appraisal or scoring part of the Form filled out for the unit in ques- tion, on the basis of the information received in the Survey  Schedule.  Preliminary Appraisals Obtained  \"Up to date, members of the sub-committee have been in-  strumental in obtaining preliminary and confidential Appraisal Forms filled out by:  (a) Two eastern companies of the Bell Telephone System;  (b) Two units of General Motors Corporation; (c) A department store on the West Coast;  (d) A paint, roofing and floor covering company of California.  \"In addition to the above, a small company in New Jer- sey considered the Form briefly without attempting to score its health activities.  Next Steps  \"When at least ten or twelve such Appraisals have been received your sub-committee will proceed to hold further meet- ings for the study and consideration of the entire subject and for the possible revision and development of a proposed offi-  cial Form for the Appraisal of Industrial Health Service.  General Comments  \"The general comments received to date from companies using the preliminary and tentative Form would seem to indi- cate that;    LQ.  (a) The original Form, as proposed, is too comprehensive and should be shortened and simplified.  (b) Under present economic conditions, and the low level of health activities in industry, it will be difficult to inter- est industries in such a Health Appraisal Form.  (c) It may be desirable eventually to de- velop two or three Forms - for large, medium and small companies, with some possible variation based on the type of organization and work involved.\"  The committee expressed its approval of the progress already made in  the development of the Appraisal Form for Industrial Health Service.  Report of the Field Director  Dr. Buck reported that surveys have been made in the following cities: New York City - Tuberculosis survey Cambridge, Mass. - Tuberculosis survey Chicago, Ill. Survey of Health Department Montclair, N. J. Hospital Survey  '  t  Income from surveys this year will total approximately $3,000.00.  Dr. Buek reported further:  \"In addition we are negotiating with Elizabeth, N. J., for a hospital survey and have had inquiries from Jackson- ville, Florida, and Salt Lake City, Utah, regarding health surveys. It seems quite sure that we will be called upon for a health survey of New Mexico about January of next year.  \"We have during the year maintained our usual close co- operation with the National Tuberculosis Association, the National Organization for Public Health Nursing, the Ameri- can Social Hygiene Association, and the American Child Health Association. We have been répresented on the Summer Round-Up Advisory Committee of the National Congress of Parents and  . Teachers.  \"The Field Director has attended and given papers at meetings of the Western Branch of the American Public Health Association in Pasadena, the State and Provincial Health Authorities of North America in Washington, and the National Tuberculosis Association in Toronto. He also participated in the Tuberculosis Institute held in Philadelphia. Invita- tions have been accepted to present the Health Conservation    il.  Contest to the Connecticut Public Health Association, to talk at the meeting of the Vermont Conference of Social Workers, and to participate in the program of the Texas Public Health Association and the Texas Public Health Nursing Association. \"  With reference to the present financial status of the Committee, Dr. Buck reported that expenditures for the first eight months had totaled  $12,646.92, and that the expenditures for the year would amount to about $18,970.92.  The total income to date, minus $5,500.00 to be held over for the completion of this year's Contest, amounts to $23,973.65. There is an addi- tional $1,000.00 to come in on field service. This means that we have a sur- plus of approximately $6,000.00. It was Dr. Buck's feeling that some of this might well be spent in rendering a limited amount of field service in connec- tion with the Contest, if the committee approve. The committee raised no objection to this suggestion.  Professor Winslow said that this financial report was an example of the excellent Management of finances by Dr. Kendall Emerson and Dr. Buck.  At the luncheon Professor Winslow stated that the purpose of this meeting was to discuss three important functions which all those interested in the maintenance of public health protection should unite in fostering:  (1)- The Stabilization of public health appropriations.  (2) The stabilization of sound health organization and administration.  (3) The evaluation of public health activities to the end that funds expended for public health shall be used in the most scientific and effective manner.  is Stabilization of Public Health Appropriations  A general discussion followed of the present problem and the part the committee might play in bringing about stabilization of health appropriations. Dr. Dublin reported on the work of the Association's Committee on Stabilization  and referred to the activities of a Similar group of the National Municipal League.  : Mr. Folks, Chairman of the National Tuberculosis Association committee, read the resolution passed at the Toronto meeting.  Mr. Kingsbury offered the Suggestion that a telegram be sent to ’ President Roosevelt calling attention to existing emergencies in state and local health organizations, and asking him to give a short radio message on the    12.  Subject at the time of the Annual Meeting in Indianapolis, or at his conven- lence. This suggestion met with the approval of those present and Professor Winslow asked Mr. Kingsbury, Mr. Folks, Dr. Ferrell and Dr. Kendall Bmerson to draft a telegran.  The following Message was approved and sent to President Roosevelt:  \"At a meeting of an administrative committee of the American Public Health Association in New York today eXisting emergencies in State, city and local public health organizations official and voluntary, were re- viewed and their serious implications recognized stop we enthusiastically share your expressed view that nothing is more important to the state than the health of its people Stop we therefore urge you to exert the great influence of your office ina radio message to the American people at the time of the American Public Health Association Convention in session at Indianapolis the week of October eighth stop we respectfully request an opportunity for representatives of this committee to present to you personally these facts and this request  John A. Ferrell, President, American Public Health Association  E. L. Bishop, Chairman of the Executive Board  C.-H. A. Winslow, Chairman Committee on Administrative Practice  Homer Folks  John A. Kingsbury  Kendall Emerson, Executive Secretary American Public Health Association  Word has been received from the President that he will be unable to deliver a radio talk, but he will send a letter.  After further discussion it was the consensus of opinion that work on stabilization of public health appropriation should be left in the hands of existing committees appointed to carry on activities in this field, and that the Committee on Administrative Practice should cooperate in every way possible.  eg. Stabilization of Sound Health Organization and Administration  It was felt that the Committee on Administrative Practice might render  valuable service in this field. Reference was made to Los Angeles, Cal., where the local medical society had set up an outline of the duties of a health de- partment. Dr. Pomeroy had sent copies of this material and his Pepiy to a  humber of the members of the Committee for personal comment and advice. It was pointed out that if the committee had had &@ prepared statement on what consti-  tutes sound health organization it would have been more valuable to Dr. Pomeroy than individual comments.  Dr. Haven Emerson stated that this is no time for inter-professional controversies. Our job is to tell people what is a good public health program and not to take part in controversies.       13.  After further discussion it was voted to authorize the chairman to appoint a small committesc to draw up a brief, concise statement setting forth the essentials of public health organization; this statement to be presented to, the C.A.P. at a meeting in Indianapolis, and when approved to be submitted to the Association for endorsement. It was recommended that the committee appointed for this purpose should be retained as a permanent committee to act in an advisory capacity on questions pertaining to health organization.  Professor Winslow appointed the following committee: Sub-Committee on Essentials of Health Organization  Dr. Haven Emerson, Chairman Dr. Matthias Nicoll  Dr. Thomas Parran, Jr.  Dr. John L. Rice  Dr. W. F. Walker  3. The Evaluation of Public Health Activities to the End that Funds Expended for Public Health shall be used in the Most Scientific and Effective Manner  Professor Winslow stated that this project was in keeping with the work already being undertaken by the committee and that further discussion of it would be deferred until the afternoon session.  After the luncheon session the committee resumed the regular order of business.  Appraisal of City Health Work Dr. Palmer presented the following report:  \"The work on the revision of the Appraisal Form has been in progress for the last three years. The completed form is now submitted for approval to the Committee on Administrative Practice with the understanding that suggestions will be presented to the sub-Committee on Revision for action on October end. In the revision effort has been made to har- monize the content with that of the Rural Appraisal Forn. Comments have been solicited from the chairman of the various sections of the American Public Health Association, from the national voluntary health associations and from other inter- ested groups and individuals. Valuable suggestions have been received and incorporated into the Forn.  Introductory Statement  This has been completely revised in order to interpret the uses and purposes of the Form more clearly. ,    Pe  General Information  Considerable revision has been made in the general statistical information requested. A new feature has been added in the introduction of charts to permit cities to observe their standing in certain particulars in comparison with other cities.  The Appraisal Form  The new revision provides for 13 independent sections, two of them, \"Other Disease Activities\" and \"Other Activities\" not being scored. The old form contained 14 sections. One of these \"Heart Discase Control\" has been omitted. Another, \"Cancer Con-~ trol\" has been merged in the section on \"Other Disease Activities.\" Another, \"Popular Health Instruction\" has been distributed over the various sections.  Although the Laboratory Section is likewise distributed over other sections in the Rural Form, our Committee decided to retain this material as an independent section.  4 new column has been provided in which the work done by private physicians may be entered.  Principal Changes  Numerous changes in details have been mad", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-byxr.4rsr_m6ne", "00000000-0000-0000-04F3-CD52EEAD97EE", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Vital Statistics: A Study of the Practice in Municipal Health Departments", "9918573882206676X118", null, "1922", "1922", "Louis I. Dublin, Vital Statistics: A Study of the Practice in Municipal Health Departments. American Journal of Public Health, 12 (1922).  This article by Louis Dublin was also a chapter in the report of the APHA Committee on Municipal Public Health Department Practices published in 1923.", "Articles", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "10", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "VITAL STATISTICS*  (Chapter from the Forthcoming Report of the Committee on Municipal Health ae Department Practice)  INTRODUCTORY  TATISTICAL work is for many reasons one of the least developed of  _ the activities of municipal health de- partments. The scienceof vital statistics is still young and its rules and practices as applied in health administration are far from fixed and formulated. It is not often closely connected with specific and urgent health measures. It has been de- veloped largely to serve as a method of control and for purposes of research. It requires often the exercise of a special technique with which many health officers are unfamiliar. As many do not know exactly how to profit from such work, they do not provide for it in their bud- gets. Nor is it always possible for those health officers who do appreciate the value of good vital statistics, to obtain the necessary funds for this activity from the city authorities, or the codperative service from the rank and file of the medical profession, who, in the last an- alysis, are the chief source of the records which make up the material of public- health statistics. Nevertheless, there has been, in recent years, much stimulation of vital statistics in American cities, and health officers are more and more par- ticipating in their use. While there is still much diversity of procedure and preat inequality of accomplishment, the field of vital statistics is coming’ rapidly ‘into its own. A real opportunity is pre- sented at this time to standardize and to adapt statistical procedures to the best service of American public health.  At the very outset, distinction must be made between the several activities which make up the statistical work of health departments.  Many health officers consider the regis- tration of births, deaths and perhaps of  cases of sickness, as the entire field of vital statistics in their departments. This is, however, only the beginning. In addi- tion, there is the important function of investigation. This involves tabulating the results of the registration of births, deaths and sickness and of analyzing and interpreting the tabulations. It is per- haps equally important to carry out in- vestigations into the make-up of the population and into special phases of the public health. Where properly ‘con- ducted, the investigations of the public- health statistician should show what the program of the health officer has accom- plished and what new activities are called for. The results of vital statistics inquir- ies should be a guide in developing the policy of the health officer. In this wise, the vital statistics division is an essential element in the health officer’s organiza- tion. It is the research or checking  agency determining what is good from  what is bad, and the degree of success which the various divisions of the depart- ment attain. Very few health depart- ments have carried out their vital statis- tics work in this spirit; but, this is ob- viously the program for the future.  - JURISDICTION  In a number of cities, the registration of vital statistics antedates the organiza- tion of public-health work. It is not sur- prising, therefore, to find occasionally that registration is outside the jurisdic- tion of the health department. Much de- pends upon the registration laws of the state. In the great majority of the cities, however, in 63 out of the 83 studied, the health department has local jurisdiction. This is true in the 12 largest cities studied, with the exception of Boston and Pitts-  *Prepared by Louis I. Dublin, Ph. D., Statistician, Metropolitan Life Insurance Co., New York City.             burgh. Likewise, in the 13 cities of the second class, with the exception of Newark and Jersey City, registration is a local health function; and in 42 out of 58 cities of the third class.  The exceptional situations arise out of the working of the state laws. In Massa- chusetts, for example, and in a number of other states, vital statistics registration is under the control of the secretary of state. The local administration of the law is carried out by city or town clerks, who report directly to the state official. In the Massachusetts cities, under this law, the health officers may receive the death certificates from the undertaker, issue the burial permit, and then send the certificate to the city or town clerk, by whom it is filed and return made to the office of the secretary of state. The vital point is, however, that in those cities where the health officer is not the local registrar, he has not direct access to the certificates of birth and of death and he receives copies of them from the town clerk only as a courtesy. They reach him late, and because of his lack of responsibility for them, he is very unlikely to use them for any constructive purpose. :  But, even in those states where juris- diction is with the state department of health, the local health officer may not be the registrar. This apparently occurs in Pittsburgh and Louisville. Confusion of responsibility and duplication of func- tion often result under such conditions.  COMPLETENESS OF REGISTRATION  As the cities are all* in the death regis- tration area, the registration’of deaths is reported to be virtually complete in most of them. Seuencities, including Milwau- kee, Kansas City, Mo., St. Paul, Omaha, Dallas, Houston, Salt Lake City and Du- luth, succeeded in registering only 95 per cent.of the deaths.  in the middle and southwest, Den-     *With the exception of Forth Worth and Des Moines. : r  +Only fifty-eight cities in this study are in the Birth Registration Area. —  Six additional cities —  ver, San Antonio, Des Moines, Fort Worth, Kansas City, Kan., and Okla- homa City, succeeded only to the extent of 90 per cent.  Birth registration, on the other hand, is much less completely carried out.t It is a newer activity in the cities of the country, and only a few of them make the claim of 100 per cent completeness. These are Grand Rapids, Utica, Lowell, Somerville and Elizabeth. The rest vary considerably in the extent of their claimed completeness of registration, from 70 all the way to almost 100 per cent. It is important to note that in the cities of the first class, there are still some, like Cleveland and Chicago, which succeeded only to the extent of between 70 and 80 per cent. Much, obviously, de- pends upon the location and age of the ° cities, the provision of the state law and the traditions for good registration that have prevailed in the vicinity.  From the comments made by the field agents of the Committee, it would appear that the best registration is obtained in those cities where the local health de- partment has immediate responsibility. With few exceptions, poor registration results when the local authority is vested in the hands of some one who is not the health officer. This applies especially to | birth registration, because the immediate utilization of the birth certificates*by. the health officer for the conduct of infant hygiene work, stimulates physicians and midwives to return complete and early certificates. On the other hand, where there is no such use made of the birth records by the health officer, registration is likely to be carried out in a perfunctory manner.  It should be realized that the collection, the recording and the prompt transmis- sion of birth and death certificates to the State authorities, are the first duty of the municipal registrar. The success or fail- ure of a vital statistics division will be determined to the degree to which these functions are carried out. The other 5  activities of such a division will be pos- sible only as these primary ones are fulfilled.  PROSECUTIONS AND CONVICTIONS  No prosecutions are on record for the non-reporting of deaths. Only a few cities reported any prosecutions or con- victions for non-reporting of births. New York City, for example, reported five prosecutions and five convictions; Norfolk and Savannah, likewise, re- ported some cases. In Boston a few prosecutions for failure to report births are on record; but warnings are sufficient- ly effective usually, because of the har- mony existing between physicians and the registry department. Similarly, in  ~ other cities, much will depend upon the  tact and firmness of the registrar and. the degree of the cooperation which he can win from. the body of physicians. TABULATIONS AND PUBLISHED REPORTS Nearly all of the municipal health de- partments studied make tabulations of one sort or another from the birth and death certificates which they receive, re- gardless of whether they carry on regis- tration or not. These tabulations are in- tended to serve either as office manu- scripts without publication, or as the source for a variety of tables in weekly,  -monthly and annual reports.  Very often where no formal printed or multigraphed weekly, monthly or annual report is circulated by the health officer,  convenient summaries are submitted to  the local newspapers, and these serve as sources of information on important facts of the local vital statistics.  All of the 12 cities in the first group issue an annual report; ten of them issue formal monthly reports, one city (Chi- cago) includes its monthly summaries with the weekly report. In two of these (Cleveland and Pittsburgh) monthly re- ports are circulated in typewritten form. In addition, 7 of these first-class cities issue either printed, mimeographed, or typed weekly reports. Twelve of the 13 cities in the second class issue some form  of report, 8 an annual report, 10a monthly and 5 issue a weekly report. All of the cities of the third class, except Camden, Kansas City, Kan., Elizabeth, Utica and Erie, issue some’ form of weekly, monthly or annual report. In a few cities this report consists often of a single sheet, giving simply the number of deaths during the year from principal causes, the number of births, and a few other items of information which, it seems to the registrar, would be of in- terest to his local public.  ANNUAL REPORTS  The vital statistics sections of the an- nual reports vary greatly from city to city, depending very largely upon the resources available in funds and person- nel. In a number of cities where a fair amount of valuable detail is shown in the published reports, the date of issue is much delayed, presumably by the lack of adequate clerical assistance to take off tabulations and to prepare the material for the printer. When these reports ap- pear, they are already out of date. But, apart from this criticism, the annual re- ports of the larger cities are often valu- able documents. Those from the cities of New York, Chicago, Philadelphia, Boston and Providence are replete with useful information. The New York re- port usually contains excellent historical tables on the course of mortality from the principal cause of death. These tables have been supplemented at various times by the issuance of condensed re- views and special monographs abstracted from the manuscript and the records in the New York Registrar’s office. A com- mendable activity in New York City is the preparation of fairly detailed statis- tics for sanitary areas of New York City.  The Philadelphia annual report gives extensive and valuable information on births by months, ward or residence of mother, by age, color and nativity of mother and by occupation of father. Facts for the causes of death are shown according to titles of the International          List of Causes of Death with respect to sex, age, calendar month and wards. The  Philadelphia report is one of the few -  which show annually the occupation of the deceased persons according to certain  important causes of death. The mortality,  from certain diseases, typhoid fever, for instance, is shown by wards of the city and by calendar year. The infant mor- tality tables are especially rich in detail. Favorable comment should also be made upon the tables shown in the annual re- port for the Newark Department of Health. The tabular matter is frequently supplemented by graphic charts and these promote the understanding of the his- torical and comparative facts in an agree- able manner. :  The reports of the. City Registrar of Providence are especially valuable for their full data on births by nativity of parents, age of mother and other items for the instructive arrangement of the tables showing deaths from all principal causes in relation to sex and age of the decedents.  Reference is made here only to some of the outstanding features of a few an- nual reports. It is impossible to discuss at any length the contents of all of them because of the great variations that exist in the types of tabulation, in the arrange- ment of the data and in the local needs for special information. There is ob- viously need for greater uniformity in the character of the reports issued by the cities of the various classes as to size. It is recommended that. the Section on Vital Statistics of the, “American Public Health Association be requested to pre- ‘pare a set of standard tables for use by ‘municipal health departments, with rec- ‘ommendations as to the best size and arrangement of .the report. _plan* ‘similar to: that outlined in the Cleveland Hospital and ‘Health Survey ‘could be used. as a guide. The types of tables on sopulation, birth, death and morbidity statistics shown therein are the kind of statistical material which should  Possibly a.  e  be in the annual reports of the larger municipal health departments. It should be remembered that the Census Bureau and the U.S. Public Health Service issue valuable statistical reports on mortality and morbidity for the principal cities, and local departments should strive to avoid duplicating these. The field for the local authorities is obviously to develop de- tailed data which will facilitate the ad- ministrative work of the health depart- ment. MONTHLY REPORTS  The monthly reports are the most fre- quently issued and the most useful be- cause of their timeliness. They contain usually a summary of the number of deaths which occurred during the preced-  ing month, by principal causes, and in a_  number of instances by broad age groups, singling out especially the deaths of in- fants under one year. In addition, there is also in most reports a table showing the number of births reported as well as one giving the number of cases of prin- cipal notifiable diseases registered. Out- side of these tables, there is very little uniformity among the published tabular results of the several cities. Some give the meteorological data of the previous month for the city in question. Others give summaries of the activities of the employees of various divisions of the department. Again, there is need for standardization of reporting of essential items, Some such arrangement as is shown in the general tables published monthly by the New York City Depart- ment of Health could profitably be adopted by the larger cities. In addition to facts on the causes of death, the cities could agree upon a form of statement of  the reports of the principal notifiable dis-  eases, and for births and marriages. WEEKLY. REPORTS  The few weekly reports which are pub- lished are decidedly scrappy in nature, and, outside of a small number, are hardly of much use. It would be highly desirable if all of the cities would issue simple, uniform weekly statements to sup-  plement what material the general statis-.  tical reader obtains from the weekly Public Health Service reports and from the Census Bureau Weekly Health Index.  PERSONNEL  Under this head, we may consider the registrar or statistician on the one hand, and the clerical staff on the other. The presence of a full-time registrar or statis- tician depends upon the size of the city,  but equally upon the interest of the health  officer in vital statistics as an aid to ad- ministration. Thus, in the 12 cities of the first class, there are 9 full-time regis- trars. In Cleveland, the registrar is on part time. In Baltimore, the health of- ficer acts as registrar, and in San Fran- cisco he is at the same time the chief clerk of the department.  In the 13 cities of the second class, 10 report a full-time registrar or person in direct charge of vital statistics work. In Seattle the health officer acts as registrar. No statement is available for the remain-  ing 2 cities, namely, Jersey City and In- -  dianapolis.  In the 58 cities of the third class, there are full-time registrars in 30. In Provi- dence the registrar is also the superin- tetident 20% )> health. =: Ine. Bridgeport, Memphis, Dayton and Oklahoma City the registrar is on part time and in one case he is the city physician. In the re- maining 23 cities, which are almost all among the smaller ones, there is appar- ently no one in direct charge of vital statistics. It is presumed that in such cases the duties are merged with those of the health officer or some physician or clerk in the department.  ~ The number and character of the cler- ical staff vary with the size of the city, with the type of registration law which prevails, but especially with the type of health administration. A health officer who is interested in modern and scientific public-health work will insist upon the  qn  tabulation and analysis of the vital data of his population, and he will at least attempt to secure an adequate staff of skilled statistical workers.  In New York City, where excellent traditions of public-health work and vital statistics have long prevailed, the staff includes, in addition to the highly skilled registrar, 5 ‘physicians who are as- sistant registrars, each supervising a bor- ough, 5 other physicians who issue burial permits, and 55 clerks. Among  the latter, 44 are engaged on registration  duty, 7 do strictly statistical sheet work, and 4 operate mechanical de-  -vices, such as card-punching and sorting  machines. There are two counting-sort- ing machines used in the preparation of the tables, which are equivalent to a con- siderable number of additional clerks.  But disregarding the item of machinery, .  the number of registration and statistical clerks in New York City is about one per 100,000 of population. In the cities of the first group, the average number of  ‘clerks is one per 100,000 of population,  in those of the second class .6, and in those of the third class 1.2.  The facts at our disposal clearly indi-  cate that there is a serious deficiency of  statistical personnel in the health depart- ments of American cities. To conduct adequate registration of births, deaths and of sickness, and to tabulate and an- alyze the data so collected, are serious tasks in most cities and require suf- ficient skilled personnel. From the re- sults achieved in New York City, and in a number of other cities where good vital statistics are produced, it would appear that a minimal clerical staff of one for each 100,000 of population was needed to assist the full-time and well equipped registrar. It is unfair and not at all economical to expect a health officer with his many duties to be the registrar and statistician as well, and al- most as undesirable to give the registrar untrained or insufficient help. Where this occurs, it is poor economy, for either          the health service must suffer through the lack of necessary guiding data, or through the misuse of the health officer’s or registrar’s time.  SALARY OF REGISTRAR  Data on the salary paid to the registrar or statistician are available for 43 cities. In some cities, as in Baltimore and Seattle, the health officer functions as the registrar and, apparently, no addi- tional salary is paid. In some cities, as in Providence, the health officer receives an additional stipend as registrar. In others a small salary is paid the registrar, which is supplemented by fees, as in Cin- cinnati and Toledo; and in a few, as in Atlanta and Reading, the registrar is paid by fees only. In a considerable number, however, little or no statistical work is really conducted and apparently no pro- vision is made to cover the salary of the registrar or his staff.  In the 43 cities where the salary of the registrar is stated, the amount varies markedly: The salary varies naturally with the size of the city. The same de- gree of skill and of administrative ability is not required of a registrar in a small city of 100,000 as in a city of 1,000,000 or more people. But, while this is gen- erally reflected by the figures, there are striking exceptions showing how di- verse is the conception of the city au- thorities of the value of such work. Thus, in cities of the first population group, 8 salaries are stated, and these vary from $1,584 in St. Louis to $5,500 in New York City. The next highest amount is in Chicago, $3,800. In Cleveland, the part-time registrar receives $1,/50. The average salary paid to full-time regis- trars by 7 of the first-class cities is just under $3,200.  In the cities of the second class, the salaries of 10 registrars are given. average for full-time service is $1,850, and varies from $1,260 in Portland to $3,300 in New Orleans. In Cincinnati, the salary. of $1,600 is supplemented by  fees. In the cities of the second class, all  The  but two, Milwaukee and New Orleans, pay less than $2,000. In 21 cities of the third class, the average salary paid to the full-time registrar or statistical chief is $1,630 and varies from a minimum of $560 in Paterson to a maximum of $2,400 in St. Paul. The part-time regis- trar in Providence receives a salary of $2,750.  It is clear from the above that the registrar in a. considerable number of cities is a clerk acting under the direc- tion of the health officer. Only: the routine duties of registrar are carried out under these conditions. It is to be se- riously questioned whether persons with adequate skill or ability to function as statisticians of health departments of large cities can be found who will accept salaries so low as those we have quoted. These are, in many instances, less than those usually paid to junior clerks in commercial pursuits. Almost without exception, the salaries paid to registrars in American cities are too low to insure the proper type of statistical service.  COST OF VITAL STATISTICS  It is difficult to give a_ satisfactory statement on the cost of vital statistics in municipal health departments. This re- sults from the great variety of procedures in American cities. As already pointed out, some of the large cities, Boston, Newark and others maintain a vital statis- tics division in their health departments and in addition support a registry office in the city clerk’s department. In the  _ great majority of cases, however, vital  statistics registration is a part of the health department’s activities.  Costs as reported to the Committee have not always been clearly classified to show what has been included. We can- not always tell whether the cost as given covers the registration work carried on outside of the health department. In any case, reports are available for 33 cities;  7 of the first class, 7 of the second class,  and 19 of the third class. In all, the per capita cost for vital statistics is 1.6 cents. )  This is also the average figure for the first and second class cities, The average is 2.5 cents for the cities of the third class. The per capita cost is, therefore, greatest in the smaller cities, and it 1s in them also that the greatest variations are, of course, found. The maximum cost per capita is found in Hartford, where 5.2 cents is expended, There are a number of these smaller cities where apparently nothing is spent on vital statis- tics. Among the larger cities, there is a little more uniformity of expenditure. In the city of New York, where both regis- tration and vital-statistics analysis are ad- mirably conducted, the cost is 1.5 cents per capita, and in Chicago 1.9 cents. In Boston the per capita cost in the health department is 3.6 cents, but this amount includes the cost of clerical work and supplies for the whole division of vital statistics, records and accounts. What the cost for vital statistics alone is, it is impossible to say. It must be remem- bered, however, that in Boston a consid- erable sum, unknown to the Committee, is expended by the division of the city register outside of the health department, so that altogether Boston ranks high in  ‘the list of large cities for its expendi-  tures for vital statistics.  In the cities of the second class, the high figure of cost for New Orleans is conspicuous, 4.6 cents per capita. In the cities of the third class, the expendi- tures for Providence, 4.9 cents; St. Paul, 42 cents: Birmingham, 4.1 cents; Hart- ford, 5.2 cents; Albany, 4.0 cents, all show high figures. The high per capita cost in Providence may be explained by the uniformly high standard of statistical work conducted there. For many years the annual reports of the Registrar have served as models of form, completeness and penetrating analysis of the depart- mental activities to the health officers of the country.  In view of the accomplishment of New York City and Chicago at costs less than 2 cents per capita, it would appear rea- sonable to suppose that an expenditure  of this amount per capita would bring the results in registration and analysis necessary for the conduct of effective health work in the smaller cities which require fewer tabulations and simpler an- alyses. Possibly the higher per capita cost of a skilled statistician in smaller cities and of a few other items of over- head will make the per cost of statistical work higher in the smaler cities which carry out this type of work seriously. It is not always possible to reconcile the high cost of vital statistics work in some of the smaler cities with the character of their published statistical output.  FEES  Fees are usually charged for tran- scripts of birth and death certificates and on file as the vital-statistics division of health departments who are usually the custodians of these divisions. In fact, this source of revenue often determines the amounts appropriated for vital-statis- tics work. Of the 68 cities which re- ported on this item, all charged fees for transcripts. For 38 cities reporting the amount charged, the fee was 50 cents in 26: cities anid 25 cents. mm. Seniesa Denver the fee is $1.00. The amount thus collected varies, of course, with the size of the city and the demand for transcripts. In New York City nearly  $50,000 was collected in 1920. But there  are considerable differences in amounts received in cities of about the same size. Thus, Chicago collected a total of $13,200 and Philadelphia only $3,157. Sometimes a large amount is collected in a medium-sized city, as in Hartford ($2,716), indicating perhaps the extent to which the public has been educated to avail itself of the facilities of the regis- trar. In this way, the amount received in fees is perhaps also an index of the completeness of registration and the re- gard which the community has for the records on file.  The amounts received are credited in 46 out of the 83 cities to the general fund of the city. There are a few (4),             such as Pittsburgh and Memphis, where the fees collected are received by the registrar. In a few cities fees are cred- ited to the health department, as in New Orleans, Indianapolis, Omaha, Duluth, Flint and Canton, and in a few the fees are credited to the state department of health, as in Kansas City, Kan., Reading, Erie, Scranton, Fort Worth and Des Moines.  SUMMARY AND CONCLUSIONS  1. Vital statistics as an_ effective agency in public-health administration has been developed by only a few munici- pal health departments.  2. Vital statistics registration 1s fairly well developed, much better for death registration than for birth registration. The conduct of registration is still com- plicated by confusion of responsibility. In some states where the law empowers city or town clerks to register vital statis- tics, the local health departments have no responsibility for the accuracy or com- pleteness. of registration. Under such conditions, registration is likely to be un- necessarily expensive and unproductive for constructive health work. The regis- tration of births is then often incomplete,  and of deaths rather inaccurate as to  statements of cause. 3. Nearly all cities issue some form of  report; the monthly report is most, fre- quently issued. The tabulation of data in these reports show very little uniform- ity as to method of compilation or as to content. There is great need for agree- ment, among health officers and municipal vital statisticians upon what tabulations are called for in cities of different popu- lation classes and upon the size and style.  4. The fevisitars are, for the most part, not well equipped for research work, and, in a considerable number of cities, are paid so inadequately as to pre- clude the employment of competent men. The clerical staffs are often inadequate in number. On the other hand, in many cities the number is sufficiently large to justify the expectation of better work than is now obtained. A minimal clerical staff of one clerk per 100,000 population, in addition to the registrar or statistician, should generally be sufficient for the con- duct of the vital-statistics work of the health department.  5. The average cost of vital-statistics work is 1.6 cents per capita. But there are altogether too many cities which spend next to nothing on their vital- statistics work, and also many cities which, although they spend considerably more than the general average, do not appear to profit particularly from the re- sults obtained.", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, "American Journal of Public Health", "American Public Health Association", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-khpr~vdut_8fqp", "00000000-0000-0000-D0EA-24A843C4980B", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Report of the Committee on Uniform Tables, appointed by the American Public Health Association", "9918573882206676X119", null, "1912", "1912", "This report reflects public health workers' quest to standardize data collection and analysis procedures.", "Reports", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "3", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "a oS : we hy ato Oo Lnes -  Report of Committee on Uniform Tables Appointed by The American public | Health Association. :     Annual Report , New York State Dept. of Health 1912.     fhe committee appointed to cooperate with a corresp onding committee of the American Statistical Association and with the » Bureau of the Census begs leave to submit the accompanying memorandum on uniform tables as its report of progress, with the request that such of the specific recommendeation as may seem advisable be adopted as rules of statistical practice. The cooperating committee of the American Statistical 2 Association, through its chairman, Professor Walter F, Willcox, Pe presents a draft of a report of the Association, with forms : of tables, which is incorporated in this report, subject to any amendments or alterations that may be made, prior to its adoption, by the American Statistical Association. :  There is also appended to this report a circular letter issued by the Bureau of the Census, and it is urged that the report be published, if feasible, perhaps as a census pamphlet, as a basis upon which definitive recommendations and forms of tables may be presented for adoption at the next session of the Association. |  ‘“ The problems involved are so far reaching and difficult 3 in their practical solution that it would seem unnecessary to —~ proceed slowly and cautiously rather than to exhibit undue haste vin the formulation of proposed forms. r Respectfully submitted,  —  Wm. C.Woodward Marshall Langton Price Chas. A,Hodgetts F.L.Watkins Committee,  Memoranda on Uniform Tables for Vital Statistics.  1. It is more expedient to discuss the general question of securing uniformity and to agree upon some general principles than it is to adopt at this time any actual forms of standard tables.  2s Standard tables should be considered with reference to their appropriateness for, first, the census reports; second, state reports and bulletins; third, city reports and bulletins.  : Se Census reports should deal primarily with states and large cities, giving only the fundamental data for small cities a | and counties. They should deal with data in the mass, for broad comparisons chiefly, and to establish standards (e.g.,corrected rates, general life tables) with which local comparisons can  be made. _Ns¥-State Depti of Hea}th-anmual Report~1022.  4, State reports should begin where census reports leave off, and study the state as a unit; and they should give special : attention to counties and groups of counties ( e.g. see Bulle- tins of Indiana, Michigan, and New York ), and should present |  data in considerable detail for small cities and villages,singly and grouped into cities of from 5,000 to 10,000 population,etc. They may even take up the individual towns or townships when such units are important, but they should hot go into the sub- divisions of large cities that maintain an efficient registra- tion service,  5. City reports, especially those of large cities, should deal with the primary units of area (city blocks) grouped into such larger aggregates ( wards or sanitary districts) as may be found advisable, and they should specialtize on morbidity statistics and their relation to mortality statistics.  6, Whatever mortality statistics are worth printing et all are, as a rule, worth printing with full details of sex, age, and color ( when colored pepulation is ten or more percent) « This refers to the primary tables.  7. \"Unknowns\" should never be\"distributed\" but should appear — as a criterion of registrative efficiency for sex, age,color and all other fmumux items. |  8S, Important general tables, and especially those for causes of death, should show ages, by sez, for each xm quinguennial period from 0-4 to 95-99. The first quinquennial peried should be subdivided into single years. A detailed list of centenarians should be given, and for infant mortality the first year of life should be stated by months, the first month by weeks, and the first week should show at least the first five days individually. For tables of occupations the periods should be: 10-14, 15-19, — 20-24, 35-44, 45-54, 5564, 65-74,75-84 and 85 and over, with special statement for children under 10 years, when child workers under that age are found. :  .9. All main tables of causes of death in reports of states and large cities should use the detailed International Classifi- cation whthout change or alteration, except the Subdivisions (but not additions or changes) of titles may be made when necessary. For minor tables in the same way, and for special purposes shorte! selected lists may be made, each title representing certain de- finite tikles of the detailed International Classification. Fach title should bear its International Classification number to assure ease of comparison and certahnty in regard to the in- ¢lusion of terms.  10. For the following diseases, tables should be presemued by calendar months and by the regular series of age periods until less than five per cent may be embraced in the final period: _ Typhoid fever :  Malarial fever  Measles  Searlet Fever  Whooping Cough  Diphtheria and croup 0 Influenza  Tuberculosis  Bronchitis  Pneumonia       N.y.State Dept.Health-Annual Report - 1912 . Ss -«  Bronchitis renumonia 7 Diarrhea and entritis By months, Weeks ahd days of age. Congenital debility, ete By months, weeks and days of age.  a Causes of death with death rates should be presen’ ed for each year since eginning of registration if pract- teable according to the abridged International Classification, | . otherwise according to the list of the International Table of the Registran-Ceneral's Report. oe  12. The first general tables constructed ghould pro-= vide for the data contained in the main tables of the inter= — national statistics of the Statistique Generale of France. | | 13. Every report should contain a table showing the dates and population, by sex and agesy. at several recent censuses ° - * | 14. Every report should contain a table showing the elements of population, by sex, age, color, nativity, etc., at — aoe CONnBUS »", "American Public Health Association. Committee on Uniform Tables", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tmpc_g5q4_xsgb", "00000000-0000-0000-A01E-115E5C3D1567", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Annual Report of the Committee on Administrative Practice, 1926-1927", "9918573882206676X120", null, "1927", "1927", null, "Reports", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "5", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "SS Tia, een PS TSE  Annual Report of the Committee on Administrative Practice  1926 ~ 1927  The Committee on Administrative Practice was reorganized and placed on a permanent basis at the Buffalo meeting of a year ago. It is now com- posed of twelve members appointed by the President of the American Public Health Association to serve for four year terms, in overlapping groups, so that three members are appointed each year; plus the executive secretary of the Association and the taree officers of the Health Officers! section as ex officio members, - sixteen members in all.  The committee, as its work has developed during the past five years, has come (with its Field Staff) to constitute essentially the technical ser- vice division of the A.P.H.A. organization. It appears to have at the moment three essential and primary functions. These are:  A. The collection of material in regard to existing health depart~ ment practice by surveys and questionnaires.  B. The critical analysis of the data thus obtained and the formla~  tion of the results of such analysis in the shape of standard forms and  programs.  C. The making available to individual communities of the results of procedures A and B by operating an information service and by making on re- quest more or less detailed local surveys and appraisals.  | A fourth function, that of organized publicity to further the improvement of health department practice by the wider use of the technical machinery now available, has been more or less forced upon the Committee; but we believe that mich of this work should be shifted to the recently organized Promotion Division of the A.P.H.A. office.  We may conveniently review the work of the past year and the plans for next year under the headings just outlined, although the first two funce- tions, the collection of data and the development from them of standard forms  and programs, must necessarily be considered together since they are intimately  interlocked.  The most important single task, upon which the Committee is now  engaged is the revision of the Appraisal Form for City Health Service under a  sub-committee cf which Dr. George T. Palmer is chairman. This Appraisal Form, which was placed in your hands in January 1926 has exhibited a usefulness be- yond our highest expectations. It has been said, and we believe, with some justice, that no single factor has ever done more than has the use of this appraisal form to develop city health department practice in the United States; and it has been extensively used even in foreign countries. The Committee has, however, felt from the beginning that there were two very real dangers in the practical application of the appraisal principle. It might, on the one hand, invite unjustified and damaging criticism of departments having low scores, and it might, on the other, lead to undue standardization and to stagnation on the part of cities having high scores. The first of these    hazards has, so far as we are aware, deen completely avoided by the use of the Appraisal Form in an individual city only with the approval and on the request of the Health Officer and by the avoidance of any comparison of total Scores obtained by various cities on a comparative basis. The second hazard, we have planned to obviate by revision of the Appraisal Form at three year intervals and the first revision will be completed during the coming year and presented to you at the 1928 Anmial Meeting. It is plamed, not only to alter items in the present Form which have been shown to be theoretically and practically unsound and to increase levels of attainment required for attain- ment of a perfect score to keep pace with current progress, but also, if possible to include certain new items not now included at all. Individuals and organizations interested in Mental Hygiene and in Industrial Hygiene, and in the control of heart disease, cancer and malaria, will be asked to submit for their respective fields standard administrative programs with appropriate numerical ratings of various activities on an objective basis; and if it  proves that such programs and ratings can be prepared they will be included in the 1929 revision.  A second project, of major importance, is the attempt to extend the Appraisal principle to cover the health machinery of rural counties. The sub-committee on this project, under Dr. BE. L. Bishop, has prepared during the year a tentative Appraisal Form for Rural Health Work which is placed in your hands at this meeting in printed form. This form was published some time azo and has already been extensively tested out in practice by the Committee in Tennessee and to some extent in the states of New York, and Georgia. County or district health officers from fifteen states have written to us for 402 copies of these forms and up to October 1, 652 copies in all had been distrib. uted. This Tentative Form will be continued in experimental use till 1929.  In order to proceed soundly with the preparation of programs for rural health work, it seems to us essential that we should have the samdé broad basis of kmowledge which was obtained for city health services in the compre- hensive surveys of 1920 and 1923. Dr. Bishop has therefore prepared a defin- ite program for a two-year study in which 50 typical counties should be sur- veyed, covering all sections of the country and all degrees of public health development. He presented this program at the Surgeon-General's Annual Conference of State and Territorial Health Officers last spring. The matter was referred to a Committee of the Conference on Standard Forms for Reporting County Health Work (Dr. Ss. W. Welch, Chairman). Dr. Bishop's committee and Dr. Welch's committee are now working on the problem with every prospect of hearty agreement. It will be necessary, however, to obtain Special funds to the extent of $25,000 if this work is to be undertaken.  In addition to the appraisal forms which cover in a general way the  whole balanced program of community health service, your committee hag pro~  ceeded actively during the year with the study of certain details of health  department procedure wnich seemed to lend themselves to analysis in the direction of uniformity of practice. Thus, a Sub-committee on Record Forms, | with Dr. Geo. C. Ruhland as chairman, has rendered a notable service during the year in the preparation, after a vast volume of correspondence and Critical study, of sets of Standard forms to be used in connection with the  communicable disease Service, the laboratory Service, the school medical in-  spection and the nursing service of thé health departments. These four sete  of forms are before you in printed form ag the Second definite evidence of  work completed by the Committee during 1927. Dr. Ruhland's sub-committee is  now proceeding with the preparation of similar forms for use in connection with tuberculosis and venereal disease. x  «a=  Dr. C. Hampson Jones! sub-committee on Standard Health Department Reports has prepared a preliminary analysis of the functions and contents of @ model anmial report which will be read before this section by Dr. Jones and Professor Hiscock, a third concrete result of our sub-committee activities presented at this convention. Topical outlines for the various sections of the annual report will be worked out in detail during the coming year.  A sub-committee on Model Ordinances (Dr. H. F. Vaughan, Chairman) is at work on a schedule of the main essentials which should be the objectives of a health code and its final report is promised by next spring.  An important special problem, that of the relation between the health department and the local hospitals has been considered by a sub- committee of which Mr. Michael Davis is chairman and his very interesting report based on a questionnaire sent out to 247 health departments and to 1365 hospitals and dispensaries will be read at the meeting.  The problem of the hospital has been forced upon the attention of your committee, not only as a cooperating agency in the primary activities of the health department but, as itself, in its very essence a part of the machinery for community health program. We have been called on during the year to make special surveys of the hospital situation in St. Louis and Scranton and have undertaken a Study of the dispensary situation in Cincinnati.  ‘In order to meet future demands of this kind the scope of Mr. Davis! sub-  committee has been enlarged and its title changed to Subcommittee on the Organized Care of the Sick. t[¢ has been asked to prepare a suitable survey schedule for the study of hospital and dispensary service and to supervise the field service of the Committee in this important field. ‘the work projected will continue to be carried on, aS it has during the past year, in Close cooperation with the officers of the American Hospital Association and  Mention should be made of one other subcommittee, that on Analysis of Public Health Procedures (Prof. A. TW. Freeman, Chairman) which has not been active during the past year but from which we hope much in the fature. It is the function of this subcommittee to attempt, step by step, the analysis of the demonstrated values of various community health activities in terms of actual life saving, ~ a problem of enormous difficulty but the solu- tion of which will alone give us the ultimately sound basis for the standards  and the appraisals now necessarily grounded in the main on group judgment and empirical experience.  A special subcommittee, with Dr. Haven imerson as chairman, was appointed as a result of action taken by the American Public Health Associa- tion at Buffalo to present to the American Medical Association the invitation to create a joint standing committee of conference between the two organiza— tions on the inter-relationships between the public health movement and the medical profession. Conferences on the plan have not yet proceeded far enough to require any further official action. Your committee desires, how- ever, to express its feeline that if the American Public Health Association does take part in any such permanent conference committee it should do so through a special Committee named by the President for the purpose and not through tae Committee on Administrative Practice. a  We may now proceed to a consideration of the third primary function of the committee, the translation of its general program into concrete action through local surveys and appraisals. It is this work which of course oc- cupies the major share of the time of our very able staff, including Dr. Walker, Dr. Drake and Miss Phillips. During the past year we have made more or less complete surveys and appraisals in Athens and Clarke County, Ga., Burbank, Calif., Cattaraugus County, N. Y., Chicago, Ill., Cincinnati, 0., Fargo, N.Tak., Glendale, Calif., Hingham, Mags. , Lowell, Mass., Quincy, Mass., Rutherford County, Tenn. , Salem, Ore., Scranton, Pa., St. Louis, Mo., and Syracuse, N. Y., while more or less definite commitments have been made to ll cities and counties in 7 states and 1 province in Canada for surveys to be conducted in the near future.  We have been invited by the State Health Departments of Michigan, Massachusetts and Ohio to undertake studies of their organization and adminis- tration, an opportunity which we shall welcome if the necessary funds for this purpose can be obtained.  In the fourth field of activity, that of propaganda for the advance- ment of the general cause of improved community health services, the committes has opened up during the year two very important possibilities of cooperative effort. The active interest of the Chamber of Commerce of the United States has been enlisted in this cause and three most helpful bulletins have been prepared in Dr. Walker's office on Prolonging Life by Community Effort and on Health and Community Prosperity which have been printed by the Chamber and distributed to local Chamber of Commerce secretaries and to health officers throughout the country. Dr. Walker was also invited under the auspices of the Chamber to deliver a series of lectures on public health before the National School for Commercial and Trade Organization Executives. We believe that local healtn officers will find the movement thus actively fostered by the U. 5. Chamber of Commerce of incalculable value to them in the future.  Continued cooperation has also been maintained with the General Federation of Women's Clubs in stimulating a health interest on the part of its constituent organizations. A schedule of community health study was pre- pared and 3828 copies distributed to the local clubs, of which 93 schedules were actually filled out by clubs from 13 states. One month's free service by the Committee on Administrative Practice was promised to the state showing the largest proportion of communities participating and this’ friendly com- petition was won by the State of Rhode Island.  The Health Officers! News Letter is issued monthly from the Office of the Committee and is now sent to all state health officers, to health  officers of cities having a population of over 30, 000 , and to all full-time county health officers.  The total budget of the Committee for the calendar year is estimated at $42,000, balanced by an income of about $19,000 from special grants and general retainers and $21,000 from payment for surveys and appraisals, leaving a $2000 estimated deficit to be made up by the association. For 1928 we look forward to a basic budget of $48,000, with $18,000 more needed if the state and rural surveys are to be undertaken.  The budget of the Committee can conveniently be considered under three main heads. It includes first of all appraisals and surveys of local comminities which are normally self-supporting. Of the fifteen cities and    yi  ~ 5 w  counties surveyed in 1927, 14 were paid for, 5 from municipal funds, 3 from funds of local voluntary agencies and 6 from funds of outside voluntary agencies interested in the local health work. This part of our budget takes care of itself and is likely to amount to about $30,000 a year.  Special studies like the rural health survey and the state health survey must obviously be supported by special gifts and will only be under- taken if those gifts are forthcoming.  Finally, there is a basic and essential general service cost of the work of the Committee which is and ought to be considerable. All the prelim inary work of accumulating data for the various subcommittees, the prepara- tion and revision of appraisal forms, the preparation of the News Letter, the conduct of Information Service, the aid which mst necessarily be furnished very frequently to health officers unable to pay for it, - all these things ba pceicane involve a large part of the time of the staff and considerable expenditure for travelling, postage and printing which brings in no direct financial re- turn. We have been able to carry this basic service cost during the past year through a grant of $10,000 from the Metropolitan Life Insurance Company, a grant of $7500 from the Milbank Fund, a retaining fee for consultation service of $1000 from the Commonwealth Fund and a similar retaining fee of $400 from the Department of Health of the City of Detroit. The indefinite continuation of grants from outside agencies is most unlikely and even with those now available the powers of the present field staff have been strained far beyond a Wise and profitable limit. Dr. Walker has rendered to this committee a ser- vice of a rare and unusual character and hig usefulness grows with the exper- lence of every year. It would be the poorest policy to break down his health or to let him slip out of our hands to some more appreciative organization.  We must give him additional assistance wWrich will require more money, not less.  There seem only three ways to finance this general service cost of ne committee on a permanent basis; by grants from outside agencies, by grants from the Association or by contributions from local health departments. Out- side grants cannot be relied upon for an indefinite period and Association grants of large size are at present beyond our financial resources. It is really the health departments of the country which profit most directly and most immediately by the work of the Committee. The logical source of support would seem to -be contributions from these departments just as the source of Support for the National Safety Council which renders to the manufacturing industries a similar service is the contributions of those same industries. If more cities would follow the example of Detroit and place in its anmal budget an item of $100 to $500 for consultant service from the Committee on  Administrative Practice, our financial problem would be solved in the one really satisfactory way.  C.-E. A. Winslow, Chairman A. W. Freeman  Haven Emerson, Vice-Chairman C. Hampson Jones  Louis I. Dublin, Secretary G. D. Lummis  E. L. Bishop G. T. Palmer . H. N. Calver W. S. Rankin  F. G. Curtis James L. Roberts  Michael Davis G. C. Ruhland  W. F. Draper Henry F. Vaughan", "American Public Health Association. Committee on Administrative Practice", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-si4z.wkzu-8hku", "00000000-0000-0000-9F39-73736AE9C213", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Third Report of the Committee on Municipal Health Department Practices, American Public Health Association", "9918573882206676X121", null, "1923", "October 1923", null, "Reports", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "8", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "| Third Report of the Committee on Municipal Health     — sea (October 91923) » > =  ent Practice of the american Public 8  a: if Sate a  In view of the need for authentic information in re     and relative values of current developments in official health proced 0,     “~ Anerican Public Health Assogiation.in 1920 appointed a Committee on that certain initial     : _ - | Municipal Hoalth Department Practice with the assurance — — w along this line yould be aided by the generous financial        studies to be made . i assistance of the Metropolitan Life Insurance Company. ‘The Committee at the  2 , ve outset Limited its work to the 63 cities in the United States of approximately . So 100,000 population or over in 1920. A survey of the administrative health 7 machinery of each of these sition was made sawing the year 1921 by member's of  (1) representatives of the comi shee on the pasis of a detailed schedule inoluding        over 600 items. Some of the general results of ~ study were presented in. S 2 os ek preliminary form at the 1921 meeting in New Yorks The complete analysia of     the material and its preparation for publication proved a time-sonsuming : | but a second report was re at the 1922 moeting in Cleveland and a mamber _ te ee OO rH em oe oe me mee oe (1) | Appreciative weucré should be made of the assistance quuerousiy furnished by the United States Public Health Service and - the  “American Red Gross in the conduct of these arrays     (2) Aaerican Journal of Public Health, All, 7-15, 158-147, Januar February, 1922. —             gard to the temlencies - -                             of infividual ohapters of the “Gommittee's report, dealing with particular phases of health department pashan have beon published aweing the past eg: The caaplete report has now at last passed through the press and has appeared during the past me a special bulletin of the United States Public Health Services v The objectives contemplated by the Committee on pnt ctyed’ Health  Department Practice seemed so important to the beaker American pabiie     Health Association that they wore given a 7 Wile of primary signi ficane 108 «AG report of the Committee on Reorganization; adopted es a basis for the     in the 2  fundamental policies of the Association at the Cleveland meeting. thas in  the section of the Reorganization Soma anaes report dealing with Inter-       actives, reads as follows:  (3) Milk Inspedation, IleV.Hisoook, AvdePeHle, Zils 577-591, daly 1922. Vital Statisties, LeleDublin, A.JePsHe, ALI, 750-757, September L922. Water Supplies, AeWeFreeman, Aed aPeHe art, 759-763, September 1922+ Pablic Health Education, GeAmumdsen, A.d.PeH., AIL, 815-825, October 19226 Public Health Nursing, GewHeAeWinslow and M. Burkhardt, Pablic Health  Nurse, October 1922, pps514~-5206 An Ideal Health Department for a City of 100,000 Population, Ge-EeAsWinslow ~ and HI, Harris, AndaPolle All, 891-907» e - fuberqulosia, Ce*E-A.Winslow and G+F.Baker, American Review of tubereulosis - December 1923, pp»960~975. 2 _ Seen on of Health Departments, ‘AaWeProenan, AadeP ella g Bilt 65-161, oe Sehool ‘Te Supervision, I.V-Hiscock and W.?.Fales, AvdaPells, Alii, 259-269, April 1923. - Venereal Disease, Ml.A.Clark. Journal of Social Hygiene, IX, 27, 1923. b                     (5) American Journal of Public Health, XII, ps987, December 1922.     es. rT        \"the objectives of the Association should be the preparation, study,  tdigation, and presentation of scientific public-health proceduresy     the best method by which such knowl edge     gan be given to the public, and the expression to the public of professional opinion in regard te such procedurege\" :  Later on under Section ¢, J       eGo the report recommends \"that there be maintained at the central office of the Association such regords as are obtainable and gan be kept current with regard to the organ-  ization, e oxpenses, procedures and results of public#health practice in —     , meat th departments in countries represented in our membership.\"  Tn line with the policies thus foxmmlated, your Committee on ituniespal, Hoal th Department Bractice first of all set ‘out to realize the ideas of a  ented Glearing house for current infomation in regard to health department  practice as suggested in the paragraph quoted above from the report of the  dommittee on Reorganization. Various possibilities suggested heneelvan. along this Lines It is most desirable that the studies begun by the Committee in the large Gities of the United States should be extended to the large dities of Canada, Cuba and Mexico, to the smaller conmaunities,~ whose need for standardization is perhaps even greater than in the case of the Large i citles,~ and to the State and Provincial health departments. It soened to the mommies, however, that the natural and logisal first Step was to provide for the noeping up to date of the information already secured at Considerable cost in regard to the large cities of the United States. ‘The United States 7 Public Health Service was, in our judgmont the logical body to eesied in this work « in view of its general program and policies and in view of the surekes wank it has rendered to so many individual communities in the ‘Line of gounsel  as to administrative health procedure, The matter was therefore presented to Surgeon-General Cuming and it is a source of great gratification to be able to report that General Cumming has agreed to establish within the  United States Public Health service an office of Administrative Health =»                    vactice and has assigned to the direction of this work Surgeon Paul Preble, — |     ‘whose experience in health survey work fits him in a unigue fashion for this. : task. Dr. Preble will outline hia plans in the subsequent discussion and  it is only necessary for us to say further that the records now in the     BS a < } ag let a possession of the Gomi ttee on Municipal Health Department Practice witt Y d eo made aval able for the new office of the United States mee ae Health           members of the inectons Public Health Assogiation. Ome of our ideals, the           establishment of a _—. for eurrent informat ,On in} rege Le departinent practice, te~ekwe substantially realized. 0 og OS eel os jet . mB Ph in regard %  mt procedures Lt was felt that something more was necessarye As : A     ‘mm addition to the Gollection and dissemination of informa  $0 ourre     we understand the nan of the members of this association they deal re a clear picture of present couditions ehiefly in order that those conditions - may be improved in the futures Public health practice is in a dynamic, not — a static gonditions The object of the health official is to improve the machinery now at his disposal go as to raise it to the highest point of efficiency and to supplement that eure ad by obtaining new appropriations sufficient make his organization $a, the requirements of the new publica health, It ‘ins seemed to your committee that the most important service which the American Public Health Association could possibly renter to its members would be to establish a direct ¢ield service for the assistance  of the executive officers of health departubnts along the lines laid down                 above. ‘The committee therefore sot out to obtain the funds necessary for     the establishaent of a service of this. sort and was once more fortunate in  ssouring the assistance of the lietropolitan Life Insur     AGG ) Company which 8 has evenniget the sum of $15,000 aoaaasary to carry on the work for ‘the year .       1925-244 It ig with the keenest satisfaction that we announce that  DreWeSe Rankin, State Health offtear of North Carolina, and, past Hrenkcant  of the American Public Health Association, has obtained leave of absence  from his state to undertake this work, which he has already attagked with  vision and enthfsiasm, and to which he will devote his full time after the  first of January. Wo more oonvinoing evidence could be found of the |  importance of this work and no better augury could be offered for its success  thon is found in the fact that Dr.Renkin has been willing to undertake it. As Field Direator of the Committee on Municipal Health Department     Practice, DrsRankin, wrth—sseh-aortrt avy, will be at - the service of the individual members of he American Public Health Association —  in aiding them through correspondence, personal visits, and spetial surveys,  : to build up the status of public health work in their individual commmitics, | Your Gomnttee has felt that it would be of inestimable value to the health —  officer who is eriving to secure moral and financial Resting sor a constructive public health program if there were available not only objective standards  by which the need for a rounded health program could be demonstrated  but also some definite machinery by which such standards acw.a be brought  to the attention of municipal officials and the general publicge We have —  felt it to be essential to the success of our objectives to create mach lnery     of this sorte As a first step it has seemed that a spirit of generous rivalry between Gommunities could be promoted by offering a series of medals $i recognition of attainment and advancement in community health services ‘the first series of these medals (gold, silver, and bronze) we suggest may be | awarded at the 1924 meeting of the American Public Health Association to the —  three cities of a. population of 100,000 and upward, which can show the most  nearly adequate coemennh te health service as of Jammry, 1924. Tho status aa  tne different sities ehould ¥ determined by personal surveys to be sonauated, ee                j a  pr  the commmity health program (voluntary as well as official). It is the  hoped that scholarships or other methods of recognizing eminent personal :  Peason that we have taken no steps to earry out our plan im detail util : of  - . We have felt that any grading of health departments on al, 2, 3 basis  2 departments whose progress has been limiteds In Pogard to this matter,     he  . reprosentative agents of the Commi tte, rated on a scoring plan which will     be presented by Dr.Rankin during the present discussion for your consideration. The scoring should take into agcount all health agencies forming a part of     purpose of the Committee that in awarding similar medals in succeeding years the selection of the winners in the competition will be based not only  on actual attainment, but also on progress since Janay, 1924, and it is  accomplishment as well as commmnity achievement, may be introduced in the titdang to develop & spirit of emulation in health services throughout the — country. | | Your Comaittee fully realizes the GL etioultios involved in such a program and the importance of so carrying it out that the health officer who desires: aid in the-stimmlation of local public opinion may regelve such aid without the possibility of inviting eri ne of any ouier health department whieh for. any reasen faile to measure up to the highest standards. It is for this  we had the opportunity at thie meeting for a full and frank Consultation wit the health officers of the country's     or the etpression of their merits in terms of an arbitrary score card would a be clearly out of the question. It is our hope that nitinately a APRA ORE i is  of departments into a small number of grades may pevhane be feasible; but for     the pResens we believe it would be bent to limit ourselves to the —  of notably distinctive serrlae without any attempt to evaluate the work of            however, we —_ to have the opinions of the z members of the Association expressed in the course of the following discussions ] | oe ‘The basis on which health department practice shall be judged is a second y rr SS     health department practice mst be extended to the large     ofan     point of perhaps even greater importance ant the nature of the recognition to be granteds It is clear that the system of grading to be used must  be sufficiently elastic to take account of looal variations in the nature of the health problems to be dealt with and of the part played in a given cormuntity by voluntary agencies» ‘The Committee has given careful thought  to these wien during the past six months but has refrained fran adopting any definite policy until your counsél could be obtained. Dr.Rankin will present his ~eem@eteviews in regard to the matter in the subsequent : discussion a we hope with your assistance that a sound basis for the work oan be laide | :  In one respect, your Committee feels that its own personue]l needs to ~~ strengthened in order to deal wi th the problems involved in the proposed program. Its membership at present includes but one mmicipal health officer in agtive service and one other member who has served in the past as health officer of a large city. We believe that several now meubers with direst  ee ftom mandeipal ra pie L       denies a nog should be — to our  , ne adi eet VE ees —— sm _— Pct Ca # ie                If the American Public Health Association is to fulfil its largest  | possibilities we are convinced that the work of standardiging and stimulating     Gities of Canada, Cuba and Mexico, to small communities, to cowities and to state and provingial |                  health departments. So soon as funds  f | E | \\\\.         are available ines tasks too should — be undertakene So far, however, as the problem primarily presented to the eoopevation of the United States Public Health service and the Metropolitan  Committee is congerned we believe that the program worked ot through     Life Insurance Company Gonstitutes an encouraging report of progress. We  feel that the American Public Health Association, in initiating and supporting —  the activities of the past three years for the improvement of the character  i  of the service which its members render to their commu nities has shown a vision _ .     and a sense of public resp onsibility characteristic of the highest type of  professional spirits; and that the program if carried forward in the future will Tesult in farereashing improvements of the public health service and in @ substantial reduction in the burden of preventable disease and deaths", "American Public Health Association. Committee on Municipal Health Department Practices", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-f6s3.uqhk_atef", "00000000-0000-0000-DC0D-798AAA66C56E", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Fourth Report of the Committee on Municipal Health Department Practices, American Public Health Association", "9918573882206676X122", null, "1924", "16 October 1924", null, "Reports", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "12", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Fourth Report of the Committee on Municipa} Health Department Practice  Presented to the Public Health Administration Section ~ of the AgPeHeAs at the Fifty=third Annual Meeting at  Detroit, Oct. aly 19 24,  General Objectives  Your Comnittee on Municipes Health Department Practice was created four years ago with the conviction that the time had come for a concerted advance in the field of American municipal health service. The fundamental scientific and snciaigeesl bases of health department practice had become reasonably well established in their broader lines; and in almost Te special contributions of real, and sometimes of unique, value were being made to the technique of administrative method, Yet there was no central clearing house to facilitate the prompt interchange of information in regard to such pioneering efforts; there was ok eseeen standard for evaluating either the program or the performance of a health department as a whole; and there was no source £5 which the individual health ofeesr could turn for counsel and for practical support in the execution of his vbese.  Your committee was created to fulfill this need, Its functions, were obviously threefold. It was necessary to establish machinery for (a) The collection and filing ef current and up <ss information in regard to public health practices and procedurese (pb) The critical anelysis and interpretation of the data ee (a) The bringing to ths individual health officer of the eBonits of such analysis in the way most Likely to be of immediate practical assistance  to him in his official work...        { i |    ‘Sie  = 2 =  In other words, the task of the Committee was to apply in its special field, that of municipal health practice, the primary objectives of the American Public Health Association as a whole, as stated in the report of the Committee on Reorganization two years ago, “the preparation, study, standardization and presentation of scientific public weRith pracedures.*  The Collection of Information in Reeard to Health Department Practice .  The first definite step along this line was taken by your committee when in 1921 it developed a carefully prepared schedule of information  covering every phase of municipal health work and through the generous  financial assistance of the Metropolitan Life Insurance Company and the  invaluable cooperation of the United States Public Health Service, set about ei the satemuet inn called for in the schedule for eightyathree cities - the United States which had a population of approximately 100,000 or more, The information, thus obtained was systematically analyzed, and in 1923 was published by the Public Health Service as Bulletin 136. Copies of eae Bulletin were presented to the members of the Association last year at our meeting in Boston,  The United States Public Health Service seemed the logical body to  continue in the future this task of collecting and keeping up to dete current  information in regard to public health procedures, We had the pleasure a year  ago, in Boston, of reporting that on the instance of your committee Surgeon General Cumming had agreed to establish in the Public Health Service an office of Administrative Health Practice under the able direction of Dr,Paul Preble for the fulfillment of this function. During the current year Dr.Preble pre» pared a new and more elesexens schedule for the study of municipal health practice which was applied under his direction by officers of the Publie Health  Service to a second survey of public health procedures in one hundred  cities of 70,000 population and over, — Sos  These surveys are all now in the hands of Surgeon Preble and are being carefully analyzed, and their important data tabulated and classified, This material will be published in a second bulletin dealing with city health practices as soon as the preparation of the information contained aS schedules has been completede The Committee is under heavy obligations to the Public Health Service for the work which Surgeon Preble is doing in keeping the studies of city health practices CUrrente  : In this connection, mention should be made of the fact that the American Child Health Association, having become interested in a similar study of municipal health activities, agreed, after — with representatives of this Committee, +o include within their studies only cities with populations under 70,000 $0 that there would be no overlapping in work and no duplication of surveys of city health departments. The Child dealth Association has made a study of some sig hty-six : cities in the United States with peneietions ranging between 40,000 and 70,000 during the last year and is now engaged in preparing its material for publication at an early date. : |  it #2 tna be seen that the collection of data — to city health  procedures or practices has been developed to a highly satisfactory degree and that 4hrough the creation of a special office within the United States Public Health Service ideal machinery has been created for ie carrying out of such work  on a permanent basis in the futures  The Evaluation of Public Health Procedures. fhe basis for any sound public health program must necessarily be sought in a study of none bicel experience, The collection of raw data as to current practice is however only a first step. The data must be analyzed and critically evaluated if they are to prove of the maximun practical value.  It is sometimes of course the case that a health officer desires simply a    = Ane  piece of concrete information, such ae ths number of cities practicing terminal funigation or the srattics of New York or Detroit in regard to the isolation  of whooping cough. More often however the problemswhich come to us require: the expression of some degree of critical judgment, as to the relative value of this or that procedure and its relative importance in the general public health field. i is inevitable that any group or committee in a comparative study of city health work, must set up in their own minds, if not on paper, certain standards of service and standards of values as bateeua different classes of service. The advantegs of committing these impressions to paper is that they may be reviewed, criticised and improved by the many who may be interested in the subject.  Such standards of health practice, immature and eeutediee thoug n they may be, not only constitute a natural evolution of comparative studies, but they become almost a matter of necessity in the development of a helpful field service by this Committee. The field representatives of this Committee, in conference with city health officers, in advising city health officers with reference to such important matters as their budgets, personnel allotment, program and reasonable objectives with reference to the more important problems acs in city health work, must give either their individual judgment on these important matters or must express a group judgment which is in reality noth ing more nor less than a standard. It is a standard however which is binding on no one but the committee itself and binding on the committee only until it is modified in the light of future experience,  It is from this point of view that your committee presented in Bulletin 136 a tentative plan for An Ideal Health Department for a City of 100,000 Papaiattets and it is tu — point of view that it hopes during the coming year on the  basis of the material collected by the United States Public Health Service, and    we Fue  by the American Ciild Health Assoc ia tion, to prepare special monographic studies of what Soeus at the moment to be the best current practice in various special lines of administrative health work. Above all, your Committee, in its contact with the health officers who  have called upon it for service, has been impressed by the need for a concise and clear form of evaluation for the activities of a municipal health department as awmle. In our report of a year ago we suggested that it might be helpful to award medals to certain cities of outstanding merit in community health service as evidenced by their showing on a searing plan to be devised by Dr. Rank ins This plan of awards was criticized in the subsequent discussion at the Boston meeting and after obtaining by letter as asinies of the health officers of the 83 large cities the proposal was definitely abandoned, The preparation of an appraisal form to be used, not for publication or for the comparative grading of cities, but for the use of the health officers and of our field agents in their service to the health officers has so far as we are aware met with universal approval.  | The aim in this appraisal form was to provide in the first place for an evaluation of the relative importance of various lines of public health activity and in the second place for an estimate of the results attained along each line of activity as measured by the service actually rendered. The : Committee began the formulation of such an appraisal form in April, 1923 when it requested Dr. Charles V. Chapin, one of its members, to evopare for it a draft of a schedule which could pe used for this purpose. Dr. Chapin complied : with this request and, in the fall of 1923, the Field Director of the Committee, Dr. Rankin, after making certain minor alterations in the tentative foc that had been prepared, brought the statement vefordine Committee for its senidien., The Field Director; taking the Committee's revised statement into the field, where he had opportunity to confer with a number of individual health  officers in regard to it, received from themmny helpful criticisms and    — Gme  suggestions, some of which were adopted tentatively for still further revisionss  In his field conferences, the Field Director of the Committee had the opportunity of oeveubites the subject of standards iu eue eel health depart ment practice to two separate representative groups of city health officials. One was a group of health officials of New England cities with which contact was made through their attendance upon the New England ipalth Institute which met in Boston early in May of this year. A second group contact was made possible through the ee of the State Health Commissioner of Ohio were, on account of a considerable number of large cities closely related ae it was sonvonpent. at the time and place of the meeting of the | Ohio State Wedical Society in Cleveland the latter part of May, to discuss 3 with a group of Ohio city health officers the question of the appraisal of city healthwork. <A third group contact was effected through the interest of Dre S. J. Crumbine, Director of Public Health Relations of the American Child Health Association who interested the State Commissioner of Health of Michigan and, tirough Dr. Olin's cooperation, was able to present the matter to a con twelve city health officers in that State. With all three groups  a temporary organization was effected for the purpose of considering standards  and, if possible, revising the tentative appraisal form of the Committee to  meet their own group judgment and needs.  At this juncture, tae Committee, in its initial efforts looking toward a statement of standards for city health department practices, found a most helpful ally in the American Child Health Association. That Association, in travelling the same road over which the Committee had previously passed,  came, through the comparative studies of the health work in the eighty-six  cities which it had surveyed and in the planning of its subsequent field  service with these cities, to the same necessity encountered by our Committee,  that of crystallizing its impressions of practices and values into a definite    re  schedules of values and attainment. aries two separate statements of standards, those of our Committee and those of the American Child Health Association, had thus been independently formulated, it was realized by the representatives of both organizations that the two statements should if pocecis be reconciled and consolidated. in conference between representatives of the two organ- izations, which lasted well over a week, tne two tentative forms of appraisal for city health department practice were reduced to one. Subsequently representatives of goyotnl of the other large voluntary organizations, such 68 the National Tuberculosis Association, the American Social Hygiene Association and the National Organization of Public Health Nursing, were conferred with and their criticisms and su est tame incorporated, so that the statement came to be, at least for the time, an expression of the group rudenent of representatives | of four of the large national voluntary health organizations, as well as of the representatives of the American Public Health Association.  It was clear to us all, however, that even this widely based group judgment as to appraisal standards for values and attainments in city health work would be of Little service unless it incorporated the practical viewpoint and experience of a larger group of city health officers than were directly represented on your committee, As a result of this conviction, a committee of three members from each of the groups of city health officers that had been formed, one in New Bngland, onein Ohio and one in Michigan, were called in to review the tentative statement of standards of values end attainments which had been prepared and were paced to criticise it and amend it in such a way as to make it conform to their composite judgment and to meet their practical needs. An agreeme nit was reached between the repranentatives of the voluntary organizations present that in this conference with the nine city health officers representing the three temporary organizations of a total of thirty-six city health officers, the voting, the settlement of all differences at judemont, should rest entirely  with the city health officers. The conference with the representatives from    a Om  the three groups of city health officers continued for four days, August i8th, 19th, 20th and 21st, and passed upon both the principles and the minutest details that had entered into the development of the tentative standards. A number of changes were made and out of the conference came the only expression that we have of vrofessional group judgment as to what should constitute standards of service and relative values in municipal health work. It was understood on the adjournment of the conferences of city health officers that the representatives would present the statement to their respective groups with a request that these thirty-six health ertueers accept it as it is until next August; that they apply it to an appraisal of the work of their own departments; that, after full epaescereises and trial, they name their representative to meet with represen Lotives from the other groups, now temporarily organized, and with those of several other groups that will be organized for revising the statement next yeare  The general lines along which the appraisal form is being developed are indicated in an address delivered by Dr. Rankin, Field Director of the Committee, in an address delivered before Conference of State and Provincial Health Authorities and printed in the American Journal of Public Health for October. Copies of the appraisal schedules in its present form can be obtained by any member of the American Public Health Association on application to the New York office. It involves, as tr, Rankin points oat in the address in question, an evaluation of each stangn “ivlavity of municipal health departments upon a basis of so many points on a total of 1000, with additional credits for special acitivities, such as malaria and plague control, industrial and mental hygiene not now universally undertaken by city health departments; while under each activity the value to be set down is dependent upon service actually rendered  as determined by specific quantitative standardse    = Que It would be fruitless to debate sue details of this appraisal form upon the floor of this meeting; but your committee urgently invites individual criticisms and suggestions in regard to it. The clearest light on its merits and demerits will come from its application by health officers to their owm organizations. The group of health officers from Ohie, Massacmsetts, and  Michigan now studying the schedule in this way is no close corporation. We  urge all of those here present who are servingas city health officers to take  part in the work, and to indicate their willingness to try out the schedule and attend next year's conference for its revision. We desire to broaden the basis of group judgment behind this appraisal form until it represents the  direct experience and the considered judgment of the entire membership of the  Health Administration Section.  We are convinced that an scoraieni form arrived at by the procedure here outlined will prove of very real value to the individual health officer in enabling him +0 develop a balanced departmental program becad upon the bedrock principle of relative values and safeguarded against the undue influence of this or that special group eoteed to some particular interest; to check up on the performance of his own bureaus and thus maintain is-depertment at the highest point of efficiency; to defend himself when unjustly attacked by showing that his department meets the objective standards of nic of opetcunl colleagues; to secure funds for expansion by indicating where his organization falls short of that generally accepted as ideal; and to secure the cooperation of local voluntary health organization along the most effective lines.  Presentation of Studies and Standards to Halth ‘A Officers or Field Service | S  The first phase in the work of this Committee was necessarily the collection  of information in regard to city health department practices, the second phase the  organization of group judgment for evaluating the information obtained, and    - 10  the third phase, which has now been approached and already begun, is that of field service, that is making the results of its studied directly applicable to city health work, During the first three years of the Committee's work» he weliect ic of information, naturally dominated the picture; during the last year the organization of group judgment in form of tentative standards was the outstanding feature and, following this, the development of a practical, helpful field service will take the ascendancy se |  Even during the past year, howevér, the Field Director of the Committee | vic ibed and conferred with the city health officials and others in nineteen cities, spending an average of shres days in cach city; vy invitation ne conferred with the health officers of two states with reference to their state problems and on one occasion was called to advise a group of interested citizens and physicians with = to the development of a county health department}; he eadeeased nine national and state organizations of health officers in regard to the sina and work of the Committee, spoke to nine local medical eScisties in regard to che relation of the medical profession and the public in health work, andalso addressed ten luncheon clubs on the subject of public health, a total of twenty=five addresses  Your —— has felt that the continuance and extension of its field ser vice was of primary and tastano importance and therefore set itself very early in the present year to seek for the financial support necessary to carry out this plan. Since 1920 the funds at our disposal have been entirely contributed by the generosity of the Metropolitan Lite Insurance Company. We felt it manifestly unfair to approach this company for further aid unless it could be shown that the tasks we had undertaken were of sufficient importance to appeal to other friends of the cause of public health, We are happy to. report that the interest of the Milbank Memorial Fund has been enlisted in this activity of the Amorkaau Public Health Association and with its support  secured we again approached the Metropolitan Life Insurance Company which    =] Le fas eatend <6 appropriate 910,000 for our work during the year 1925, a similar sum being contributed — the Milbank Funds  With this financial support it will be possible during the coming year not only to retain the services of Dr. Renkin as Field Director but to provide him with competent assistance sufficient to enlarge materially the scope and volume of his work, Furthermore the service which can be rendered to members of the American Public Health Association along this line will be further extended by an agreement which hes been reached with certain of the great sectional voluntary health agencies represented in the National Health Council under which field representatives of these obenaatioe Wiki be available as consultants i so phases of health eduinistepil such as tuberculosis or child hygiene or public health nursing for those health officers who may desire their aid,- in exchange for similar services rendered by Dr. Renkin and his staff to the organizations in question,  Your committee believes that the posgibilities of this field service under such direction as that of Dr. Rankin are almost unlimited. The extent +0 which it develops will depend on the extent to which the individual members of this section call upon it in a futuree Your Sxcities is reasonably confident thet if your demands exceed the capacity of the staff available more baste eas be found for expansion when they are needed, If you desire to ‘chow wheat other health officers are doing and how that knowledge can be applied to your local conditions Cell upon Drs Rankine If you want the effective support of professional opinion in securing support for needed local developments call upon Dr. Rankin. this committee is your ponmit ses directly responsible to this association of efefosaicucl workerc. The members of the Field Staff sre your employees organized for your pereee The upbuilding of the public health service of this country along the lines of sound and. enlightened progress is primarily  the problem of the professional public health workers themselves, It is a    legitimate source of pride and setisfaction that the lead in the solution of this problem has been undertaken by the body to which it logically belongs - the  w  American Public Health Association.", "American Public Health Association. Committee on Municipal Health Department Practices", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2xer.2i8t.pywb", "00000000-0000-0000-7BA1-326F1C171562", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Fifth Report of the Committee on Municipal Health Department Practices, American Public Health Association", "9918573882206676X123", null, "1925", "21 October 1925", null, "Reports", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "12", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Fifth Report of the Committee on Municipal Health Department Practice. i  The objectives and policies of the pastes on Municipal Health ppetinent Practice have been sufficiently cuhlined in the four previous veneers which | have been made to the American Public Health Association.~ It need only be repeated here that its ideal is to realize the primary aim of the Association as a whole, \"the preparation, study, standardization and presentation of scientific public health procedures, by the eckisstion of information in re- gard to current administrative health practice, the analysis of the material obtained to derive standards of organization and achievement and the transla-  tion of these standards into terms of concrete achievement through an informa-  tion bureau and field service placed at the disposition of all members of the | Association,\"  During the past year the Committee, at its own request, was reorganized and enlarged so as to make it more fully representative of the practising health officers of the country. As at present constituted it includes six city health officers, two state health officers, one representative of the ‘United States Public Health Service, three professors of health administration and three representatives of voluntary health agencies. Hight states, in is dition to the District of Columbia and the Dominion of Canada, are represented in its membership. : i  The funds for the support of the work of the Committee during the first four years of its existence were generously contributed by the Metropolitan  Life Insurance Company. During 1925 the cost has been borne equally by the  i. Presented at the Fifty-fourth annual meeting, 5t. Louis, Mo., before the General Session, October 21, 1925,  &. American Journal of Public Health, 1922, 12, 7, 138; 1924, 14, 184; 1925, iD, <2,    OT  Milbank Fund and the Metropolitan Life Insurance Company. Both these organi- zations have consented to contime their aid to the Committee during the coming year; but it is felt by all concerned that the work of the © mittee is in essence a primary, and indeed a fundamental, activity of the /iserican Public Health Association itself and that the Association should next year begin to take a substantial share in its support,- with a view to assuming, as soon as possible thereafter, complete financial responsibility for what is logically a bureau of its administrative office.  Surveys of Health Practice.  Three general surveys of municipal health practice in the United States have now been conducted; the first in 1921 (covering 83 cities of over 100,000  population) by the Committee itself with the cooperation of the United States 3 : oe Public Health Service and other agencies;’ the second in 1924 (of 100 cities  of 70,000 population and over) by the United States Public Health Service;  and the third, also in 1924 (of 86 cities between 40,000 and 70,000 population)  by the American Child Health Association. The United States Public Health  Service has asked the Committee on Municipal Health Department Practice to  take charge of the analysis for publication of the data collected in its 1924 survey; a the full report based on this study will appear during the coming winter.* The report. of the A.C.H.A, on the smaller cities has just been is- —s The Committee does not plan in the immediate future to undertake on  its ow initiative any further comprehensive and detailed surveys of munici-  pal health practice in the cities of the country as a whole. It believes  that this type of service, whose value has now been completely demonstrated,  can most logically be carried on by the office of Administrative Health Prac~  tice, created in 1923, in the United States Public Health Service. :  3. Published as Bulletin 136, U.S.P.H.5., 1923.  4. Several chapters of this report are already in print; see American Journal of Public Health.    aS  The Committee will, of course, feel free to conduct surveys of indi- vidual cities when called upon to do so; and for this purpose the Committee is preparing a revised survey schedule suitable for such studies,  The Committee feels that by the use of the Appraisal Form for City Health Work (to be diseased in a succeeding paragraph) it should be possible to secure from the city health officers themselves annual reports of the more Significant phases of health department achievnsst. which could be analyzed and published promptly so as to furnish year by year a timely picture of cur-  rent progress,  The Committee has peceived urgent requests to undertake a comprehensive preliminary study of county health service like that which it initiated for city health departments in 1921. If the Association desires us to undertake this task, and if Surgeon-General Cuming's approval and cooperation can be secured, we shall be glad to do so, If, however, this enlargement in the scope of our labors is authorized, the name of the Committee should be changed a eatnsitne Eapetjoe of tg tnartons Public Health Association; Without the qualifying word \"Municipal\",  A second problem in the field of health practice which has been brought forcibly to our attention is the nsed for the most convenient and exa.t records and in their dealings with the public. At the request of the secre- tary of the Association a special eukeoomrbttes on Health Department Forms, including several co-opted members not members of the general committee,  has been created to deal with this subject and much valuable material in the files of the Association office has been placed at the disposal of  this sub-committee,  ge ay oe  Standardization of Health Practice.  The major work of the Committee during the past year has been devoted to the perfecting of the Aepenneai Form for City Health Work, whose history was discussed at some length in our report a year ago.  The aim that me have had oe has been to devise a brief Appraisal Form which would sicid a reasonably accurate picture of the health services actually performed in a city as evidenced by certain typical sample activi- ties, It was to be based not on money expended or personnel employed, which indicate resources rather than performance. Nor was it to be saece om mor- tality rates, which are affected by so many racial and industrial factors as to make comparisons between various cities so frequently misleading. It  Was our idea rather to measure the immediate results attained,- such as statistics properly obtained and analyzed, vaccinations performed, infants in attendance at instructive clinics, physical defects of school children discovered and corrected, tuberculosis cases hospitalized, laboratory tests performed; with the confidence that such immediate results would inevitably lead on to the ultimate end of all public health work,- the conservation of human life and efficiency.  The preparation of such an Appraisal Form involves, first of all, the selection of such typical activities in the various fields of Gosdie <oue vice as are susceptible of definite and quantitative measurement; secondly, the establishment of standard values for a maximum score, based on the ac- tual performance of those cities which have attained outstanding results in the various fields in question; and, thirdly, the weighting of the various rated activities so as to give a reasonably clear picture of a bal- anced health program.  As stated in our report of a year ago, \"We are convinced that an Ap- praisal Form, if it can be arrived at by the procedure here outlined,  would prove of real value to the individual health officer in helping him , to develop a baianced departmental program based upon relative values; to check up on the performance of his own bureaus and thus maintain the depart- ment at the highest point of efficiency; to defend himself when wnjustly at- tacked, by showing that his department is organized in accord with the views of his professional colleagues; to secure funds for expansion by indicating where his organization falls short of that generally accepted as ideul; and to secure the cooperation of local voluntary health organizations along the most effective lines. \"'  The task of preparing an Appraisal Form is, however, one of the great- est difficulty and delicacy and we have felt it necessary to proceed with the highest degree of caution in our — As pointed out in our last re- port the Committee began its consideration of the Appraisal Form problem in the spring of 1923 when Dr. C, V. Chapin prepared the first draft of such a form for its consideration. In the fall and winter of 1923-24 Dr, W, 5. Rankin, then Field Director of the Committee, devoted himself with the greatest energy and enthusiasm to the development of the appraisal plan and brought into consultation in regard to it a large mumber of health offi- cers and representatives of voluntary agencies. In particular, the American Child Health Association, in connection with its comparative studies of health work in eighty-six cities (referred to above) and in the planving of its subsequent field service with these cities, came to the same viewooint held by our iendt tos: that it would be desirable to express its finding in the form of a definite schedule of performance and values. After two separate Appraisal Forms, those of our Field Director and those of the American Child Health Association, had thus been independently formulated, it was realized by the representatives of both organizations that the two forms should, if possible, be reconciled and consolidated. In conference between representatives of the two organizations, which lasted well over a  week, the two tentative forms of appraisal were reduced to one. Subsequently,       « G «=  representatives of several of the other large voluntary organizations, such as the National Tuberculosis Association, the American Social Hygiene Asso- aiation and the National Organization for Public Health Nursing and other members of the National Health Council were invited to submit criticisms and suggestions.  Finally, in August 1924 a committee of nine ay health officers, representing groups of city health ict who had been enlisted in the ee study of the Sceaiaedl Fort in Ohio, and Michigan, and in New England, were called together in New York for a four-day conference, at which the Appraisal Form was again revised in a form later printed and issued: by. the American Public Health Association for experimental use for one year.  After these lengthy preliminary sonterengia, after one full year of trial use by a group of nearly fifty cooperating city health offiners and after the sstd cation of the Appraisal Form to the study of the data for 186 cities collected by the U.S.P.B.5, and A.C, HA. surveys, a second gen- eral conference was held in New York last month in which the Committee on  ‘Municipal Health Department Practice, the group of nine health officeu.j,s representing cooperating officials in New England, Ohio, and Michigan and the American Child Health Assosterion and other national voluntary health agencies all took part. On the basis of recommendations made by this con- ference your Committee has once more revised the ieprasaal Form and believes that it is now in sufficiently satisfactory shape to be issued with the ap- proval of the Committee as a basis for the appraisal of city health work.  This revised Acpoasaal Form is therefore wanenied as an appendix to our report. It will naturally require further oe in the future as knowledge advances and practices progress; but we delieve that in its present form it should be given trial for a period of at least three years. in order to secure data which shail have comparative value. We purpose to  make a continuing study of the Appraisal Form by careful analysis of the    scores actually obtained by the cities of the United States during the next year, by activities and items and in relation to budgets, personael and disease rates, in order to be in position, with the aid of sther eae interested, %~O recommend whatever changes may be necessary in the form in 1928,  We celieve that the general ability of the Appraisal Form idea has been abundantly demonstrated by its first year of trial use. In a publication issued by the American Child Health Association there were reported the fol- lowing specific uses, as demonstrated by its experience with the cities of 40,000-70,000 population; and our own field agents report exactly simi iar reactions among the larger cities. The term \"rating schedule\" as used in ‘the following quotation refers to the tentative Appraisal Form described above, -  \"As a brief Sor carat appropriations of the Department of Health.  - Ts several cities the ratine schedule has been used to show in a comparative objective manner certain deficiencies in the department of health, and the expenditures which would be required bo secure a full credit in place of these deficiencies. When peanented in this manner to the appropriating body of the city, as a vart of the health officer's budgetary request, the health department Was, in one instance, the only department of the city goverment to receive an increase (amounting to 14%) for the coming year. Jn another instance in the face of a strong movement for retrenchment throughout the  _ eidy goverment, ihe health budget, presented in the light of the rating schedule, was the only departmental budget to remain uncut.  \"As a basis for a health program. The value of the aiteae sche iuie - as an outline of a well-balanced health program has ennenked to several health officers to such an extent that their future programs are definitely based up- ‘on achieving the standards set in the schedule. * * * * oe te eee eS NAs a iets for an Anal Report. It has occurred to one health officer  to incorporate in the annual report of his health department the detailed  2 gee ae oa rn fer Re ET —  rating of his city, for the 83 items included in the schedule, and to publish the score attained, with full discussion of many of the items, Another health officer has largely improved the character of his annual report in the light of the Appraisal Form,  \"As_a means of interesting a Mayor or Chamber of Commerce, In a city which had recently scored itself the health officer was studying the ating when the mayor of the city happened to enter the office of the health depart- ment. Upon being shown the low rating of the city in eoputherss prevention the mayor inquired what could be done to improve the situation. When the health officer explained that he could secure a full score (30 points) if he had $175 with which to buy toxin-antitoxin, the mayor promised to make the sum available from special funds. Other health officers have taken the rating of their city to the mayor or the chamber of commerce and by comparing the lo- cal rating with other cities have awakened a new interest in the city's health record.  \"As_a means of arousing and cementing the interests of the Departments of Health and Fducation and of the private agencies in each other, A striking outgrowth of the use of the rating schedule in one city where the findings were reported in a special meeting, was the development of a new sense of the  interdependence of all health agencies on each other and the formation of a  joint health committee on which all agencies were represented under the leader-  ship of the health officer. In another instance the rating schedule brought to the heaith officer for the first time the realization that he shou.d co- operate with the department of education in its health work if his city was  to secure the fullest return from its health activities. school medical  inspection was promptly started.\"  Such evidence as this has materially strengthened our conviction that the A, praisal Form is likely to fill a large and important place in the history of public health development. Cn the other hand we realize very  fully the hazards involved in any such plan. In a problem so complex and    = | so diversified as public health administration, so materially affected by climatic and social factors, and in which the relationships between public and private agencies are so vattous: no mumerical appraisal plan can fail at times to yield an unscientific and inaccurate picture. If the Appraisal Form were to be used by outside lay agencies for the arbitrary comparative rating of cities, gross injustice might result. The primary safeguard of the scheme must lie, however, in its application: It has oe prepared and issued by a comnittee representing the professioral health workers of the ee for the use of the minicipal health officer in he self ooprasadt  of his work. We believe that there should be no appraisal of the health  -  work of a city on this plan anoepe 5ate the approval of the health eificers concerned; and that in no event should there ty any publication of the appraisement of the ann ieee of a city except with the consent of the health officer. |  The Committee has approved the official publication of an. Appraisal Form only after long consideration and much discussion. Certain nembers of the Caranetas seriously doubt the advisability at this time of setting up any formal standard eee health service. Certain others have felt that there is at present no adequate scientific basis for an attempt to formulate ay numerical expression of the relative value of various health activities. A majority of the Committee, however, have felt that ic SeGeeE utility of the method more than outweighs its inherent inaccur- acies and disadvantages, and have approved the schedule with the hope that in tis next. three years experience and study will permit the further p< cfec- tion of the schedule and the establishment of methods of evaluation which will obviate the objections which have been offered he the present plan.  In the field of stagierdatéen, as distinct from fact finding, the ee represents in a sense a primary step. It indicates a general  i ~ 10. balance of activities and it measures actual service as indicated by typical items, susceptible of quantitative statement. Ultimately, however, we fe6l that the work of our Committee mist logically and necessarily lead to the preparation of more detailed standards for appropriations, personnel, |  ea SS elie equipment, policies and SEPERRUEES for each major division of the benkee  spss  ers     Se nomic: a none oreenen  eee department, - in fact a picture of a model and well-balanced om depart           ment such a5 Wa\" attempted in our 1923 report, but on @ much more extended scale. The three surveys now seine should make it possible to approach this task with greater success than has been possible before. With the aid of men- bers co-opted on the sections representing the special fields concerned we plan to procaed as rapidly as possible with this important task.  Field Service of the Committee. |  Finally, there remains for consideration what is perhaps the most important of all the functions of the Committee, the bringing to the individual health officer of such direct aid in the conduct of his daily ‘work as may be furnished by a properly organized field service. Our plan -for this service received a heavy blow when early in 1925 Dr. W. S. Rankin,  our Field Director, resigned to undertake the direction of the medical work  of the newly organized Duke Foundation. On the first of May, however, we were fortunate in securing the services as Acting Field Director of Dr. C.st Clair Drake, whose past administrative experience as Director of the Illinois State Health Department weil fitted hin for this task.  The outstanding items of the summer's work are as follows:  Twenty-seven cities were appraised on he basis of inf ormration gathered through personal visits.  Thirty-two cities were appraised from inforration secured from the survey, supplemented by information furnished by the local health officer.  One hundred survey schedules were prepared in duplicate. One copy was sent to the local health officer with the request for    i  insertion of supplementary data. One was retained as permanent office record.  The health officers of practically all the larger cities have deen addressed twice by letter for the purpose of  a. Advising them of the aims, purpose and policies of the Committee.  b. Securing supplementary information for the appraisal.  Permission was received from 21 cities to use their local city  graph on the cover design of a special city health number of the Survey Graphic.  Copies of the tentative appraisals of 37 cities have been made, preparatory to sending to the local health officer for correc- tion and the supply of supplementary information.  Although the summer has been largely given over to the appli- cation of the appraisal form to specific cities, a service call was made to Manchester, New Hampshire, and invitations are at hand to visit:  Knoxvilie, Tenn. Toledo, Ohio Portland, Oregon  Trenton, Nd.  Waco, Texas Houston, Texas  San Antonio, Texas Dallas, Texas Forth Worth, Texas.  On September 8th, Dr. W, F. Walker, who had long experience in health ad- ministrative methods as a member of the city health department of Detroit and who had in addition unusual experience in the survey and appraisal of cities as Research Associate of the Arerican Child Health Association was appointed  ield Director and will give his special attention to the cities of the east- erm and central states. Or. Drake, as Associate Director, will for the present work in the southern states and provision will be made as soon as resources per-  mit for special service to the West coast. Its field officers will be prepared  to offer detailed information as to this or that administrative practice, such as the length of time to isolate a particular disease, the simplest of-  fice practice in filing reports of nuisance inspections, the exact status    = if =  of the serum treatment of scarlet fever, the accepted ratio of staff numses to surervisors; general counsel as to the development of a rounded and con~ plete health progran for their communities, counsel based on a knowledge of what is being done by other health officers throughout the country; or the effective support of an organized nation-wide professional opinion in se- curing needed appropriations or facilities fron city authorities or in mobilizing public support for their policies. As pointed cut last year, \"This Committee is your Committee, directly responsible to this Association of professional health workers. The upbuilding of the public health service of this country along the lines of sound and enlightened progress is pri- marily the probleu of the professional public health workers themsel: 2s.\" It is to aid in the realization of such an ideal that the Committee on Health Department Practice stands ready to be of aid.  In view of the enlarged staff and facilities of the Committee it will be possible during the coming year te offer te the health officers of the country a larger measure of consultative and advisory service than has  heretofore been vossible.", "American Public Health Association. Committee on Municipal Health Department Practices", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-84d5_hp59~wv5h", "00000000-0000-0000-9199-D116B55D7CDB", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Review of Work of Committee on Evaluation", "9918573882206676X124", null, "1930", "circa 1930", "This subcommittee of the Committee on Administrative Practice investigated the necessity and effectiveness of various public health practices, as well as epidemiological data gathering methods and analysis.", "Reports", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "12", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "- With approval, and by testing a new procedure that appeared theoretically promising.  Some et least of the current and generally approved practices of health depart=  methods which will give at least as good results with less expense to the  a” E", "American Public Health Association. Committee on Administrative Practice", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kvk5.mxpi-izc7", "00000000-0000-0000-AE81-0D4B78D1BBD0", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "A Decade of Progress: Tenth Annual Report of the Committee on Administrative Practice", "9918573882206676X125", null, "1930", "1930", null, "Reports", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "11", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "~o~  ‘preparation, study.  and field service.\" whe original coumittee had 8 members, of whom 6 are  of an Appraisal fora for the quantitative measur     A Decade of Progress. /730 ommittee on Aduinistrative Proctice  | eieunns of public | ctan S Yale Sehool of Medic ine     Just a decade 0s in the your 1920, the Auericun Public Health Avsoctation appointed a Committee on Maniei ped Health Depo rtacnt Practiced, for “the dardization and presentation of selentifie public :     health procedures, by the colleetion of informtion in regard to current : administrative health pmetice, the anslysis of the snterial obtained to derive ctandards of organisation and achievement and the translation of these standanie into terms of |        onerete achievement through an information —     still on ite active roll and the initiattor by a generous gift of 10,000 a your from the Metropolitan Life Insurance jompany. This was the entire anmal budget of the Gommittee for ite firat  of ite work was mde possible            quinguennium. | , » yours later (in July, 1925) our first aystematic study of the       genleabion of the 83 largest cities of the United States, in ecogemtion with the U.S.Pablie Health Service and ether interested aguuahits was gublished as Bulletin 156 of the United States Public Health Service. | This volume wes not only the first qmanaansiee comparative review of health | practice in the world but it included also the first attempt at an outline | of the essentials of an tdend eity health sseustaation, ih this sane yoar,  Dre .S«Rankin became Sela director of the Committee and began the preparation —          performance.           in servies) and initieted ite survey Gltles.     in 1925 the name of the aomuittce wae changed to Coamittee a correspond with the broadening of ite intereste to include state                its work. Dr,Haven Emerson served av chairmen for the year 19252 DrsWaF Walker was appointed Field Director Seytesber 1, 1926, It is with deep  0 DeeBalkerte resignation te take effect next April. His five yor 2  and to the eawe which it las at heart ani credit for the resent aceomplishsents                      Assoetati on made publication of ite Surve        the 100 largest oltice of the United States and the Gomnitt invaluable volume on dome     madty Health Ory anlantion by Profcesor 1, V Hlecowk,          efforte at active development of public interest in Laproved adatnistrative                yoo™N  health prictice began at thie time in cooperetion with the Federation of       ntative Appraieal Yorn for rural areas was lewaeds 1 $7500 from the Milbank  and paid field service had so developed thet the Committee budge’        Form for Clty Health Work which wae ieeued Jenusry 1,1929 with the inmelucion of cancer and heart  The task of 1920 wae the revielon of the Appraisi wl     diseace at major setivities ami with a new acor ing plan (to encourage      « gonparigen by setivities rather than by total scores) ae well as many minor  improvements, In this year, too, the State Health Departme     nte of Mascachnestt s, Michigan and Ohie were surveyed and a study of rural health practice wae — Hd, the budge’  ched with a grant from the Commonvealth F ag 16,000. Thie was the last of the sight years for which the            | Metropolitan Life Insurance so generously contributed te the work of the     Financed by a group of insur       cities, sponsored by the U,5.Chamber of Comic TOs »        mee conwmies and conducted under the technical  direetion of the Coumittee on Adainietrative Preetice. The budget of the        Tommittce wae tnereaged to $65,000 ineluding ae major items $29,000 fr Commonwonlth Pund for the rural study, nearly $21,000 from payments for field 020,000 from the Che 0            er of Commerce for teehnical ateletance in the Health Conservation Contest and $1e 509 from the Milbank Fund. During the carrent year (1950), with a budget of nearly $99,          9, the Comalttee hea eontimed ite etedies of rural hoo lth practice and ite cooperation in the Healta        Gonservation Contest. It has undertaken a questionaire analysis —                               Child Health ami Welfare. | \"his wrief review of the major activities of the Comittee on Adminietrative Practice as a whole would be most tnoomplet     to the work of ite subcomaittees which have ande eontritutions of the first —     importance in their special fields, The first of tenes subeoundtteee        commit teer aa fol lowe:  Subsomaittee on Record Formas. G.C.Ruhland, Ghairmar     abcomnittee on Organised Cave of the Sicks M.il,Davis, Chairmen subcommittee on Rural Health Work. “E,l.Bishop, Chairman ubecumlttee on State Studies, ¥.P. Draper, Chainenn Subcomalttee on &ppraisal Form for City Health Work. ¢.7,.Palmer, Chairman | Wonoal of Administration. 4.%.Veughan, Chairaan oumittee on Howlth                        Conservation Contest. lel, Diblin,Chalrmar beoamittee on Nursing. Sophie Nelson, Chaiman -  malty Health Organimations ly I.Dubling «Chad  Subcommittees on Health Department Reports, J, leRies, Chai rean  sub@omalttee on Evaluation of Public Health Activities. Haven gmerson,  Every one of these subcommittees ie active and functioning and as                   ivan of the general qmuittee for nine of ite ten years of Life, the writer mst pay a tribute to the colleagues who have curried a major ehare     ittee on Adainistrative Practice is sul generis in     aging devotion of ite meabers to the solution of their                   Common problemss     The mjor enterprise of our Committee during ite ten years of service     has, of course, been the development of a model plan for manieipal        misation (ae embodied in our 1923 report ond in ty Health Organization) ani the preparation ef        the Appraisal form for the quantitative messurement of the services rendered by such on organization, It took considerable courage to undertake such an | enterprise eight x       APS aS05 There wae a good chance thet the attempt would  be regarded as agadenie and Saneaetiont and would die aborning; and there |  vad an altornstive poosibility that, if sesepted, 1t might lead to the  over~staniardisation which is om of our characteristic national vices, Our venture of faith has, we believe, been more than justified by  ite resulte. The plan of Som nity health orgs     nization presented in 1923 bae been accepted az generally sound hovever, it omy be modified     te eult speeial lemi circumstances. The buiget of 2.36 per capita worked out at that time closely approximates the actual practice of the best organised ¢ommnitics today. The Appraisal yorm has proved iteelf beyond any queetion a generally sound measure of quantitative performance.  the dang |     or of oversstabilization has been mot by providing for revision of the Appraisal Yorm every three or five years, & revision whieh, like a Republican revision of the tariff, iv always in an upward direction. the standaniigations gation plan for       n gense of the health officers hae prevented undus they have uaed the Appraisal Form and the model organi     self-appraicel to cheek up on the balance of their programe, for the     interpretation of those programs to the public and as powerful arguments for needed expansion, We can speak of these results without undue vanity for after 411] it ie the health officers of the country who have supplied          coeemreiasiciierecueemmemnanciriescreeeeueren ueiries ee a  the smteriale for the model hea  - fetrative health preetice through     ith program and appraieal and it is ‘the health officers who have used these instruments with such effect. The  enntetes on Adminietrative Pract     the health officers have pooled theiy reservoirs of experience, It is these health offieers who have been the real forees in     trative health practice froma haphazard political expericent to a standurdined selentifie procedure during the pact ten years,   +Glesely allied with the Gevelopment of the plan fer model health organtsation and of the Appraisal oma, has been the service rendered by Dre Talker and hie staff in the provigion at cost of speeial intensive ee        Committee, carrying ite infiuence from Quiney,Mase. to South Paeadena, Oslif, and from Marlon County, Oregon     and Hone lulu,?.B,  Pus, the Comalttes has moved rather eteséily forvard in ite teske of eolleeting : yenetiene of deriving therefrom standards of organisation and ach levement and translating these standards into terms of eonerete achtevenont through ite information and tleld service.  are in the liet ae well ae Montreal, Aenads     and analysing material in regard te current administrative     A Subsidiary and sompuhat different type of responsibility has, through force of ciroumetances, been forced upon the Committee during the past four years. The efforte onde by this association to mise the level of edmin~ out the country attracted the interest not     only of the insuranee companies and the foundations but of powerful national       so  agencies interested in civic vettersent. In 1926 Dr-Valker was able to enlist the active eupport of the General Federation of Yomen's Clube and of the UsS Chamber of Commerce     1» Both theee organisations have taken at       active part in promoting the cause we have at heart and beth have initiated * eompetitions in the field of local health  mong their constituent group Progress « Dr. Dabl in will describe the cutetanding success of the Health       Conservation Contest in whieh 108 ¢ities took part laet year, and which has already led to an increase in local chambers with active health committees from 100 to 226, Tt is difficult to eetiate the value to the health officer of euch a mobilisation of support as is here represented, The opportunity offered and the work had to be done; and, although particular programs wore so elesely tied up with the technical problens        fe outside the priaary functions of the Gomalttee, these     vey and appraisal that we have been fe reed to ear  ty them forward, ef the Gomaittee on Administrative Practice        then involves She continuation of three major old activities as foll@rne: fae The continued study of current municipal health practice with a view to the imeediat        uheomaittees on Record Porm, on Orgenised Care of the Sick, on Manual of Administration, on Health pepartaent Reporte and the like,     ig lon, at cost, of survey and eonsultation service to  individual comaunt ties with regard to their specific problems.     @. The provision of teehnical aid in th promotional activities unde extaken se of Commered,     ia qoperation with the General Federaticn and the CGhamb                Pee     nigh we hope and believe the Comittee can attack during the degade w       sigh ie to com.  The first of these fs the oroblem of rural hygiene. During the past fifty yesrs health hes cone to the eity and death rates in the crowded tenements have been cut aluost in half. On the farm and in the village , gh the splendid efforts     ugh maixmecle hee occurred. [6 Le trus timt throu     of the United States Public Health Service, the State health officers and the Rockefeller Youndation we have 500 full tive county hoalth unite in : nities are, however, without  operation in the United States, The other 2000 cov        any effective health oach        foundatione have been leid. Most of these fall time eunties work with a     lic health nurse eorvi       budget of 25 to 60 cents per capita ond with one pub  20,000, 40,000 there are not apre then a desen rural     counties in the United Stetes which have such health aachinery as would be     considered adequate for an urban areag and yet ve mow that the actwal needs of the form dweller are as great and probably greater than those of his city gousin, The development of modem public health service for the rural areae  ie the major health problem of the future,= not only in the United States but     It wae this conviction that led us in 1927 to aek the Goomonwealth Fund  ‘er a grant to make the flret comprehensive, comparative study of rural     health service. The field work of that study is now complete and Dr. Bishop  and preFreesan will report te you ite progress, I need ine re Ly add ag Cha trean     of the Committee that, if you approve, oe plan te make the furtherance of fural health work a major feature of our program for the fubure, in the belief that the equality of opps     rtunity of which Amrican tradition boasts will not                of the actual etatietien] rewulte achieved, Present stand  be & reality until the child in » rural county bas his @qual chance Pinally, we have a second             to believe muy be Thie eegond new task ie the attempt to evaluate the real wignif~          the ease.     ieance of cur administrative health procedures vy detailed selentifie study ands of health organisation and appraisal are based upon existing practice, in general  upen the practiee of the upper quartile of the cities which have been sur oy de  They vest upon the as@umption that the ends surrently sought               currently used to attain theese ende are sounds Im wany inetances they surely are. We may be reasonably sure that 1% is well to lemunize children age inet diphtheria and to pasteurise mile Yet even with reapest to euch obvious  @lements in our program it would be hard to elite a es     y of regulte obtained ties we conclusive ae to convinces the sceptical of the value        in varlous comenn! of the measure and to make clear to the health officer the particular admin« ra to many phases of health adainistration we have no exact evidence whatever x pon whieh to justify our empirical practices.     istrative procedures mst likely to yield maximum results, With regs        «TS may seem a somewhat illogical process te spend ten years in setting staniards and then to try and find out whether thove etandards are seientifi~ Cally justified. Yet I believe it was wise to do just what hae been done. A  clear picture of actual adminictretive practice and ite empirical foundatione  ‘Was oesential to more refined analysia; and preeticss, for which we convincing     statistical jurtifieation ean be adduced but which are indleated as valuable  on sound a wierd grounds, Will long eentimue to bulk large in health                                        ~10-  inistretion.s It ie full time, however, that & beginning should ve made  in laying a flrmer bagie for our work. If you approve, we hope to begin with       a few relatively simple problems, such ao scarlet fever isolation (with reayect     wh of the ivolation period for eases of various elinteal types)         diphtheria immnization (with respect te the wee of toxeid and toxin~a titoxin : at werlour age perlode and under various stakaivtenhios procedures) preneta! service (with the hope of ooneuring the actus tal Clinics ani prenatal mursing and the conditions under which they may be made  offs     i veeulte sitained by pre     tLive) routine inepeetion of echool ¢hildren (with a view in particular te the results of inspections made w doctor, nurse and teacher, reepeetively) and pasteuriaation ef milk (with reference to the actual rezaltes obtained by     injstrative procedures). Theee five studies alone will take the greater part of two years and the whole tack stretches inte the indefinite fut     aro, Ye believe, however, that it is the next great task which aust be fomoved from a basis of a       etrative health practi ioe iss step by step, to be     priori empiricism to     that of demonatrated eclontific     evidente.  Thie Le our accounting of our ten years’ stewardship and these ave the     poesibiiities we see for future service. If we have in some meagure         you it is for four reasons, We have had gensrous financial suppert. $6000 came from the general budget of the Assoctetion and $254,¢     ning of ite work tho Committee hae apent over $660,006            wuranoe companiot, foundations and cities and counties directly served by ute We have had an extraordinary commlttee sembership, sixteen men ani women serving with unparalleled ardor and enthusiasm and gaining in capacity for ,  service with the experience of each year, %@ have had the splendid leadership                         members of this astociation and of the health officers of the country.      t be done under any system of     : We are dealing with essentially research : problems which on freedom to openi the funds we receive for specific a purposes fc 1” those specific purposes aad theough personnel chosen and J     ag sOreL ynekted for those specific purposes. So far we have been given euch freetens of the health adminis ‘vesr actual fruit. So far we have hed that support.     our work cannot be done without the active interest     traters who alone con make our studies ant etaniardes  yo O™~  If the Aesoulation feels our task ie done we ghall be clad +. rest from cur labors. If it desires thet thie tack ghell continue, a reasonable measure of autonomy aml a gencrour wesmare of support from the health efficere of the country are essential te the future of the Committee on Aduinietrative Practice.", "American Public Health Association. Committee on Administrative Practice", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e6qa.fccf~9ewi", "00000000-0000-0000-8A15-2AF591B560AF", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Committee on Administrative Practice Program for 1933", "9918573882206676X126", null, "1932", "01 November 1932", null, "Agendas (administrative records)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "6", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "-gervice:  PROGRAM FOR 1933  The Committee on Administrative Practice of the American Public Health Association hopes to carry on the following activities in 1933:  (1) Field Service = The Commi ttee will during 1933 offer the following types of f", "American Public Health Association. Committee on Administrative Practice", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fiwn_jxpq_f5s8", "00000000-0000-0000-70D3-42A8D48AE15F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Activities of the Committee on Administrative Practice, 1932", "9918573882206676X127", null, "1932", "[1932]", null, "Reports", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "5", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "—_  ON  soe . Activities of the COMMITTEE ON ADMINISTRATIVE PRACTICE of the AMERICAN PUBLIC HEALTH ASSOCIATION ,'9°%  The Committee on Administrative Practice was organized in 1920. Its function is the analysis, evaluation and standardization of public health practices. The activities of the Committee are carried on directly by its membership which is composed of outstanding leaders in public health work in > this country. The present membership includes:  Professor C.—E. A. Winslow, Chairman, Yale School of Public Health, New Haven, Conn. Dr. Haven Emerson, Vice Chairman, College of Physicians and Surgeons, Columbia University, New York, N. Y. , Dr. Louis I. Dublin, Metropolitan Life Insurance Company, New York, N.Y. Dr. E. L. Bishop, State Commissioner of Public Health, Nashville, Tenn. Dr. Charles ¥. Chapin, formerly Health Officer, Providence, R. I. Dr. Michael M. Davis, Julius Rosenwald Fund, Chicago, I11. Dr. Allen W. Freeman, Johns Hopkins University, Baltimore, Md. Dr. Joseph W. Mountin, U. S. Public Health Service, Washington, D. C. Miss Sophie C. Nelson, President, ational Organization for Public Health Nursing, Boston, Mass. Dr. George T, Palmer, American Child Health Association, New York, N. Y. Dr. W. 5. Rankin, Duke Foundation, Charlotte, N. C. Dr. John L. Rice, Health Officer, New Haven, Conn. Dr. Robert H. Riley, State Commissioner of Health, Baltimore, Md. Dr. George C. Ruhland, Commissioner of Health, Syracuse, N. Y. Dr. Henry F. Vaughan, Comnissioner of Health, Detroit, Mich. Dr. W. F. Walker, Commonwealth Fund, New York, N. Y. Dr. Carl E. Buck, Field Director.  The Committee fulfils the functions for which it was appointed through its Field Service and the work of its sub-Committees, of which there are some fourteen at the present time.  FIELD SERVICE  The first activity of the Committee was a study of public health undertaken in 1920, in cooperation with the United States Public Health Service, of all cities in the United States having a population of 100,000 and over.  (See Public Health Bulletin No. 136 - \"Report of the Committee on Municipal Health Department Practice\").  In 1924 a similar study, also in cooperation with the United States Public Health Service, was made of all cities having a population of 70,000 and — over. (See Public Health Bulletin No. 164 - \"Municipal Health Department Practice for the year 1923\").  A full-time field director for the Committee was appointed in 1925 and the services of experienced staff members hayebeen available to states, cities, and rural communities for the purpose of public health and hospital surveys. The number of surveys made in the past eight years is as follows:  4 states; 43 cities; 30 counties; 77 total.       These surveys are always carried on in npopamatien with the local heelth department and have the endorsement of the state health department  concerned. They provide a complete stock-taking of public health facilities  in the community, an analysis of the data obtained, an evaluation of the work which is being rendered in relation to the needs of the community, and finally a report with recommendations for correcting deficiencies noted and planning a coordinated program. (See “Exhibit I\" for typical report of survey and appraisal ). |  SUB-COMMITTEE ACTIVITIES  It is impossible in a brief report to present all the work which has been accomplished by the sub-Committees of the Committee on Administrative Practice. However, mention should be made of some of the most important activities undertaken.  Health Conservation Contest  In 1929, in cooperation with the Chamber of Commerce of the United States, the Committee inaugurated the first Inter-Chamber Health Conservation Contest. The enrollment for that year was 149 cities. The interest in these annual competitions has increased steadily. There are 266 cities already en- rolled for the 1932 Contest and before the end of the year it is anticipated  that the number will be larger.  Consultant service to cities ieee in the Contest is provided through the field staff of the Committee on Administrative Practice. (For detailed report of the Health Conservation Contest see \"Exhibit II\").  isal £ City Health Wo  The first Appraisal Form for City Health Work was issued o expesinantal form in 1925. This was somewhat revised and a second edition printed in 1926. After three years use this sub-Committee prepared a third revised form which appeared ready for use Jamuary 1, 1929. (See \"Exhibit III\").  For the past year the sub-Committee has been worsing on another revision which willbe Tew for use in 1954.  The use of the Appraisal Form is advocated and demonstrated by various means. Health officers are encouraged to use it as a means of checking up their  own activities and in reporting public health practice and progress. It is  used extensively for teaching purposes by universities.  Appraisal of Rural Health Work This gub-Committee drafted the first Appraisal Form for Rural Health  - Work in 1927. A second edition, very much revised on the basis of information  gathered in an intensive survey of rural health work, was released for distribu- tion January, 1932. (See \"Exhibit Iv\").    ——  The survey of rural health work, mentioned above, brings together for the first time complete and comparable information of health department practices in rural areas. The final report of this study is now in the hands of the printer and will be ready for distribution at an early date.  , The sub-Comittee on Rural Health Work is now concerned with the development of record forms to be used in rural communities.  Record Forns  The sub-Committee on Record Forms has concerned itself with the development of suitable record forms for use in city health departments. Two  printed reports have been issued. (See \"Exhibits V and VI\"). Arrangements  have been made with a commercial organization for printing the forms approved by this Gommittee for sale to health departments desiring large quantities of  the forms. A third report of the committee will be issued in 1933.  ~  Organized Care of the Sick  Under the auspices of this sub-Committee two studies have been carried on, one in 1927 on \"Relations between Heal th Departments and Hospitalsti (See \"Exhibit VII\"), and the other in 1929, dealing with \"The Care of Comnuni- cable Disease Cases in General Hospitals.\" (See \"Exhibit VIII\"). In 1931 the committee prepared \"Twenty-five Questions Which Public Health People Should Ask About Hospitals and Clinics in Their Commbnity.\" (See \"Exhibit IX\"). At the present time the committee is concerned with the development of a survey schedule and appraisal form for agencies furnishing organized care of the sick.  omnunity Health 0 ion  In 1927 the Committee on Administrative Practice brought together in one volume entitled “Commnity Health Organization\" model plans of organization of a health department for cities of 100,900 population and over, for cities of from 2,000 to 100,000 population and for a rural community of 30,000 population or less. One of the major accomplishments of 1932 was the preparation by the sub-Committee on Commmity Health Organization of an enlarged and revised edition of this book. (See \"Exhibit XxX\"). 2  Evaluati on of Administrative Practices  Through generous grants from the Commonwealth Fund studies were inaugurated in 1921, under the auspices of this sub-Committee, on Diphtheria Imminization, Toxin-antitoxin and Toxoid, Scarlet Hever Control, and Prenatal Car ee. =  Diphtheria Immunization = The work of the first year covered e study in the epidemiology of diphtheria in relation to the active immmization of  certain age groups. (See \"Exhibit XI\").  In 1932 an analysis was made of all the accurate data on diphtheria immunization which could be obtained. A letter was sent to all health officers | of cities of 25,000 population and over, and to state health officers, calling    attention to the inaccuracy of records on the present stetus of diphtheria im- __ munization, and putting forth a plea for more careful and accurate local = appraisals of the diphtheria problem. Enclosed with the letter was a short pamphlet on \"What Should Each Community Know Concerning Its Diphtheria Problem?\" a set of suggestions concerning methods of taking cross sections or otherwise estimating present immunization status by individual age groups anda table through the use of which children immunized in past years can be translated  into their present age. (See \"Exhibit XII\"). Studies have been made in Niagara Falls and Albany, New York, of the relationship of immunization to births (births being used as an index of child population) in the epidemic areas  as compared with other areas of the city.  Toxin-antitoxin and toxoid - Laboratory tests have been conducted to determine whether toxoid is more efficient than toxin-antitoxin, themuber of antigenic units, the number of doses, and the interval between injections required to develop a negative Schick. (See \"Exhibit XIII\").  Scarlet Fever Control - The first year of the study was spent principally in the development of methods of determining what factors enter  into the problem of scarlet fever control. (See \"Exhibit XIV\"). Studies during 1932 have been undertaken to ascertain the importance of carriers and missed cases in the transmission of scarlet fever. Contacts have been carefully examined on the day of report of the first case, on the second day, and at the end of the first, second, fourth, and eighth weeks. As a result of these examinations and by culturing, numerous missed cases and carriers heave been discovered.  A very careful analysis of 10,666 eases of scarlet fever has helped to  substantiate many of the tentative conclusions included in previous reports  and in addition has shown that, at least as far as this series of cases is concerned, more than 50 per cent of all scarlet fever deaths occur among children under five years of age and over 80 per cent among children under 10 years of age, and that the case fatality rate of scarlet fever cases under one year of age is 22 as compared with a fatality rate for all ages of only 1.  Prenatal Care - The problem considered by this study was: \"To what extent are the favorable results in lower stillbirths, neonatal and maternal mortal-— ities apparently obtained from the use of medical and nursing service in the prenatal period, due to the character and intensity of the service, or to the period of pregmency over which the service was rendered?\" Complete and careful studies have been made of all deliveries in one year at the New Haven Hospital and of 1000 cases at the Boston Lying-In Hospital. The sub-Committee has not made  a report of the progress of this work, but it will do so before the end of 1952.  nt H th Depa t Practi  The program of this sub-Committee was started in 1932 and is desiged — to bring about the establishment in the office of the Committee on Administrative  Practice of a file of current information concerning practices of health departments.    6.  Medical Participation The sub-Committee on Medical Participation has as its objective the  coordination of interest and activities of health departments and organized medicine. Because of a lack of funds the committee was unable in 1932 to  undertske much active work except as individuel members of the committee had  the = of presenting the subject to interested groups. (See Program for 19338).  Additional sub-Committee reports are presented in \"Exhibits XV,XVI,XVII®", "American Public Health Association. Committee on Administrative Practice", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-53j6_rquh.k4a2", "00000000-0000-0000-877B-85568F8B1263", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "The Improvement of Public Health Work through the Stabilization of Health Budgets, Sound Health Organization, and the Evaluation of Activities", "9918573882206676X128", null, "1933", "20 September 1933", "Draft of an article that addressed the many challenges faced by public health workers as budgets for public health activities declined during the economic depression of the 1930s.", null, null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "9", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "a . u Ano %  i 5 bs dot pt =  Lar — fut f f en dy. -O        % aw  THE IMPROVEMENT OF PUBLIC HEALTH WORK THROUGH THE STABILIZATION OF HEALTH BUDGETS, SOUND HEALTH , : ee atten AND THE EVALUATION OF ACTIVITIES / :  During the past two or three years health appropriations, never adequate to do a completely satisfactory job, have been cut severely.  Some departments of health lost most if not all their child welfare activi- ties, others a majority of their public health nurses, and still others such im- portant functions as tuberculosis and venereal disease eontrol. Suen eliminations of essential health activities are bored if continued, to eventually adversely affect the public health. Such reductions are clear cut examples of false economy for which we are bound to pay many times over in the future.  There are three important functions which all those interested in the maine  tenance of public health protection shouid unite in fostering:  ay)  Gg The stabilization of public health appropriations. WA IG \"os     (2) The stabilization of sound health organi gation and administration.  (3) The evaluation of public health activities to the end that funds expended for public health shall be used in the most scientific and effective manner.  The first of these functions can be achieved only by a widespread educational campaign aimed to acquaint the public, and most particularly those. officials who are entrusted with the appropriation of funds, with the importance and necessity of maintaining adequate health machinery and of the false economy in reducing es- sential. public health appropriations.  The average individual, including legislators, knows something about highways, he sees them and he uses them, he sees the parks and recreation centers and uses  them, he sees school buildings and uses them, he sees the policeman on the corner  and knows what he is there for. All these things he is interested in because    they affect him personally. He may have heard of the health department, probably not much more than that. It is doubtful if he has ever actually seen any real public health work going on. If hé has any impression at all as to what consti- tutes public health work he is more than likely to picture it as embodying primar- ily garbage collection and tacking up quarantine signs. He does not see that public health work affects him personally in any way and he therefore quite naturally lacks interest in it.  Even granting, as we must, that public health work is far more difficult to dramatize than highways and public schools, nevertheless the average individual 's complete lack of understanding of what public health means to himis a severe crit- icism of our methods of general public health education.  The keynote of our general public health education programs must be to in- dividualize public health if we are ever to attain the measure of success which the importance of the work merits. This seems to be the crux of the entire prob- lem. Public health protection must be made the problem of the average individual. He must be made to see that public health does affect him personally, intimately, definitely, and in an important way.  How can this be done?  Obviously all the various avenues of publicity may be made use of - radio, talks, newspapers, pamphlets, and magazine articles, and the program should be nationwide in scope.  These are, of course, merely the avenues of approach. How shall the message or messages be prepared and presented? This presents a much more difficult prob-  lem but we may perhaps formulate certain principles which might prove helpful: (1) The talks and articles, particularly radio talks, should be short.  (2) They should be simply worded and their keynote must be an in- dividual personal appeal. We must minimize the general appeal to civic mindedness and impress the average individual, to whom our remarks are addressed, with the fact that our message affects him personally.    (3) Particularly in radio talks, the dialogue is apt to be far more effective than the individual talk.  (4) We should not attempt to cover more than a few pointe in any Single talk or article.  (S) We should bear in ‘mind the fact that our usual audience is already interested in and has some knowledge of our message. In this program we must constantly endeavor to reach a dif~ ferent type of audience ~- one that has as yet no special interest in and knowledge of our message.  (6) We should recognize the fact that with very few exceptions public health workers are not properly trained to themselves do the job which we wish done. We should, therefore, wherever possible, avail ourselves of professionally trained people:  (a) Professional actors for radio dialogues,  (b} Persons trained in advertising and journalism for the preparation of talks and articles.  With these general principles as a background, with the aid of professionally trained people, and with a nationwide well planned program, we may look forward to | at least come measure of re  The second function, that of stabilizing sound health organization, may be approached through an educational campaign, perhaps a somewhat more direct campaign than the one aimed to stabilize health appropriations, in which the American Public Health Association sends directly to legislators and legislative bodies its pro- nouncements on what constitutes sound health organization.  Such pronouncements would include at least the following definite statements:  (1) The health officer should be directly responsible only to his board or to the chief executive of his community.  (2) All public health work should be coordinated under the leader-~ ship of the health officer and the health officer should have no other responsibilities. Health and welfare activities should not be combined in a single department.  (3) (a) The health officer should be employed on a full-time basis and should be well trained for his position.  (b) There should be full-time well trained personnel for all executive and administrative positions.    (4) The health officer should have supervision over the entire community public health program (whether or not he actually earries on all the integral parts of the program) and should provide such personal health services as are essen- tial and cannot be adequately provided through other channels.  (5) Preventive and curative medical activities are very closely allied and should be inter-related as closely as possible but:  (a) Curative medical activities such as the care of the indigent sick should be carried on by medical insti- tutions, hospitals, universities and the like. Al- though welfare and charity organizations may well finance such activities they should not themselves carry on medical work.  (o) All preventive medical activities should be carried on under the direct supervision of the health officer. This does not mean that the health officer need neces- sarily be in direct charge of all preventive services but rather that he should have such supervisory juris- diction as would protect the public health and ee duplication of effort.  Now we come to the third function in which we should also all be interested; the evaluation of public health activities to the end that funds expended for pub- lic health should be used in the most scientific and effective manner.  It is unreasonable for us to expect appropriating bodies to provide adequate public health budgets unless we can show that funds appropriated are being used in as Wise and scientific a manner as knowledge permits. It is probably safe to say that there is less public health money wasted than in perhaps any other govern- mental department but on the other hand it is probably true that there are a great many health departments which could make a more scientifically effective use of some of their funds.  There are two main objectives in evaluating health activities and the rela-  tionship of health activities to each other:  (1) To show appropriating bodies that we are using funds in as wise a manner as possible;    (2) In communities where, in spite of efforts, appropriations remain decidedly inadequate to make the health dollar just as effective as possible.  City and Rural Appraisal Forms have given us excellent measuring rods for the general adequacy or inadequacy of health programs and the degree to which these programs were well or poorly balanced. They also provide some actual evalu» ation of services rendered but with the exception of the weighting factor which is applied to each major activity there is nothing to evaluate the relative importance of the various component parts of the public health program nor is there in these Forms any means of evaluating the effectiveness of individual procedures.  Are we spending too mich or too little for a given health activity in rela- tion to other essential activities of the program?  Are the procedures used in carrying on an admittedly essential activity so designed as to give maximum results for effort and funds expended?  These are questions of great importance which every health department ought to seek answers for.  Today our deh, and one of our principal jobs is to take out the \"slack\" in our programs, to differentiate between essentials and non-essentials, to judge the relative importance and effectiveness of the various factors in and phases of pub- lic health work. Obviously this cannot be done by any rule of thumb method for eee differ both in their problems and in the eelative importance of their problems. Malaria control may be of paramount importance in one community and of no significance at all in another, and so on. It does mean, if we are to do our job well, that we must consciously or unconsciously appraise and evaluate the component parts of our program and in many even the details of procedure within a Single activity. We must first clearly define our major problems and then study  the relative effectiveness with which our activities are meeting these problems.    For example, we must determine first, whether or not all the activities of our pro-  gram are essential and secondly, having established the fact that a given activity  is essential we must examine the various procedures employed in carrying on that  activity to determine their relative importance and effectiveness.  While, as previously stated, it is obviously impossible to determine precise-  ly how any given community can make its health dollar more effective there are  perhaps certain fundamental principles and general considerations which any com-  munity can profitably make use of.  Three fundamental principles readily occur to one:  (1)  (2)  (3)  That we should exert our major efforts to those activities which give the greatest promise of effectively protecting and promoting child health, particularly of young children. This would immediately classify communicable disease control, pre- natal, obstetrical, and infant work, and milk control as es- sential activities. Since water supply and sewage disposal affect persons of all ages attention to those activities would, if this principle is accepted, be essential parts of our program.  That our major efforts should be confined, as far as prac- ticable, to those activities the results of which are reason- ably definitely known. Thus diphtheria immunization, smallpox vaccination, typhoid fever control, the protection of the water supply and the pasteurization of milk become integral parts of our public health program.  To, insofar as it is feasible to do so, carry on those activi- ties which are likely to produce maximum results at minimum cost. While naturally this group of activities will in most instances be synonymous with those activities the results of which are definite this principle would tend to eliminate (during this period of depression) elaborate epidemiological communicable disease control methods which although effective are very costly. For example, it would probably be foolhardy for a department of health today to institute the elaborate malaria control measures which were so effectively and neces- sarily carried on during the construction of the Panama Canal. As at present carried on, much of our school health work  falls within this category.  Bearing in mind these three fundamental principles, let us discuss certain  general considerations. Some of these are:    If we have been spending an unduly great amount of time and effort in inves- tigating nuisances most of which have little or no public health significance, this work can be eliminated without doing any great harm.  If we have any inefficient employees this is obviously the time to get rid of then.  One of our major considerations should be to maintain, at almost any cost of curtailing other activities, our maximum public health nursing staff for without  these important emissaries in the home our opportunity of accomplishing results  is tremendously reduced.  If after examining our program on the basis of the three fundamental prin- ciples previously referred to and having eliminated all but essential ma jor activities our budget reduction has been such that we are obliged to still further reduce our work, what is our next stent  The next thing to do would seem to be to study carefully the procedures and methods used in carrying on essential activities.  Such an examination and study might conceivably bring to light practices which could be either eliminated or altered in such a way as to effect a saving without impairing the general effectiveness of the service.  If, for example, our nurses are calling on prenatal cases eight or ten times | during pregnancy, this number of calls may perhaps be reduced to three or four, depending of course on the needs of the individual patient, without increasing our maternal or infant mortality.  Infants may be coming to our well baby conferences routinely every two weeks. We may well change this rule and have the clinic physician determine the fre- quency of visit based on the condition and progress of the individual infant.  This will very likely reduce clinic visits to 5 or 6 visits a year except for    special cases. The tendency seems to have been to try to have an unnecessarily large number of visits.  Preschool clinics or conferences often attempt to have their children come in every month or every other month. Pediatricians usually do not succeed in having their children of preschool age come in more than 2 or 3 times a year. This is as much &$ W6 can expect and is probably as often as is necessary, except of course in special individual cases. Preschool service in Bonerel. while potentially pro- ductive of excellent results and worth while, particularly in the splendid educa-~ tional effect of such a project as the a Round-Up of the National Congress of Parents and Teachers, is less well defined both with respect to procedure and re- sults accomplished than some other forms of service. It would seem that because of this fact both prenatal and infant service would take precedence over it.  Probably nowhere in the entire public health program, with the possible ex- ception of nuisance investigation, is the public health dollar less effectively spent than in school health service. There are, of course, notable exceptions to this general statement but considering the country as a whole, in spite of the re- markable potential benefits of a well conducted school health program, we have spent more money on school health services with less to point to in the way of tangible measurable results than in any other major phase of public health work. We are not referring particularly to school nursing service, although that could be greatly improved and is probably the poorest done of any public health nursing service, but rather to the so-called medical examination of school children. The story of the reasons for the ineffectiveness of school examinations, which might more appropriately be termed \"inspections\" (and we are referring here to the routine examination of elementary school children) is altogether too long to be  told in this brief article. Suffice it to say that while it is our claim that the  primary object of the school examination is to educate parents to the value of a    périodic health examination it has failed miserably in so doing. Although it is  true that we have succeeded in having large numbers of physical defects corrected We still continue to find approximately the same percentage of physical defects in school children of a given age as we did ten or fifteen years ago and moreover comparatively few parents have accepted and put into practice the principle of  having periodic health examinations for their children of school age.  Let us examine our milk control progrem. This is unquestionably an essential  activity but one in which not infrequently there is a good deal of lost motion. If milk control work of the past twenty years has been at all successful, and we believe that it has been, it has convinced both farmer and distributor that it is good business to produce good quality milk. We no longer have to employ police methods to accomplish results. If in our program we are attempting to visit each producing farm 2 or 3 or more times each year perhaps making a detailed score each time, would it not be wise to reduce the number of farm inspectors and divert time thus saved to a more productive field? If each farm has had one thorough es tion to assure us that equipment and methods meet satisfactory standards it would seem practical from this point on to devote, as far as the work in the country is concerned, our major effort to checking the milk at the country loading or dis- tributing stations, through temperature, sediment, and odor tests and bacterial examination or reduction or methylene blue test, going back to the individual farm from which the milk came whenever milk of unsatisfactory quality is found.  | These are, of course, only a few of the things which a study of procedures and methods may reveal. In general, we may say that rules of routine procedure, such as visiting the home of a patient regularly once a month, which may have been valuable at the time they were instituted, if continued too long and followed too  religiously, often result in much wasted effort.  It is perfectly clear that in order to make the best use of our public health  dollar we must know our problems and our program thoroughly.  September 20, 1933", "American Public Health Association. Committee on Administrative Practice", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-q6ih-hytu~3tqs", "00000000-0000-0000-F16C-7DD9F742F859", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "City Health: Where Does Your City Stand?", "9918573882206676X129", null, "1925", "01 November 1925", "This special issue of Survey Graphic featured articles by sixteen notable public health experts, using data gathered in American Public Health Association surveys between 1920 and 1924.", "Articles, Journals (periodicals)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "83", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "CITY ‘ile HEALTH                30 cents a copy November, 1925 $3.00 a year     duty ILLIAD ( ea 3 Dy  ISCONSING Eide lu NCALIFORNIZ iia MICHIGAN       Pee  = Be            Book fie atte     irs |              Among the largest one-man shovels in the world is this tremendous one, used on the Mesabi Range in Minnesota. It picks up 16 tons of ore ai a bite, which it deposits in a car—all in less than a minute,  ca ary day’s work at every gulp  Surgeons use a tiny A hand shovelful of ore weighs 21 oe they examine pounds, and a man can handle 200 an ear. Miners use G-E. shovelfuls in an hour. But here is motored hoists tg re- . ° te  move tons of ore from a giant that picks up, in one gulp, a mine. Wherever there ;  is dificult work to be more than a man can shovel ina day!  done you will find that the General Electric  Company makes some- _And the G-E motors that animate thing electrical that will . . help. the giant never get tired.     ~ GENERAL EL        CTRIC              No. 2) 30 cts:, $3 00 a year; foreign postage, 50 cts. extra; Canadian 30 cts. Changes of address should be mailed us two weeks in advance.  5 ill ‘be sent only upon request. Entered as second-class matter, November ‘ 25 1 Yi Apeabtanon for mailing re special rate. of postage “providedfor in~Seetion 1103, ‘Act of October 3, 1917, authorized December 21, 1921.  ‘Sec’y, Ann Reed Brenner. ‘Treas., Arthur Kellogg.     SURVEY GRAPHIC, published nionthly: and copyright 1925 by SURVEY ASSOCIATES, Inc., 112 East 19th Street, New York. Price: this copy (November, 1925; Vol. VIII,  When payment is by check  1921, at the post office, New York, N. Y., under the Act of March 3, 1879. Pres., Robert W.  de Forest. ere i emcee rs etna                       Typical Interior of Block at Sunnyside, Long Island City. Fifteen Minutes from 4and Street, Manhattan, by subway: 5c fare. Garden Homes for 1000  Families.  Sold—not Rented—at about $10 per room per mosth. Only 30%  of the land is built on—70% left for playgrounds and gardens. All houses are only two rooms deep, giving a maximum of sunlight, fresh air and  cross ventilation.  Here is a unique  BOARD OF DIRECTORS  Alexander M. Bing President  Dr. Felix Adler John G. Agar Leo S. Bing William Sloane Coffin Thomas C. Desmond Douglas L. Elliman Prof. Richard T. Ely Frank Lord V. Everit Macy John Martin  rs. J. M. Proskauer Mrs. F. D. Roosevelt Robert E. Simon  ARCHITECTS Clarence S. Stein Henry Wright Fred. K. Ackerman  investment opportunity  Buy stock in City Housing Corporation and  secure a safe investment and a steady 6% in-  come, at the same time helping build healthy homes fir people of moderate means  ITY HOUSING CORPORATION is a limited divi- dend corporation, with ideals of better homes and communities. Large scale building operations and pur- chase of land, together with better planning and group- ing of houses, enable it to sell well built homes for less than current rentals; at the same time much needed playgrounds and gardens are provided, insuring the healthiest environment.  Dividends, which are limited to 6% per annum, have been earned since the company was organized and a substantial surplus accumulated to assure the safety of the investment.  CiTy HOUSING CORPORATION  A Limited Dividend Company— Organized to Build Better Homes and Communities  587 Fifth Avenue, New York City Authorized Capital, $2,000,000 in shares of $100 each  CITY HOUSING CORPORATION, 587 Fifth Avenue, New York City, N. Y.  Please send me full information:  6.2  Name  eee eee eee ere sere eee reese eee seer revere eee eeenee  (12 answering advertisements please mention THE SuRVEY. It helps us, it identifies you.)  109             No Such Free Homes  As St. Rose’s Home on the Lower East Side of New York, or Rosary Hill Home on the Westchester Hills, an hour away from the city, exist from North to South in the United States.  They are for cancer-cases that are destitute and pronunced incurable; and absolutely no pay is received from relatives or the State.  The reasons are beneficial to the patients, and the public mercy is the softest mercy for them, as many letters of harsh indif- ference prove.  Please help to complete our Fund for a fire- proof Country Home for our present mem- bers of 60 or more such men and women of all Creeds and Nationalities.  MOTHER M. ALPHONSA LATHROP, O. S. D.,  ‘Treasurer, The Servants of Reliet  Rosary Hill Home, Hawthorne, N. Y.                          STATEMENT OF THE OWNERSHIP, MANAGEMENT’, CIRCUU,A- TION, ETC., REQUIRED BY THE ACT OF CONGRESS OF AUGUST 24, 1912, of SURVEY GRAPHIC, published monthly at New York, N. Y., for October 1, 1925.  State of New York, ; ;  County of New York, SS.  Before me, a Commissioner of Deeds, in and for the State and county aforesaid, personally appeared Arthur Kellogg, who, having been duly sworn, according to law, deposes and says that he is the business manager of the Survey GrapHic, and that the following is, to the best of his knowledge and belief, a true statement of the ownership, management (and if a daily paper, the circulation), etc., of the aforesaid publication, for the date shown in the above caption, required by the Act of August 24, 1912, embodied in section 411, Postal Laws and Regulations, printed on the reverse side of this form, to wit:  1, That the names and addresses of the publisher, editor, managing editor, and business managers are: Publisher, Survey Associates, Inc., 112 Fast 19 Street, New York City; Editor, Paul U. Kellogg, 112 East 19 Street, New York City; Managing Editor, Geddes Smith, 112 East 19 Street, New York City; Business Managers, Arthur Kellogg, John D. Kenderdine, 112 East 19 Street, New York City.  2. That the owner is: (If owned by a corporation, its name and address must be stated and also immediately thereunder the names and addresses of stockholders owning or holding one per cent or more of total amount of stock. If not owned by a corporation, the names and addresses of the individual owners must be given. If owned by a firm, company, or other unincorporated concern, its name and address, as well as those of each individual member, must be given.) Survey Associates, Inc., 112 East 19 Stas New York City, a non-commercial corporation under the laws of the State of New York with over 1,700 members. It has no stocks or bonds. President, Robert W. deForest, 30 Broad Street, New York, N. Y.; Vice-Presidents, Julian W. Mack, 1224 Woolworth Building, New York, N. Y.; V. Everit Macy ‘“Chilmark,” Scarborough-on-Hudson, N, Y.; Secretary, Ann R. Brenner, 112 East 19 Street, New York, N. Y.; Treasurer, Arthur Kellogg, 112 East 19 Street, New York, N. Y.  3. That the known bondholders, mortgagees, and other security holders owning or holding 1 per cent or more of total amount of bends, mortgages, or other securities are: (If there are none, so state.) None.  4. That the two paragraphs next above, giving the names of the owners, stockhelders, and. security holders, if any, contain not only the list of stock- holders and security holders as they appear upon the books of the company but also, in cases where the stockholder or security holder appears upon the books of the company as trustee or in any other fiduciary relation, the name of the person or corporation for whom such trustee is acting, is given; also that the said two paragraphs contain statements embracing affiant’s full knowledge and belief as to the circumstances and conditions under which stockholders and security holders who do not appear upon the books of the company as trustees, hold stock and securities in a capacity other than that of a bona fide owner; and this affiant has no reason to believe that any other person, association, or corporation has any interest direct or indirect in the said stock. bonds. or other securities than as so stated by him.  [Signed] ARTHUR KELLOGG, Business Manager  Sworn to and subscribed before me this 25th day of September, 1925.  [Seal] MARTHA HOHMANN, Commissioner of Deeds, City of New York. New York Co. Clerk’s No. 148. New York Co. Register’s No. 26032. My Commission Expires May 20, 1926.        4  $1.00—New Edition $1.00 Mind in the Making  The Relation of Intelligence to Social Reform  By James Harvey Robinson         James Harvey Robinson, author of  “The Mind in the Making,” and Joseph  K. Hart, associate editor of Survey  Graphic at Frederic C. Howe’s School  of Opinion at Siasconset, Mass. Mr.  Howe and the Atlantic Ocean are be-  hind the School and do not show in the  picture. They were snapped red-handed  in a plot to make people use their minds  as well as their fingers and feelings. | James Harvey Robinson: “As an old stoic proverb has it, men are tormented by the opinions they have of things, rather than by the things themselves. This is eminently true of many of our worst problems today. We have available knowledge and ingenuity and material re- sources to make a far fairer world than that in which we find ourselves, but various obstacles prevent our in- telligently availing ourselves of them... . In short, how are we to rid ourselves of our fond prejudices and open our minds?”  Joseph K. Hart: “Few people ever knowingly begin a book that is likely to challenge their prejudices and make them uncomfortable. . . . Reviews help in this. by giving readers advance notice of books to be avoided on pain of being stirred into thought. A single book might start a new age if it were widely read. Such a book is Prof. James Harvey Robinson’s ‘The Mind in the Making’.”  H. G. Wells: “When I come to reckon up this American visit, I think I may well feel that the encounter of most importance and likely to have the greatest lasting effect upon me is meeting and talking to Prof. James Harvey Robinson and reading his fascinating book.”  The new Survey Associates Edition of “The Mind in the Making” at $1.00 is printed from the plates of the original edition, of which 100,000 copies were sold at  $2.50 each. Cloth bound. 238 pages including the final chapter, “‘Some Suggestions in Regard to Reading.’’     SURVEY ASSOCTATES 112 Fast 19 Street, New York I enclose check (or money order) for $........ r which  fo please send me ........ copies of The Mind in the Making at $1 each.  Name  0000099000980000090C95000009000000000000000000000000000  11-1-25-        (In answering advertisements please mention THe SURVEY. It helps us, it identifies you.)  110       ‘A-62 — The Bradford. A prize - win- ning ~ design. A-75 — Dinner i bell. Solid          A - 145 — B-160 — Lady         long and brass. A min- Christmas Elizabeth. 2%” wide. iature mor - candlesticks. A-94—Cake tray in brass. Not hammered. A-21 — Colonial peas English din- It was never tar and pestle. 38” high. 942” long and 7” wide. Special...... . .$3.00 roomstick. Solid ner bell. BS priced so 2” high. Price, the B-94—Sandwich tray. Same as A-94, “except brass. 4” high. 8%\" hig  low .. $2.75 sellesces: Ga.ee pair .. $1.10 flat, without.base. Price. vos stee ss Peed Special, each. ‘SI. 95 Special. o$t. 0                     A-63 — Bud vase. Ham- A-142—The pro- A-29 — Brass mered brass. A-78—Card-tray. In brass or A -{62 — The  verbial Cheshire wall - sconce. About 6” tall. copper. A very fine piece for Renfrew. Solid Cat. Solid brass. Back is 9%” A good prize the reception-table. It is brass. 3%” long 344” long. Its long. Arm suggestion for A-165 — Can- 34%” high and 6%” in di- and 2” wide. pedigree dates extends out your whist dleholder in ameter. It could also be Carries the sem- back to ‘“‘mer- B-21 — Dolphin candlestick. 3 5”. Specially party. Speci- brass or cop- used as a candy tray. Not lance to a coat rie old Eng- Brass. 4” high by 4” in di- . price d, ally priced per. 4” high. hammered. Specially priced ofarms. Special- land.’’ Special- ameter. Price, each....$2.25 . each .. $2.95 95c. Special .. 95e  ......... eee «see. $2.00 Jy priced...$1.25 ly priced...$1.10 :         A-161 — Jacobean winding candle- sticks. 1144” high. Strikingly artis- A-37—Prize -win- tic and tastefully     A-134—Bud-vase candle- sticks. 744” high. Can-  dlesticks are popular A-4—Russian tea-set. Hammered brsas or copper. The kettle is 6” high and ning candlesticks. different in de- with men, Price, holds 10 large cups. The tray, hand-made, is 18” long and 12” wide. The 6” high. Price, the sign. Price, the DAT ........ sees $4.95 creamer and sugar-bowl are good mates. Featured, the set................ $18.00 pair ....... $3.50 pair ....... $7.50     A-9— Tulip candle- sticks. 11” high. A tulip cup, a slender  body, and a flat a.149—Dutch coffee - -pot. Hammered brass or ECC eas copper. Made| Zor a party of ae: 64\" high, * Y i y . ecial.....$4. : pair ......... $5.95 eo eas SrRO\" CUDS gees: vs A-24 — Lion candelabra. os 11%” high. A very inter- A-!— Foxboro can- A-159—Eagle candelabra. Solid brass. h ip d b d esting object for the living dlesticks. A simple Height, 14%”. Width, Oaes di It W room. It will add a mellow colonial creation. would make an appropriate wedding roug t f rom sou rass an copp er tone, where one is needed. They are 914” high gift, and is something attractively A wonderful value for very and more massive different from what the bride cus- Created by the little money. Specially than they look. tomarily receives. Specially priced, priced, each ......... $3.95 Price, the pair..$5.25 each ........ eerie -50  a reneccegrenpranereren ART COLONY INDUSTRIES pepesepasnpancnaneitte  Americas leading metal craftsmen, 34 Union Square, N.Y.City (In answering advertisements please mention Tue Survey. It helps us, it identifies you.)  111                  Some of the agreeable things people are saying —  “Altogether a remarkable issue, per- haps one of the finest numbers, not merely of Harpers, but of any magazine that has ever been published in the United States.”  TIME.  “The neu: Harpers has just come in. My sincere congratulations. The format is superb, and I like the new cover im- mensely.” .  H. L. MENCKEN.  “I am delighted with your new maga-  zine in every way.” T. W. LAMONT.  “T can’t resist making myself heard in the cheering gallery for the new Harpers. ...I don’t know when I have seen so much literature spread on a single table of contents.”  ANNE O’HARE McCORMICK.  “You are off to a fine start with the new Harpers. The September issue is about as good a magazine number as I ever saw. Congratulations!”  VILHJALMUR STEFANSSON.  “I read Dr. Fosdick’s article yester- day. It is as good as can be, and ex- tremely well written—as well written as it is logical—inseeing and far-seeing. Fle is a man of great ability and you - have done well to get him.’  SIR GILBERT PARKER.  “I want to send you my congratula- tions on the September number of Har- pers. It is a great magazine, and I should think that any author of creative literature would take especial pride in seeing his material in such good company and in such good form.”  FREDERIC MELCHER.  We are continuing our Special Anniver- sary Offer to new subscribers. For only $3.00 you may have Harpers for one year (The usual price is $4.00). Simply sign this form ‘and we will bill you later. (Or, if you  prefer, enclose your check.) Mail -it-'toy, Harper & Bros., 49 E. 33rd St.. New York.  Pa aR aR Lae aR a AR ARRON IRC INU aU a  Pea ae aR RU aN EN  y  Phenomenal  Success  of the New  HARPERS MAGAZINE     The indispensable periodical for alert minded and sophisticated America 5  HE new Harpers Magazine has achieved an unqualified suc- cess. The September issue—its first appearance—was com- pletely sold out in ten days. And from all over the country enthusiastic and wholly spontaneous letters continue to pour in, in gratifying appreciation of the vigorous, youthful, provocative per- sonality of the new magazine. Moreover, the editorial forecast promises for the coming months. even richer material. ‘The best in fiction from America and England ; fearless and vivid discussions of significant phases of American life;  important points of view from and about Europe; brilliant criticism  and absorbing problems involving the human equation. —  In short, the new Harpers will triumphantly fulfill the promise of its first numbers just as it has already established itself as the periodical most sympathetic to the modern educated mind—a periodi- cal of rapid wit, clarity of judgment, independence of thought, and high literary flavor.  In the November Number  LAW MAKING AND LAW ENFORECENT, by Arthur T. Hadley ° I BELIEVE IN MAN, by Dr. Harry E. Fosdick THE HOME-TOWN MIND, by Duncan Aikman THUNDER ON THE LEFT, Part III, by Christopher Morley WHERE THE NEXT EUROPEAN WAR WILL START, by Frederick Palmer THESE AMERICAN WOMEN, by Rebecca West THE FETISH OF THE JOB, Anonymous AMERICA’S POLITICAL DECLINE, by Frank R. Kent THE PROMISED LAND, by Hendrik Willem Van Loon THE WAYS OF THE WEEVIL, by Henshaw Ward THE ONCE OPEN ROAD, by Charles Merz STORIES; . by Walter De La Mare, Roy Dickinson, Philip Curtiss  ‘Harpers  PPP PPPOE POPP RPP PPP PG  Add coos coceeeeesseesseeeeteeenee MAGAZINE BRR TINS ne SORE REE:  (In answering advertisements please mention THE Survey. It helps us, it identifies you.)  112    First—Last—Always  MEN have been known to go for months without shelter, for weeks without food and for days without water, but no one can live for more than a few minutes without air.  Breathing is the first necessity of life—yet few of us know how to breathe to develop our bodies and to improve our health. If we could be always in fresh air taking plenty of exercise, our usual undirected, instinctive breathing would naturally develop to give us better health. Nature would take care of us. But the condi- tions in which we live, the stress of present-day life, cause us to accumulate an excess of poisonous waste products in our bodies. To help dispose of these we should go beyond instinctive breathing and at frequent times during the day mentally direct the breaths we take.  Count Your Breaths—  How many breaths a minute do you take? Stop now with your watch in hand and for 60 seconds count them. Fifteen to twenty short, top-of-your-lungs breaths? You are not breathing deeply. Occasionally you should take six or eight long, leisurely breaths a minute—so deep that the dia- phragm is expanded and the ribs are barreled out. Several timesa day stop what you are doing, stand straight with head up, shoulders back and breathe—al- ways through the nose, of course.  Try it this way—inhale, one, two,  )     Baby’s first cry! However it may sound to grand- mother’s ears, it is music to the baby’s mother. Under the spell of her eager imagination that thin  little cry is a call for her. But what he really is cry-  ing for is air. Inthe Land of Unborn Babies he had no need to use his lungs. But here, in the great wide world, his first need is air and through every moment of his life he will demand air.  three, four; hold, five; exhale, six, seven, eight, nine; relax, ten. This will give you six breaths a minute—dquiet, unhurried breath- ing. After a time your uncon- scious breathing may become deeper and you will begin to feel a new and delightful sense of buoyant power.  Good Posture First—  When you stand or sit with shoulders rounded and chest con- tracted you squeeze your lungs and make deep breathing impossi- ble. Lift your head, raise your chest, straighten your spine, ele- vate your ribs and you cannot help “breathing for health”’.  Deep breathing exercises should be taken night and morning. Empty the lungs as fully as pos- sible with each breath. This is  important. because fresh air re- moves harmful waste matter in  the blood.  That ‘‘Stitch in the side’ —  Have you ever felt a stitch in the side when running? This is a warning—not always that your heart is weak, or that you have indigestion, as many persons sup- pose, but sometimes that your lungs are unaccustomed to being filled to their full capacity. Most of us rarely breathe to the bottom of our lungs. One-third of the lung cells of the average person is unused. These cells tend to col- lapse and stick together. When the air is forced into them, it sometimes causes pain.  Your health demands that you should breathe properly. The blood circulates all through the body distributing material to build and repair the tissues, picking up waste products and fighting disease | germs. The turning-point of its journey is in the lungs where it deposits the waste and takesa fresh  supply of oxygen from the air.  Without deep breath- te ing of fresh air there ‘SS  cannot beamplesupply of oxygen. Without sufficient oxygen there cannot be adequate growth or repair of any part of the body, nor vigorous warfare against disease. Begin today to breathe deeply —hbreathe for health.                      1  “CC oo ng ya? 4  Bierce     About one out of six of the total number of deaths in the United States each year is caused by diseases which affect the lungs. Pulmonary tuberculosis and pneumonia claim more than 210,000 victims annually. Ten years ago the: death-rate from tu- berculosis was sixty per cent higher than itis today. Only a short time ago it was thought that fresh air must be kept away from patients suffering from lung troubles. Today it is known that fresh air is one of the main aids in getting well—and . this knowledge has helped to produce the     6 marked decrease in tuberculosisdeath-rate.  Deep breathing must be studied. There is more to it than the taking of a full breath. The diaphragm and abdominal muscles must be strengthened by exercise and the .  posture.  Defects in the air passages should be cor- rected if one is to breathe most effectively. Wise parents should keep careful watch over their children’s noses and throats to see that they are not afflicted with adenoids or diseased tonsils.  body must be trained to maintain correct  The Metropolitan Life Insurance Company “has prepared a booklet giving simple and interesting health rules, including scientific advice about fresh air and proper breath- ing. These rules, withthe simple breathing exercise given above, can be followed by anybody who wishes better health. Send for a copy of “How’'to Live Long”. It will  be mailed free. HALEY FISKE, President.           Published by  , et €     METROPOLITAN LIFE INSURANCE COMPANY~NEW YO  Biggest in the World, More Assets, More Policyholders, More Insurance in force, More new Insurance each year (In answering advertisements please mention Tur Survey. It helps us, it identifies you.)  113                      THREE INCOMPARABLE CRUISES WITH CONGENIAL VOYAGERS  Around the World Cruise  A World trip of 30,000 miles on the magnificent S. S. Laconia, visiting a score of Far East countries, each like a different world, is a supreme experience and becomes travel glorified. !  The Clark management (Mr. and Mrs. Clark accompany the Cruise) makes this an ideal opportunity for world travel. | Cruise sails Jan. 20, 1926. $1250 to $3000. With private bath $4000.  Another Mediterranean- Norway Cruise  The Mediterranean-Norway Cruise of 1925 has proved such a success that we are already announcing a similar trip sailing June 30, 1926—same ship, the new S. S. Lancastria, same itinerary, same rates. The prices, $550 to $1300 ($1700 with bath) are phenomenally low and we will have, as this past year, an immediate rush of ap- plications, especially at the lower rates.  It only costs $10 to make a reservation. © It is certainly the banner Summer Cruise.     The Mediterranean Winter Cruise  Twenty-one successful Mediterranean Cruises give an assured guarantee that the 22nd on the beautiful new S. S. Transyl- vania will be just as conspicuous a success. This Cruise of 62 days of princely travel furnishes the acme of enjoyment and in- tellectual inspiration.  This Cruise includes a superb ship, the famous Cunard service and cuisine, con- genial fellow-passengers, inspiring lectures and services, elaborate shore sight-seeing. This cruise represents the climax of travel, Sailing date, Jan. 30, 1926. $600 to $1700. ‘With private bath $2000.  \\\\  Illustrated book and ship diagram sent free on request. Address:  CLARK’S CRUISES, 112 East 19 Street, New York City        (In answering advertisements please mention Tur SuRVEY. It helps us, it identifies you.)  114                      ADVENTURES on the BORDERLAND     Richard C. Cabot  ARMERS who have burned old zigzag Be fences have discovered their most  productive land in the strips which have lain fallow between fields.  So with the professions. Adventures aplenty and abundant human harvest may be reaped on their frontiers.  There is needed only the adventuring mind and the seeing eye to discover what those harvests may be.  Dr. Cabot writes a series of exploring arti- cles. As a physician, he is an authority on the human heart; as a social worker, he pioneered hospital social service; as a teacher, he holds the chair of social ethics at Harvard.  In the first article, which he calls ‘“‘A Plea for a ‘Clinical Year’ in the Course of Theo- logical Study,” he proposes a definite plan for training theological students “‘in the difficult wrestle of personal relations.” He believes that both teachers and pupils would find re- freshment and invigoration if, in addition to their regular theological studies they had been ‘on the firing line together” in the face of ‘‘the hopeless discouragement written on the face of a chronic sufferer or the profusion of sophis- tries fired at one by the hard-pressed sinner.” The minister, like the doctor, must “look after the minds, the emotions, the wills, the souls”  Medicine The Min istry  of ETHICS  By RICHARD C. CABOT, M.D.  A series ot articles in Survey Graphic in which the author of “What Men Live By’ discusses the Penetration of Ethics into the Professions of  Business  Social Work Education  of humankind. Ministers trained for such ser- vice would, he believes, “become far better preachers and for this reason, as well as for their greater experience and helpfulness in the great common problems of human personality and human association, they would be more prized, sought after and rewarded by their congregations and by the public at large.”  Equally vigorous and exploratory articles, freely illustrated from Dr. Cabot’s experience,  will traverse the fence-lines of the other pro- fessions.           Here is a series to send to your thinking friends, particularly those among them who are Doctors, Ministers, Teachers, Business Men, Social Workers. p—---------- CUT OFF HERE——-——~—__.______]| i SURVEY GRAPHIC, if ew] 112 East 19 Street, New York. | I enclose $........ for which please enter 6 months’  trial subscriptions at following new subscribers: Name ..  Address Name Address  Name  Te ew ee eC we ne We Se 8 8 9 00 0 0 8 0 we ce ee 6 oa cs ne crriCA NCCI CCI MON Mo Deo cor olerie legit eel sl ale eae are SO ee ee wee we oe oe woe eee te Poof? 22°999090000000000000000000K  CIPOO9T\"9ZF992999 900000000000 0000  ee     Address     115  $1 each (yearly $3) for the  cc cee ww ww ee ee mere rrr err reer eccece  TT ee em meme mee eee ese er ccc cove cee          THE SURVEY’S DIRECTORY OF SOCIAL AGENCIES     AMERICAN BIRTH CONTROL LEAGUE—President, Margaret Sanger, 104 Fifth Avenue, New York City. Objects: To educate American people in the various aspects of the dangers of un- controlled procreation; to establish centers where married persons may receive contraceptive advice from duly licensed physicians. Life membership $1.00; Birth Control Review (monthly magazine) $2.00 per year.  AMERICAN CHILD HEALTH ASSOCIATION—3270 Seventh Ave., New York. Herbert Hoover, President; Philip Van Ingen, M.D., Secretary; S. J. Crumbine, M.D., General Executive. Objects: Sound promotion of child health, especially in cooperation with the official health and education agencies.  AMERICAN COUNTRY LIFE ASSOCIATION—K. L. Butterfield, president; Henry Israel, executive secretary. Room 1849, Grand Central Terminal Bldg., New York City. Emphasizes the human aspect of country life. Annual membership $5.00 includes ‘‘Rural America’ (monthly bulletin) and Annual Conference Proceedings.  AMERICAN FEDERATION OF ORGANIZATIONS FOR THE HARD OF HEARING—Promotes the cause of the hard of hearing; assists in forming organizations. Pres., Dr. Gordon Berry; Field Secretary, Miss Betty Wright, 1601 85th St. N.W., Washington,  AMERICAN HOME .ECONOMICS ASSOCIATION—Lita Bane, executive secretary, 617 Mills Bldg., Washington, D. C. Organ- ized for betterment of conditions in home, school, institution and community. Publishes monthly Journal of Home Economics: office of editor, 617 Mills Bldg., Washington, D. C.; of business man- ager, 1211 Cathedral St., Baltimore, Md.  AMERICAN PEACE SOCIETY—Founded 1828, labors for an inter- national peace of justice. Its official organ is the Advocate of Peace, $2.00 a year. Arthur Deerin Call, secretary and editor, 612-614 Colorado Building, Washington, D. C.  AMERICAN SOCIETY FOR THE CONTROL OF CANCER—Dr. George A. Soper, managing director; 370 Seventh Ave., New York. To disseminate knowledge concerning symptoms, diagnosis, treat- ment and prevention. Publication free on request. Annual mem- bership dues, $5.00.  AMERICAN SOCIAL HYGIENE ASSOCIATION—3870 Seventh Ave., “New York. To promote a better understanding of the social ?thygiene movement; to advance sound sex education; to combat }prostitution and sex delinquency; to aid public authorities in the «campaign against the venereal diseases; to advise in organization wf state and local social-hygiene programs. Annual membership dues $2.00 including monthly journal.  CHILD WELFARE COMMITTEE OF AMERICA, Inc.—730 Fifth Avenue, New York. Telephone: Circle 9623. To secure Mothers Allowance laws in states now having no such provisions; to pro- mote desirable amendments to existing Mothers Allowance laws, to harmonize them with the maximum necessary protection of - @ependent children; to secure proper laws affecting adoption, boarding out and placing out of dependent children; ‘so far as possible to secure home life for normal children in preference to sending them to institutions; to aid in the enforcement of Mothers Pension and kindred laws. States Council of Child Welfare Com- mittee of America comprises representatives of practically every state. Committee publishes digest of laws and educational material on Mothers Pension and kindred topics. Invites requests from responsible public and private organizations for aid in Mothers Pension problems. Sophie Irene Loeb, President; Governor Alfred €. Smith, Honorary Chairman; Marg", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ump9.us4a-9huh", "00000000-0000-0000-4EEC-2E7D18FEE5B6", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Remarks Before the Committee on Public Health of the Massachusetts State Legislature in Consideration of an Amendment to the Vital Statistics Law", "9918573882206676X130", null, "1914", "05 March 1914", null, "Testimonies", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "5", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "a | — 3 bi enc ota % : * : : : ac Q\\\\ Sein Cae . | 4 < i: ON A \\\\  oe  oss Remarks before the Committee oh Public Health of the Massachusetts  State Legislature in consideration of an Amendment to the Vital oe Statistics Law. Held at the State House, Thursday morning,  - | March 5th, 1914,  = x by ; Louis I. Dublin, Statistician, Metropolitan Life Insurance Company, New York, NY.  gi re yy &é       : Gentlemen: I will limit myself te one point; em which is in keeping with my own experience in managing a Large statistical office in New York. I desire to snehanine the great practical utility of the proposed Amendment. At the present time the vital statistics reports issued by the Secretary of State's Office are available only a year after the elo se of the ealontiex year which they deseribe. I am afraid that these reports are more ornamental than useful. I take it that the greatest value of statistics lies in their _ practical application to affairs. You want to make your statistics work. To be most useful, the figures should describe current conditions and should be in the hands of those who are doing the practical health work of the State. (The statistics should be one of the machines at the disposal of your Health Board with which they may do their work. This is to my mind the real reason why you should make the change — that is comtemplated by the Amondzent and place a qualified registrar of vital statistics in charge.  You spend a great deal of maney supporting a Board of Health. This Board is engaged in controlling the infectious diseases, especially tuberculosis, typhoid fever, scarlet fever and other diseases dangerous te the public. health. Naturally the Board wants to tout the efficiency of its work. | They desire to know whether the diseases which I have mentioned are increasing in prevalenee or decreasing. At the present time such information as comes to the Board of Health comes either voluntarily from the cities and towns, or the informa-  tion covers only a portion of a whole population. It is only  mas          Yabulatione ef U. & Bureau of the Census and Office of the Secretary | of the Coomonmvealth of Massachusettes for the year 1 9 1 0.         ; 4 { a  : typhoid Fever 421. 124 413, 42-2  Medalen | 902 U6 240 Tek | ~ Seariet Fever 272 800 884 16 Whooping Cough 286 8.5 | 183 5.4 - “Diphtheria @ croup 705 208 679 2002  pS daha SS a  Tuberculosis (all forme) 6,504 16207 6,086 179.9 Cancer (all forms) 3,159 9304 3,028 90.0     TOTAL (ALL CAUSES): $4, 302 1,605.8 $4, 407 1,616.3 through the courtesy of the Secretary of State's Office that  they can learn what conditions prevail threughout the Commonwealth.  I believe that that is the wrong way to do it. It is unecenom feal and ineffictent to do it in thet way. What they need is a statement of the deaths when they occur in order that the Board, which is responsible for the health werk of the state, may know what is trenepiring. Nothing discourages a Health Board so much as to get information toc late. Thet is exactly what is happening. My thought then, is that your vitel statistics should be tied up to the Department that ean best use them <= the Department of Health. I believe that it is the only Department of the Conmon- wealth that has a right to collect then,  One more word. It is extremely important that vital statistics be accurate. Inaccurate statistics are worse than no stativtios at all. I hava very carefully examined the re- porte that are now issued by the Secretary of the Commonwealth and I am impressed with the fact that they show defects which arise from their separation fron the Department of Health. I am distrib- uting among you a sheet showing a number of discrepancies in your reports as compared with the Gensus Bureau tabulations for Massa- chusetts for the year 1910. You will see that the figures for Ty- phoid Fever agree pretty closely. On the other hand, the Census Bureau records 152 more deaths from Measles than appear in the Cormmenwealth re- port. In the same way, the figures for Whooping Cough are very dif- ferent in the two reports. Your rate for all forme of Tuberculosis is seventeen per hundred thousand higher in the Cenmonwealth report than it appeare to be according te the Bureau of the Census. These discrepancies reflect on the care with which your figures are pre- pared. IZ am inclined to believe that the death certificates, which  sits, ‘ fiamantins 3 Sm cS a ee Pe Nothing denot  give the information in the first instance, are not as carefully questioned by the Secretary of State's Office as they are by the Bureau of the Census. This explains the differences in the rates which appear in the two reports. I am certain that your reports will be much more in agree- ment with those prepared by the experts of the Census Bureau when you have appointed a competent registrar of vital stetistics, as this Amendment provided.  Under the new regiméd, when physicians make  out birth and death certificates they will realize that  _.- there is a responsible person in charge at the State House and  a their reports will be much more complete and satisfactory.  gut       lizes a system of vital statistics so much as  oe “i by  “ the knéwledge by physicians that there is no person, “ technically qualified, in charge of the machinery. The re-  ports then détericrate and you get the form of a statistical  repert and lese the substance.", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-i82s.tff2~rhey", "00000000-0000-0000-949E-2437C5BBDF9A", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Lecture Notes for a Course on Vital Statistics", "9918573882206676X131", null, "1919", "1919", "Notes for a new course in Vital Statistics taught by Louis Dublin at the Yale University Medical School", "Lecture notes", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "23", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "LECTURE I~ TO THK =NTINE CLASS - FRBRUARY 15, 1919 aT 3:00 P, Mm, 7 S100 P.M,  Introduction te a Course in Vital Statistics  Seientific pursuits are a form of sonduct, Certain conduct re- actions grow out of fundamental impulses and instinets of our human nature. In aidition to the impulses, the instinete, of solf-preservation and for the repro« duetion of our kind, there is the impulse, or instinct of self-improvement and advancement, One phase of this latter inetinet is the impulse of curiosity ,« curiosity as te: as 1. ‘hy things happen  2. How things happen, and hew they can be made to hapren diffor-  ently  3, How can the impulses of self-presorvation, repreduction and  improvement be adjusted to the immediate circum stances of our life  4, How the circumetances and institutions of life can be ordered  so a9 to harmonize with the limitations of our human nature  5. How ean the foregoing onds be achieved with the minimum of  effort, along lines of least resistance Our very survival depends upon the outcome of these curiosities,  The _ee we inquire about with the least success in our daily life are, perhaps, the various aspects of life,+ the funetioning ef living creatures, Our ordinary,undisciplined conception of the phenomenon of living substance is not unlike the visionary, Syston ef life and ite CuVA FORME, given  te ue in the first chapter of the book of the prophet EKaekial: we see \"the wheel in the midd Le of a wheel, we heer the neisee of wings, of great waters, and of hosts\"; we see, too, \"the bow that ie in the cloud in the day of rain.” The facts of life may be studied in twe reneral waye- with the bisloriet and the physicisn we may minutely examine the etructure and functions of individual organ- isms, their wenener under certain conditions of health and disease, Thies plan of satisfying ie various ‘orm of eur curiosity we may term the Anteneive method of research, Or, second, we may desire to gurvey a passing throng of single  events whose individual charactere we know in general, ‘This latter, or extensive       ees Se  method of deriving knowledge from inquiry inte the facts for groups of persons, thinre or events is, in eeasence, statistics, “hen our curiosity is confined to living creatures, the extensive methed of research may be termed vital statistics, | A further point: the intensive er single case method of gathering knowledge through observation and reflection makes it diffieult to establish } definite principles and policies witth respect to cause and effect of health and disease, For instance, suppose we wish to determine whether the administration eof five grains of phenacetin te a patient suffering with lebar pneumonia, on the ninth day of the illness, is sound therapeutically or not. Our everyday experience tells us that we cannot draw general conclusions with safety from specific ine stances, and so, whether we are conscious of it or at, = withhold judgemont until we have “accumlated experience” with phenacetin in the ninth day of lebar | pneumenia, In other words, instinctively we statiatify; vital statistics is, a therefor, one of our ways of leoking at life, an observational adjunct to the — | mind of man, In consequonce of our human attribute of curiosity, and when that curiosity concerns itself with groupe of persons, things and events affecting life, wo are all, more er less, vital saattarigtane. It becomes our duty in 3 oa thie course te learn how to become fairly preficient vital atatieticians, Let  us see what are the sorts or kinds of knewledge of the facts of life the aspiring  vital statistician aime to develop. aden  THE FOUR SORTS OR KINDS OF STATISTICAL KNOWLSDGE     As a result of our compliance with the reasonable requirements of atatistical science in general, we way expect our researches te produce one or several of these classes or categories of knowledge: I, Smpirical knovledge, i, e., the facts of direct observation of groupe ef persons, thinge and events. Such statements of facts are of necessity crude, however well we may observe the technical requirements for gathering such  facts. Thie empirical knowledge we may term ¢:        i. @., the conclusions we arrive st, after following the precese of reasening which is known ac industion, from the crude data collected in category 7, | It i¢ understood, also, that before we preceed with our presenss ef reasoning upon our crude data, we test, craduate ond in ether ways refine the — observations. This, and the sueseeding elasses of knowledge in our esience, may he described as gnslytis gtatisties, Much of the statistical knowledge of a higher sert which we have available coneicte of the eecond category of facts. 111, Conclusions derived by the process of reasoning known as de- Sustion from. s .  te reneralized knewledge developed from other eourees ae a check or sontrol,  Theee sonclueione are referable     TV. Conclusions derived by deduction from assumptions or hypotheses, but not at present referable te observed facts, because of the feeble state of  our facilities fer cbserving living things. nhe  PRACTICAL EXAMPLES OF THE KINDS 98     - an Oxemple of the sort or kind of knowledge in category I, ¥e may mention the data published in myriads ef reperts, bluebooks, annuals and ; digests. The gethoring, tabulation and publication of such material has a methodology of ite ow, a set of criteria ae te what econetitutes the geod and the bad in ‘eseriptive, crude stalisties; in fact, seme statisticians of @xperience, but of narrow Gutlook end with little sympathy fer the aims and ideonls of the profession of statistics, conceive that thie is virtually all — there ig in statistics,  The data in bluebeoks, reports and annuals,- data such as the health offisee and registration bureaus of our onl compile, sre but the bricks- erude building material~ with which the trained statistician ef fertile mind erecte the edifice of truth, A mere pile of bricks and a yard filled with lumber de not constitute a monument of architecture. ‘The discerning, analytical statieticion bears the samo relation te the mere compiler of statistics, that the architect and artist bears to the artisane who make bricks and who cut lumber, And 90, our crude facts in category I are but the materiale for the statistical logician. Statistical analysis, the deriving ef generalized knowledge, the discovery of abiding truth, is really applied Logis; it applies the process of reasoning to the observed facts, and endeavors to. explain why things happen, how they hap pen and hew events may be made to happen or not to happen.  QOIC IN BTATISTICE       A large element in the professional equipment of the vital dtinttntSitie is the eapeekty to reason, to avoid fallacies. A study of applied legie is earnestly te bé recomended te aspiranta for competency in vital etatisties, Along with applied logic ae erdinarily understood, one should study the logis  of chance and of error, a careful weighing of probabilities and a liberal con«          struction upon sources of error in arriving at statistical conclusions, ‘The etatistician should have a wholesome aul for the haserds of thought upon phenomena subject to the laws of chance. Categories III and IV of the sorts or kinds of statistical knowledge useful in understanding the life of man, are almost exclusively disciplines im deductive reasoning. Those of ue who have opportunity to enter these immer vaulte for the treasures of thought should realize the service wa may render mankind by vitealising vital statietics, by making us view certain phenomena in a new light, by widening the realm of science and Lifting human thought sbove the mere reutine of statiatical book koeping and computing. The function of statistics is te otudy the status and destiny of man, net only to delve inte tomes and tomes of volumes, sheete and other impedimenta,  PRACTICAL OUTLINES OF VITAL STATISTICS  while, from the viewpoint of the biolerist, vital statistics compre- hende the extensive study of living creatures, it would be well for us, practical-— ly, to pidttviieriae and limit our inquiries te the life processes of mankind, more especially those processes which engage the time and attention of the physi- eiam and public health worker,  The vital statistics of the health worker are the facta of:  a. The structure, or composition, of pepulations vb. The functioning, or movement, of populations  The word “pepulation” carries with the sencept of “group” or “mass*, and the study of populations, or vital statistics, stands contrasted with the usual run of studies in medicine, which deal with the intensive observation of individuals and parts of individuale, Vital statistics is sometimes known ae demology or demography. :  ‘ DISTINCTION BETWEEN SOCIAL AMD VITAL STATISTICS | Ye may pause at this point to make clear a number of distinctions,  Some groupe ef studente are interested in the reactions of one mind upon  sare is Pe mea  another, in human associations woieck exist for purposes of mutual advancemont, defense, social ferce amenz wen, They are students of the relations ef man te man. Facts showing populations in their associations, as im families er other social units, are called social statietics, because those data form tho basis of an inductive sociology. The grouping of individuals, and the movements or functioning within these groupe, with respect to matters of health, disease, vital functioning, is in the province of medicine, amd strictly spoaking, alone constitutes vital statistics. By thus limiting ourselves, we make it possible te engage our time only with problems concerning the health worker.  a, Statistics of  The modern census ie practically our only imetrument for determining     the etructure or composition of populatione, how they are composed with respect to race or color, sex, age, sccupetions, ete. (Then give resume of Knibbes’ historical treatment of censuses herewith) noted students to “American Consus Taking”, “The Story of the Censue” and to Bright ad Hunt*s “History of the United States Census, 1790-1999,\"     After giving thie much, preceeded to indicate the importance of the figure for the total of population, its pies in computing death rates, etc., some sources of error in census enumeration technique, the arithmetic increment methed of estimating intercensal population.  Next pointed out the importance of color or race, nativity and country of birth etatisties in public health work, | Lectuse IL « te the graduate students ~ Feb. 15, 1919 © 6630 © 6.30 F. Me  Introduction to @ Course in Vital Statistica     Statistical Laboratory Practies     I « The atatiotical worker personally Quaid fications for statistical wowk ‘1. Undorstanding of tho aims of statistics Se Sense of proportion, normal reactions to life 3. Montel qualitios .  nooutmation » by discipline and practice. gnore alight sources of disturbance. = Steady supply of work. Overly arrangonent. Hygiene of vorke  | Loatedpinttsh « Speed and cloarness in acquiring Que Handwriting and sheot technique. “Hye mindedness,” “earmindednesae” Accuracy — 4n conputation « “addition mindednoss,* uitiplication mindedness.\"  waickonse « Working hard for short periods of  %ino, verous, working at Lower prossure for longer poriods of time. Effective dispo she tion of time and energy. “Stroes workers,” “duration workers\" in statisticse                    4. Physique Right habite of work, thought and life S$. Capacity to visualise  To abstract - to grasp all the issues in a given situatione       o2e  /  II = Working matorials Paper e tools « standard Lottoring - filing and ene     “gable anatony The nuaber by which a table is ontered is called the orauason The tabular resulte found are called the pagpongonta. ‘here yeqponhae & are  converted inte argmients, table io onlled \"ve roraod fora.\"     A double entry table is one with two argunonte; triple ontry, one with threo, ofte ae  Puldngs of tables; tetale, underscoring, captions, gharacterietics |  of geod heading « uses of black, red, grecn and purplo ink.  Short “text™ tabica, vorcus, long \"roference\" tabloss     Peta xka  ef — stotistichane  | oe Table numbering « Roman ond araubidee  Gave practical oxanpicd of aloves Students uade copious notede    YALE SCHOOL OF MEDICINE COURSE IN VITAL STATISTICS  A Lecture on Varioue Statistical Derivatives of Public Health Data  In Lecture IV we distinguished between two principal sorts of statis- tical processes: A, crude, descriptive statistics, i. e., of the collection, editing and tabulation of the crude, sales facts for groups of persons, things and events, and B. analytic statistics, or the graduation, testing and development of crude data, and the establishment of the various sorts or kinds of knowledge therefrom, It will be recalled that in Lecture I,we held that statistical processes leading to an extensive view of Nature produced, in general,  four sorts of knowledge:  ‘I. Empirical knowledge: or the statement of the facts of direct ob-  servation just as we find them  II, Generalized knowledge: or, conclusions reached by induction from the observed facts, properly graduated, tested and otherwise analytically treated by the processes of mathematics and logic  III. Generalized knowledge, by deduction from hypotheses, end referable as to validity to the facts of categories I and II IV. Generalized eviadin by deduction from hypotheses, but not at  _ present referable to empirical or generalized knowledge     Further observations on the sorts of stetistical data known as I. Empirical knowledge We are now at that point in our study of statistics, where we may  recognize the sub-classes composing the body of empirical or observational knowledge of groups of persons, things and events. Statement of these sub-classeg ie necessary in order to avoid dangeroue crossing of the currents of thought, te give us a safe base from which to explore the field of vital statistics and in which we may seek refuge when befuddlement overtakes us, as it will from time to time. The difficulty in the statistical thinking (%) of so many of us, is not that  we become confused end speak in strange and divers tongues, but that we have often  no means of knowing when we utter mere verbalisms, false conclusions, confounded  issues, etc, The following simple classification of what we may develop under  category I of our classes of knowledge will be found to be of value:  I. Empirical statistical knowledge: the facts of direct observation  of groups of persons, things and events  Sub-claes a:  Precise, concrete description of a group or of groups, i.¢., the number  of persons, things or events in kind or sort classes (things sorted by quelitative  ‘characters: number of deaths from cancer, the number of cases of measles, the  number of beech=leaves, etc.); and, for each kind or sort class the grouping of  cases by size or other numerical characteristics, if such grouping is possible  (deaths from cancer by age, beech-leaves by lengths, etc.). Under this head we  place most of the laboratory operations of statistics-~ schedulizing, tabulating,  mechanical devices of many varieties, systems of nomenclature and Classification,  ete, Sub-class b: Knowledge of abstract qualities of groups.  It is evident that the elementary concrete knowledge of how many units  of certein sorts or kinds, and perhaps of certain sizes, there are in a given  thease meets only a limited demand, We find it inconvenient, and nearly always  impoesible, to compare offhand 2; 00 or more long and intricate tables. Something  must be done conveniently to express in concentrated form an idea back of a table    for a given phenomenon. Take, for instance, a table showing the number of deaths from lobar pneumonia at the various ages in life. If we plot the deaths on a grephic chart, we may get a feeble idea that there are few deathe, relatively, from this disease in the early and very late ages in life, and that there is a tendency for such deaths to concentrate between 40 and 60 years. Such a concep- tion is, however, too loose for the practical discussion of the age aspects of pneumonia,  We need means of discovering the abstract type characteristics of verious\"kind\" groups, after we have classified their members into size or other numericel classes. Now, whet type characteristics of such groups are there?  A list follows:  1. Type or central condition of a kind or sort group classified in size or numerical characteristics, i,e., cancer deaths  grouped by age  a, Arithmetic mean, or, arithmetic average  b. Median  c. Mode  d, Geometric means; harmenic means (rarely, if ever, used)  The type, or central, characteristic is that abetract numerical quality about which the individuals or members composing a group tend to cluster.  é, The degree of individual diversity or dispersion of the individuals or members of the group with respect to the typical condition of the croup  a. Standard deviation  b. Mean deviation  c. Quartile deviation  d, Semi-quartile range  €. Coefficient of variation  3. The degree of symmetry of the distribution of individuals comprising a group  @, Skewness b. “Normality” of symmetrical and asymmetrical \"a@istributions\"    The foregoing statement outlinee the so-called \"shorthand\" of statistics. These type characteristics are abbreviations which help us to form ideae. Without them statistics would be only a dreary waste of tebles, a useless display of descriptive technique.  Sub=class c: We may develop another series of abstract ideas with respect to these groups of persons, things and events for which we have just pro= vided various “shorthand” type characters. We may know numerically the extent of association or contingency between different events of like or unlike sort-or-kind character (association of attributes, coefficients of correlation of variables).  z Sub-class d: The reliability, validity or probability of conclusions on empirics] observations.  a. The theory of probsbilities  b, The method of least squares, the theory of errors and ob- servations     The lectures given se far in this course have dealt almost entirely with Subeclass a of empirical knowledge in vital statistics. Let us now die- cuss sub-class b.  The Abstract Qualities of Groups  In the first place, there is more or less of a nomenclature of groups end classes to be understood before we come to the abstract qualities of such na. When the objects and individuals of a given kind-or-sort category are measured, and then sorted according te size classes, this display is called a “frequency distribution,” The size=classes should be uniform, ‘The distance on the graphic scale to denote the limits of a particular class is called the “class interval.\" Thus, deaths in the kindeor-sort category \"cancer\" when  grouped by five-year age-periods are said to form a \"frequency distribution”  with age-class intervals of five years. The number of deaths in each five       yeer age class is termed the \"class-frequency,\"™  It is nearly always best to illustrate graphically the frequency dis- tribution you happen to be dealing with. This may be done as follows:  Lay off vertical and horizontal scales on the graph paper. In ordinary practice choose the horizontal base for the class-intervals and the vertical scele for the class-frequencies (or number in a given class),  Point off on the chart the several values in your frequency series or distribution. Connect the pointe with a continuous line. This picture of your series is called a \"frequency polygon.” If however, you draw through each point @ line parallel to the base, and as wide as the class interval, and connect the ende ef these lines with the base by perpendiculars, thus forming a progression eof rectangles, you will have drawn a \"frequency histegram.\" The outline of your \"histogram\" has the appearance of a rising and falling succession of steps.  Whether you draw a “frequency polygon\" or a “frequency histogram,” you will have only e crude and approximete picture of the variation of, sey, canter deaths by age. If you had the following “ideal” conditions present:  a. Seme number of lives exposed to risk in each age class  b. Perfect registration, certification end classification of the data  Cc, An “optimum,” adequate, number of exposures st hand from which to draw valid conclusions  d, An infinitesimally small “class interval\" Such an \"ideal limit\" to your frequency polygon or histogram would be called a \"frequency curve,” Note this definition of a “frequency curve,\" The term is sof often wrongly used to @note \"Frequency polygon.” Only when we provide by means of the philosophy of the theory of probabilities and of errors end mean squeres, “ideal” requirement \"c,\" and by the integral and differential calculus requirement “d,\" can we really provide data suitable for graphing so-called  \"frequency cuves,” There is entirely too mich loose talk about frequency curves,  What we need in statistical analysis is a true appreciation of our deficiencies as philosophers snd mathematicians, and to know when to quit mouthing attractive phrases we understnad only imperfectly. The average \"statistician\" used to menufacturing tables, is too prone to handle the statistical dynamite of higher analysis, without knowing how really unfinished is much of the theory of frequency curves. Let us understand this pit»*fall a stick to simple frequency distributions and their pictures,=- polygons and histograms. The rule-of-thumb use of the Pearsonian, Charlier, Poisson, Laplace end Lexis formulse, for frequency curves by the average statistician of the descriptive,\"empirical\"” scheol is after the equivalent of the wielding of a sharp razor by an infant, In vital statistics especially one must have an appreciation for the limits upon the contents end tynodieitins of the average statistical cranium,  1. Typicel or central condition (size= characteristic)of a kind- or=sort catercory  A number expressing size-type shculd have the follewing characteristics: I. Rigidly defined, II, Based upon all observations made III. Readily understood, because possessing obvious and simple properties, IV, Calculated easily and rapidly, V, Not markedly affected by fluctuations of sampling,  VI, Shall lend itself readily to algebraic treatment. Yale School of Medicine  Course in Vital Statistics     Lecture III «= by Dr. L. I. Dublin «  during week beginning March 3, 1919 and handed to Supplementary Notes on Lecture IV (Mr. Kopf)  CC.  Exercises ~ To be prepared  Mr. Kopf, March 8, 1919. for methods of verifying addition,  iat aeahierataaimerteieentiit ee aida  Table 1  See  Number of the population in each subdivision of the United States, The  following  two errors in the \"po  the errors.  totaling, by  out nines,\"  Division and State  United States New England States Middle Atlantic States : E. North Central States  We North Central States  South Atlantic States EK. South Central States W. South Central States Mountain States  Pacific States  Increase in the population of the United States  Census year  vable contains one error in the total of population and puletion per square mile\" figures.  Locate  Verify addition by (a) reversing the direction of (b) summing pairs of columns and by (c) “casting  Population 1910  91,962,266  6,552, 627 19,315,892 18, 250, 621 11, 637,921 12,194,895 8,409,901 8,784,534 2,633,517 4,192, 304  Population of the  United States  Land Area in Square miles —  2,973,890 61,976 100, 000 245,564 510,804 269,071 179,509 429,746 859,125 318,095  Table 2  Population per square mile  1910  30.9 105.7 193,42  74.43 ©  2408 45.3 46,8 20.4  Sai 13,2  1890.  1900 Ze 40 alee 90.2 7548 154.5 127.1 65.2 54.9 2063 17.5 38,8 3209 42,0 35,8 15.2 11,0 1.9 1.4. 569  726 *  Increase over preveding census  Number  1910 91,972, 266 1900 75,994,575 1890 62,947, 714 1880 50,155,783 1870 38,558, 371 1860 31,443,321 1850 23,191,876  15,977, 691 13,046,861 12,751,931 11,597,412 7,111, 050 8,251,445 6,122,423  There are two. errors in the ™ ceding census\" in foregoing tables  Percent.  2140 2047 2000 30.1 2206 3546 3549  number\" figures for “increase over pre Locate by sub dt raction teste ~ 2m  j Table 3  =  Percentage of population of-the United States in urban and rural areas respectively, 1880 to 1890  Class _ 1910 1900 1890 1880 Total 100.0 100.0 10040 10040 Urban 4643 i ms. 2 aS  Rural 5307 : 5945 6349 70.5  How many errors in the “urban” percentages of the foregoing table can you find? What errors are there? Check by —e percentages.  Problem 4  Required: to estimate the total population of the United States,  having given (a) the area in square miles and (b) the number of persons per square miles  2,973,890 = Land area in square miles  = 30.9 = Population per square = 26765010 69216700  918932010 (actually enumerated, 91,972,266)  Check the ecouoeing multiplication in two ways (1) by the method of unitates and (2) by casting out nines.  ee ; - Problem 5 Color, or race, nativity eit parentage of the rea of the United States 1910 si Class of population Number Percent. Class of population Number Percent. Total : 91,972,266 100.0. Tote native 78,456,380 8543 : Total foreign § ‘£_13,515,886 14.7 White 81,731, 987 88.9 Total white , 81,731,957 88.9 Negro 9,827, 763 10.7 Native 68,386,412 74.4 Other colored 412,546 0.4 Native parentage 49,488,575 53.8 Indian 265, 683 0.3 Foreign parentage 12,916,311 14,0 Chinese 71,531 0.1 Mixed parentage 5,981,526 645 Japanese _ ae, \"187 Gel Foreign born 13,345,545 . 14,5 All other 3. 175 :  Verify the: figures for \"nuaber of the population\" by reference to Vol. I, Census of 1910. Verify the first two percentages in each percentage  eslum by actual division. Check your ee Es (3) wee method of unitates and by (2) the \"casting out nines\" methode    Yale Seheol of ledicine     Course in Vital Statistics     Lecture IV (2nd Lecture in Laboratory Technique)  a Review Lecture II briefly (lst lecture in Laboratory Technique). be Then take up for Lecture IV the following |  OFFICE OPERATIONS IN STATISTICAL wORK  1, Twe general divisions of practical laboratory work in statistics - (I. Methods of crude descriptive statistics and II Metheds, results and aims of analytic statistics). Crude deseriptive statis ics collects the data under category I of statistical knowledge, i. 6, the ungraduated, crude facts for groups of persons, things and events (see notes of Lecture I), yt. statistics aime to graduate (\"smooth\"), criticize and mathematically and logically analyze crude descriptive statistics, to produce eategery II of statistical knowledge. It aims also te develop data for the verification of conclusions derived under category III ef the forms of statistical knowledge,  and in the present state of our technical resources aims merely to suggest lines of investigation inte knowledge under eategory IV.                             Throughout the history of statistics, there has been more or less conflict between the “descriptive” and “analytic” schools of statistical method, The German School of Minster = Conring = Achcnwall originated the conception  that statistics consisted of an exhaustive colleetion of crude facts about the status of markind. Another “school” = the “analytic” group = Graunt, \"the School of Political Arithmetic,\" Derhanm and Silsamileh, held that the induction of principles from “representative” data (eften of small dimensions) and the deduce tion of principles from assumptions or hypotheses was allesufficient, The modern conception of statistics accords each of these \"Schools\" a place in the seientifie investigation of mass phenomena.     ae Planning of statistical inquiries; drafting ef schedules for collection of data; collecting the information by personal eanvass or by abstragtion from record and library or filed sources; eriticien ef schedules returned by investigators; editing and “coding\" of iteas on the schedule; writing of statistical cards, or punching of tabulating ecards, if schedules are not adapted directly for sorting and counting.  be Sorting of handwritten cards and of perforated cards; tabulating handwritten cards either by “tallying” or by counting; “tablesanatomy\"; tabulating perforated cards; arithmetic operations upen tabulation sheets; arithmetic =~ 2-«  operations to discover crudely the “type characteristics\" of the groups dise played upon tabulation sheeta; miscellaneous operations leading te analytie work in statisties; graphic illustrations of statistical data, crude or refined, Filing and finding data. Eleuentary library practice.  II. Analytic Statistics  here is no very sharp line of demarcation between crude descriptive statistics and analytic statistics. Some of the more elementary principles of statistical analytics are often incorporated inte erude descriptive work. Drawing distinctions between the two general groups of statistical laboratory operations as closely ae possible, however, we ‘aay out= line the following subjects under class IT,  ae Inspecting crude tabular results and deciding what metheds of “smoothing” or other adjustments of roughness in the material are necessary, and which methods apply particularly to the case in point. The proper discharge of this obligation assumes considerable practical experience with the particular gubdject matter being dealt with, and a fine discrimination in the cheice of graduating and critical “machinery,” ugually mathematical in character. When these graduations, adjustments and other critical tests have been applied toe the data, further analytic processes (also usually mathematical in character, sometimes exhausting the resources of the higher nathomaties), leading to interpretation of the results are necessary. Reasonable proficiency in the highor mathematics, retained by constant practice with this form of statistical analysis, is to be assumed. ‘the subjects to be cultivated are:  Differential and integral \"“Infinitesinal calculus,” by Irving Fisher calculus     Graduation, interpolation, sunma- “ortality tables,” by George Francis tion of series, application of Hardy theory to errors to statistical \"Institute of Actuaries Textbook,\" by data King, volume 2 \"Higher Algebra,” by Hall & Knight “Higher Algebra,\" by George Chrystal \"Method of Least Squares,\" by Maxwell Merriman “Theery of Zrrors and Methed of Least Squares,” by Walter Woolsey Johnson “ortality Laws and Statistics,\" by Robert Henderson — \"Theory of Observations,” by Thiele  Theory of probabilities \"Mathenatical Theory of Probabilities, \" ~ by Arne Fisher, volume 1 “Theory of Probabilities,” by George Streeter (Spectator Coe, Ne Yo Ce)  Inductive logic “Induetive Logic,” by John Grier Hibben “Principles of Science,\" by W Stanley  Jevons “Pr", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6ass_ew5u.e9kf", "00000000-0000-0000-9A28-DC945AA3EF40", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Notes for a talk on Nosology and Practical Office Cause-of-Death Classification Procedure", "9918573882206676X132", null, "1919", "12 April 1919", "Notes for a talk on disease classifications in vital statistics work, for the Yale University Medical School.", "Lecture notes", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "3", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Notes for talk to Yale Medical School ‘lass, April 12, 1919,     Ze ba Ont A. oe pee a ae Re mance . tie Qeke “a Bate Tee Crate eo Clee o- asec  1, Define nosology.  2. Explain difference between a nomenclature and a practical working list or “classification” of causes of death.  | 3. Explain necessity for a working list of causes of death for practical — office use, Explain why an alphabetical index will not suffice (erysipelas and St. Anthony's fire, e«e-e<c= Barlow's disease, Yerlnoff's disease and scurvy --=<= locomotor ataxia and tabes dorsalis), Read at this point paragraph marked a,  page 12, Manualg alse translation of paragraph marked a, page 3, French text.  4, The International List of Causes of Death (more especially the Census manual idapting it to American usage) is our working list in this country, It is a not a scientific nomenclature, Read here paragraph marked a, page 13, Manual. ed  a History of International List, Give synopsis of the marked paragraphs, page 7, Manual, Comment on growth in extent oye of its use and on the great importance of its adoption by  England and Tales, See marked paragraphs, sage 8, Manual,  p= Comment briefly on the history of neselegy as covered by  pages 13 to 17, Manual, Comment on adoption of the Inter-  national classification by the U. 5, Census Bureau and its use by state, municipal and corporation offices interested  i : in mortality statistics, S  b = Brief comment on the Census manual itself showing how it covers thousands of modes of statements of eauses of death which are not in the original French text (Bellevue, oe French and Census terme). 4 c « Why it is necessary to have beth a tabular list and en alpha- betical index, Tabular list is the real classification, | | The index is simply a guide to the use of the tabular list, os . Mere reference numbers are meaningless, Cive examples, ’ ge Refer to paragraphs marked b and c, page 12, Manual.  i  d « The International List is a list of causes of death arranged, for the most ourt, according to the goat of diseases rather than their pathology or etiology, Thus, it is, in the main, © i : 2. arranged on an anatomical basis. (Example ~ serebrel hemor- a T <= rhage: nervous not circulatory). Give exceptions te this j 7 eA (cancer, t.b., violent causes, otc.). Explain what Ys a? Bertillen claims to »e the advantage of the anatomical basis,  Criticise mildly. | | Ls  Explain that the list is divided, primarily, into two grand — divisions, First, disease; second, viclence. 11 of the 14 general classes are devoted distinctly to morbid conditions. Class 12 relates to old age. Class 13 relates to external < causes, (ecarbolie acid and wood alcohol). The final ~S elass, No. 14, is called Ill-tefined Diseases but under this — - heading are tabulated many deaths which are probably due to = vielent causes but which were not reported in such a way as —~— to be identified as deaths so chargeable,  @ 8  ea  NR teil a aes  S et ° cee eg 2 \\\\ Sere ae =e Se ai Se ee ee a s ~ Site iia oe.” ——_ = . Ge Sag ima ices wage Ot Sgt Se EE le gee *          ain  f - Explain what a general disease is and what are included in titles 1 to 59, Do the same for the Diseases of the Ner~= vous System and for the other classes, (Same for violence, suicide, homicide and accident), Explain some of the deficiencies of the International List and point to certain rather misleading title headings (cerebrai spinal meningitis, A.AeP., Titles 46, 50, 78, 103, 146, 148, etc.)  / Vt — g + Explain the difference between the e0tatie’ and abridged lists  pe oN and why it is necessary to have each, Explain also the = | elasticity of the International List, Give examples, The —_———___ Cancer monograph; pneumonia; Titles,173 and 175, /G co —y = FS we o,  5, Importance of statistics of deaths by cause and consequent importance of their scientific classification. op : a= This was recognized even before the coffection of mortality x statistics by Census enumerators was discarded, The Crone Ife directions to these men emphasized the fact that the state~- 2 ment of cause of death was the most important item on the for 2 This was a step in the right direction even though the hopelessness of securing accurate statements by means of enumerators is thoroughly recognized now and was quite generally conceded then,  b = We measure the comparative healthfulness of communities more by their death rate for some of the preventable diseases than by their gross or crude rate; hence, the importance of impressing on the medical fraternity the necessity of statin apy Ot hee each gase Le a fact ES Toe ie! c= aa of a societies which need accurate statistics of causes of death for the study of many problems, Interest of the sanitarian who uses statistics of the various prevent« able diseases in his work far more than he does the crude death rate, Legal importance of correct statements of cause of death, Life insurance, Pensions,     *, Practical office cause of death classification procedure,  eee        le Metter te be classified. The henlth officer or vital statistician 4 in classifying causes of death obtains his material for such classification usual» ly from certificates of death. These statements are made by physicians, usuelly; also by coroners who, in some instances, ere physicians, elso by midwives and in a varying percentum of cases by other informants. This oo is often faulty  ee on rats Bieted fo ce mpt caper es used jes EGER Nake ete y sce; ‘,  4 8  yell in 2. Explain a is ae by \"editing\" causes of death (20: nthe Th A        3. The first step is tc be sure that the cause of death, es stated— the physician, is not incompatible with other statements on the certificates de® | (Here Gistribute samples of \"Ceuse of Death Check Liste\") < Refer to Nose 42, 1265): A 127, 128, 129, 130, 131, 132, 134 te 141 inclusive, alse te 70, 71, 164, 105, 151,\" 6°\" 152, 153. Point out that all above refer to imptssible statements of cause of | 0 death with reference to sex or agée Rae afl!  a« Refer to the titles of the International List which have ab« esi 1a  Pe . solute age limitations. Refer to those which have absolute of #7. sex limitations. = =e. x  f ee el  4 ie te:  \\\\ we pe e on yi {a            | ght a ace =  {Oe ge  tee z Co  See         ie  b = Improbable statements of cause of death, (Go over the numbers indicated under \"cards to be verified\" on the E a Census cause of death check list, Take a number of FM we e specific instances and bring out clearly why these are ; la suspicious reports, Tell Census Bureau experiences in connection with several of them, Emphasize necessity for weeding out a° verifying such statements, Cite ' instance in which a certain city complained to the Census Bureau because it was charged with a death from rabies, When this was traced down, it was discovered that this death was, in fact, caused by Pott's disease which had been “edited” 23:instead of 32, Tell about the negro Virginia physician who reported more cases of relapsing fever in his practise than had eccurred in the wnole country for years, Comment on ane almost universal courtesy with which physicians supply © data on sm request which they have failed to supply on the original certificates, Mention certain exceptions, For example, the physician who replied \"This man is dead, what in h-=1 difference does it make what he died of\" also the physician from the mountain 3 = districts of Kentucky who replied ‘cussing’ the new demo~ pe . ‘ cratic administration for asking \"fool questions.” 4  P pwr  iw NA 4 Ptew I Mit - vet be Uo = AA E ffi — i ow a a. Linc fea § a mnt  POY\" ~ Cede _ Cra ae a Er oe =  F 4 E 7, Gea I o. a  | ae Pe c. ee g dl be flat Rime po     PAT TERRE Te TINE REY IR NS AG IN  NDT TENTS METER TORE  OTE YY TT  ite HOE: ay { ,     .  . — Carte | alee hosed ti fine * LAK = ude _ a ame Co Cane — Queer == * ~~ aA A — aye 3) Ylonse Opt sete  ee im The  ew r Anco  = tos:  oe 7. ey | r Be x Conn Roe fo Te  ; a = 3 Conor (Rope Opec et Ory  Le pete  / CZ e@ ob = 4 ad 7 a A = a 7 fase Cte — ( — Ye nels aes ee Nn — age | / ned ia 1 io 1; eter pet £ Ont | } i Vy fad p- Ie _—- | p/P) oo z fia - : hi f Lx 7 | p34 Z£, f aire ee | / eh }  v  ihe 7", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fp3z-ujge.ntwb", "00000000-0000-0000-65DD-6E3DFBA50B72", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Robert Levy", "9918573882206676X133", null, "1925", "12 March 1925", null, "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "aie BB atl Lal iD  Mareh 12, 1925  Dr. Robert J. Levy 7 | i 31 East 72nd Street New York, N. Y¥.  ly dear Dr. Levy  ‘This 4s a belated reply to your letter of February 24th.  A statistical discussion of the problem of the 24 cardiac cripple is likely to be rather barren at this time for the very good reason that there are so few data of en authoritative sort available. iI suppose you folks are tired of hearing me quote death rates as I have been doing these last few years. I know that you want e stetewent on the prevalence of heart affections of various types and thet is whet I should like to contribute to a meeting of the State Medical Society. But, where aw I to obtain the fects. I do not know of any worthwhile contribution which can be tapped. The only source that I know of that would be worth working ever is that in the hands of your New York City Society. As I remember it, Dr. Wyckoff has several thousand records of his clinic eases. If they were codified as tc type of lesion and ready for tabulation, I should be very glad to have the clerical work done. That might be a very interesting contribu- tion. Would you leek into this matter and let me know what the state of affairs is and also whether you could accept cur offer of cooperation. I should like very much to use these figures for the Syracuse paper. Otherwise, I doubt very much whether eg there would be any justification for a set paper on this subject. i shall appreciate your help very much.  sincerely yours 4  : 2 2 3 Statisticien", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Levy, Robert J.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mvjr.yxhk~382i", "00000000-0000-0000-330B-715DAC86CC3D", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Louis Dublin to Robert Levy", "9918573882206676X134", null, "1925", "23 April 1925", null, "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "April 23,1925  Dr. Robert Levy, 31 East 72d Street, New York, New York  begun te work over the material for t he : . aspects of the chronic cardiac cripple. & exceeds SPE cen Saab GE cesenuiiie aun eek Gh aA. ce ee qurinddtie auatathatien. As I wrote you under date of March 12th there is eo little that is available statistically, that we are still in the realm of opinion and impression. Since that date I have gone throug the material which Miss Ling was geod enough to bring, dah regeunante. I understand, the work of Dr. Syckoff, I have studied the charts and carefully examined the text all of which ia interesting, but frankly, it does not tell us very much that can be used in ovaluating, statisti - eally, the present problem of the chronie cardia ppl thing, I am mot clear as to the definition of the very tern. ‘Mor does be. Wyckoff's material attack thie problem direetly. There are includ- ed a considerable number of cases that ere in the incipient stages and they could not be called crippled, and as for those that are more or less impaired they are a special group who are selected by the very nature of the institution to which they go. i doubt very much whether we can transfer tho findings of the | inte an ovaluation of the anount of chronic cardiac trouble in the comunity by lesions or degrees of impairnent.  ia el 0h ak cin a all ld dink ii ieee wee  do anything that is going to be of any real use to you at this symposium. I om mot attempting to crawl out of my engagement. On the contrary, I should be so happy te do anything that would make my contribution of sone value. But it is my deliberate opinion that it is still premature e@bienpt to evaluate the character of the problem. Before one can do hak 14 wanda be meseneary thet « qned dual of prisare eark be deus o0 the part of your Agsociation to establish definitions, to collect data more and more, and incidentally, to keep in ciose touch with patients eS ee ee ee on ee ee ue noe  ion from @ll sourees inte one view. That ) be done  Vik and ok Ge sean Ae  - Tam emtirely open to your suggesti d guix : and I ahoulé be glad. to talk the Shing ever sith you at your seaven- ience. But frankly, my best opinion at this time is te drop me from your programme mich ae I regret it.", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, "Levy, Robert J.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-necj_tjd7~m42w", "00000000-0000-0000-D78C-8E2049752AD5", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Letter from Thomas B. Wells to Louis Dublin", "9918573882206676X135", null, "1926", "10 June 1926", null, "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "ESTABLISHED 1817        —     HARPER & BROTHERS PUBLISHERS  NEw YORK AND LONDON  “Wnt 11926  49 EAST 33” STREET, NEw YORK,N-Y. dune 10, 1926.  ae  yo  Dr. Louis I. Dublin,  The Metropolitan Life Insurance Company, Madison Avenue & 23rd Street,  New York, New York.  My dear Dr. Dublin,  = In the current issue of the Review of Reviews I have come across an excerpt from your report published in the journal of the  American Heart Association. The subject of this report is one that al-  ready interested us and, to be frank, we have made more than one attempt  to secure for Harper's Msgazine an article on the subject. The two men that we approached both said that they did not feel competent to handle the theme properly. I wonder if you could not be persuaded to do ib.  Dr. Haven Emerson made a statement in the Survey  some months ago that ten per cent. of the beds in the hospitals through-  out the country were now occupied by heart cases. The picture that you give is even more appalling, but what I am interested in chiefly is to find out the cause of this remarkable increase in diseases of the heart and whether the condition as you present it is purely American or is to be found in the hospitals throughout the world. If it is only an American condition it seems to me even more significant and interesting, and people should know more about it. Is there something in our American life that accounts for its increase? How much has it to do with the way we eat and drink and exercise? If all these matters could be covered in a semi-popular paper of not more than four thousand words in length, I think it might exert a very salutary influence. While we do not print illustrations in Harperts Magazine we could use charts such as the Review of Reviews has reprinted.  I shall be very grateful to you if you care to con- sider the idea of doing such a paper for us and I think that its pub- lication in a magazine of our sort would be most helpful to the work of the American Heart Association. As you probably know, we have always made a feature of our scientific articles, so that it will not be necessary to write down to your readers, as might be the case if you  ~                ESTABLISHED 1817  Dr. Louis I. Dublin........Page 2.  HARPER & BROTHERS  PUBLISHERS :  NEw YORK AND LONDON  49 EAST 33” STREET, NEw YORK,N-Y.  June 10, 1926.  were preparing an article for some more popular magazine.  Hoping that I may have the pleasure of hearing  from you in regard to this matter,  TBW/RD  Very sincerely yours,  Editor, Harperts Magazine.", "Wells, Thomas B. (Thomas Bucklin), 1875-1944", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-sqcr~3wpy_2qn9", "00000000-0000-0000-8EBF-17CFFCA4F6B1", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Memorandum from Alfred Lotka to Louis Dublin (in re: correlation between deathrate from heart disease and deathrate from Scarlet Fever, Diphtheria, and Rheumatic Fever", "9918573882206676X136", null, "1931", "17 July 1931", null, "Letters (correspondence)", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "2", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "Dr. Dublin  In re: Correlation Between Deathrate from Heart Disease  and Deathrate from Scarlet Fever, Diphtheria and Rheumatic Fever        I have made a preliminary study of these correla- tions and append below a statement of the results. I wish, however, to preface these results with a word of caution. Correlations in time series of this sort must be viewed with a good deal of suspicion. There is no satisfactory technique for measuring the reliability of the results. This is a well known fact and is especially discussed in Rietz's \"Handbook of  Mathematical Statistics\" page 163: \"There is great danger that coeffi- cients based on time series may be wrongly interpreted. For instance a high coefficient may result if two series fit their secular trends badly and the badness of fit in the two cases is similar.\"  The probable error of the coefficient of corre. lation in .time series does not furnish a test for significance. Here again I quote Ré&tz, page 163: \"The significance of the-probable error of a constant computed from time series is not know.\" UThe coefficients of cor- relation computed for several cases from the data for the United States 1910 to 1928 are as follows:  lag r l. Scarlet fever Heart disease 0 ~ e224 15 2- Scarlet fever Heart disease + 5 013 + .18 3. Scarlet fever Heart disease + 4 «35 # 215 4. Rheumatic fever Heart disease +1 324 14 5. Diphtheria Heart disease - 3 299 + 012  Attention is particularly drawn to the last item in which merely by the way of illustration a computation was made correlating diphtheria in a given year with heart disease three years prior. Note that this gives the highest correlation in the series, namely, .55, yet this is an absurd way of comparing observations for it would imply that heart disease in one year causes deaths by diphtheria three years later. This example was | done merely to show how unjustified it is to conclude from a relatively high correlation obtained in this manner that there is causal relation between the two phenomena correlated. The correlation coefficient of e35 such as observed in example three would, in itself, be regarded as fairly important. For example, this is the degree of correlation observed between stature of brother and sister, (See Pearl, Medical Biometry, page,310)but as just — the case of time series, such correlation cannot be taken ser- iously. . —  It is easy to explain why there is no very clear cut  relation between the deathrates from the several diseases mentioned above  and that from heart disease,in spite of the fact that we have every reason to believe that actually these diseases are frequently followed by heart impairments. The fact is that such heart impairments, if they become the reported cause of death at a later stage, do so at all kinds of irregular intervals varying from perhaps a few weeks to fifteen or twenty years or even more, The consequence is that the picture becomes blurred beyond recognition.  This is my provisional report. If after consideration of the facts submitted it is desired to proceed to complete analysis of the figures, considerable additional work would be required to cover both United States and Metropolitan figures and to go into some detail in the way of trying out varying lags between the deathrates in question. I have every reason to believe that the samples given above are quits representative of the results that would be obtained if the work were extended in greater detail °     July 17,1931", "Lotka, Alfred J. (Alfred James), 1880-1949", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dxdm-bh4s_hhkf", "00000000-0000-0000-8C6C-2E009C473546", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Memorandum from Donald Armstrong to Louis Dublin", "9918573882206676X137", null, "1931", "20 July 1931", null, "Memorandums", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "2", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "ore                                         MEMORANDUM July 20th, 1931.  DOCTOR DUBLIN  - STATISTICIAN  ‘ really do not know what to say about the significance of these corre-— lations, nor about the desirability of further work along similar lines. Items  3 and 4 appear favorable. Has a diphtheria and heart disease relationship  been worked out with a positive lag of several years?  This observation occurs to me, if I interpret the tables correctly. For the Registration Area, there is a marked decline running fairly consistently throughout the entire period for scarlet fever, diphtheria, and rheumatic fever. This is true of the period from 1910-18 as well as of the later years. On the other hand, there was very little, if any, décline for heart disease from 1910-18 inclusive. In fact, the 1918 rate is the highest in the series, though I suppose that was partly influenza. Nevertheless, it might appear that we had to experience a consistent decline in these other cayses for a considerable period prior to the appearance of a parallel decline’ in heart disease, The same general relationship seems to hold for our industrial pol- ieyholder experience. Does that mean anything, and is it worth further inves-— tigation?  In addition to the above inquiries, I am convinced, in the present more or less hysterical state of the public and of the professional mind concerning the apparent menace of heart disease, that there are several points that might legitimately and advantageously be made: :  1) We are certain that “heart disease\" is a complex of various etiological  factors and pathological conditions, most clearly understood when analyzed by age groups. . r 3  #2) There is a younger age group in which heart disease is largely rheuxuatic in origin, though probably in part related to other acute infections. Here there has been a recent mortality decline, concomitant with and perhaps, chronologically at least, subsequent to a decline in certain probable causa- tive factors. You can best judge what stress, if any, might legitimately  “—~——____be_placed upon the apparent correlations. In any case, the situation with  reference to this segment of our heart disease problem, is reassuring and hopeful.  3) There is a second middle age unit in which syphilis is the principal _ factor. Is it possible to determine the trend of mortality there? Apparent-— | ly, the age limits should be approximately from thirty to eixty, according to} Carr, Wyckoff, Lingg, Collins, Church, and others. Certainly, this phase of |[ the situation is not hopeless, as syphilis is surely to a large degree, with | adequate resources, therapeutically, if not prophylactically, controllable.     4) There is, finally, an older age group, largely arterio-schlerotic, or degenerative, where the rates are no doubt rapidly increasing -- an increase ©                  ae is ineritenle because of the onton of the eepleticn. This increas  is also, in part, the logical sequela of successful disease preventive and  _ death deferring efforts at younger ages. It is, in fact, the reward of ac=  - complishment. Also, for many it furnishes a prompt and ‘painless exit, and  has, from this angle, a beneficent aspect. To some extent, the mortality  here may also be in part controllable or deferrable, through personal hygi mental hygiene, the periodic health examination, and similar efforts at con = servation, and the adjustment of ‘physical and emotional strain.  5) Finally, from a mortality point of iow, £ believe 1950, ie our a ience, showed for the first time in several years, « turn downward in the curve. 1931 apparently is going to approach 1920.pretty closely. Is there any indication here that inspite of the inevitable accumrulation of cardiac/ - hazards in the older age group, the saving at younger ages may be going to stabilize the picture, if not to start a. downward et A , ie  6) In any case, it is Jiportant to distinguish between these oe To talk about heart disease as being the greatest cause of death, and being an = increasing menace is misleading, because the observation hast necessarily be based largely, if not entirely, upon this inevitable natural process reflect- ed in the older age groups. The problem must be split so as more specifical ly to indicate those phases which have a genuine public health ees rem :  _} and that reflect promising methods for more complete control, at see o* greatest economic and social significance.  If we can decide what siestHicance to eles upon the statistics, can we not develop something that would have timeliness’ and orientation value?  .  Denna. heeds: Me a Fourth Ving roni dent”", "Armstrong, Donald", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9py9~rgck-eifs", "00000000-0000-0000-51E4-81ACE01FA75A", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Memorandum from Alfred Lotka to Louis Dublin (in re: Dr. Armstrong's suggestions for the investigation of heart disease in relation to predisposing causes)", "9918573882206676X138", null, "1931", "25 September 1931", null, "Memorandums", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "2", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "Dr. Dublin  In res Dr, Armstrong's Suggestions for the investigation of Heart Disease in Relation to Predisposing Causes     The topic might be discussed in either one of the two following ways:  1. By some attempt at numerical correlation measure- ments.  26 By general descriptive discussion.  Discussion of (1). The method (1) has been tested and it has been recommend- ed that no reliance be placed on correlation coefficients obtained by comparing time series of heart disease and certain other diseases, with an arbitrary lag introduced by trial in such manner as to produce the highest possible correle- tion coefficient. It does not seem desirable to reopen this question, the chief objection to this method being that the sequelae of such diseases as rheumatism, whooping cough, etc. are so spread over a series of years after the prime cause that no correlation study can be expected to effectively detect the true cause and effect relation which undoubtedly exists; any correlation coefficients  that may be found by this method are very apt to be due to something quite dif- © ferent than the relation that is really being investigated.  Discussion of (2) We have good material on which to base a discriptive discussion, gathered together in our work sheets for the book on mortality.  A really good descriptive discussion is valuable even though it may not involve correlation or similar technique. Dr. Armstrong, in his memorandum of August i3th expressed interest particularly in the trend of heart disease fatalities at ages 30 to 60. As the work sheets now stand, we cannot give data on precise- ly this age group but we can give data at 35 to 65. The sheets before me cover ae Organic heart disease; b. Endocarditis and valvular heart disease;  ce Chronic myocarditis; andd. Angina pectoris. :  Among the items enumerated above the case of chronic endocarditis and valvular heart disease is peculiar in showing for the period 1921 to 1930 a marked decrease at ages 35 and over, whereas all the other causes listed show increases, and in some cases very great increases.  The effect of the influenza epidemic seems to be clear. ly visible in all those series which we have been able to carry from the year 1911 to 1929, There is a sudden drop in deathrates for the year 1919 as com. pared with the year 1918, followed by a tendency to return to the old level or to exceed it, except in the case of chronic endocarditis and valvular heart disease.    a £2  : As to the relation between heart disease and certain predisposing causes, these sheets, of course, give no clue. A discussion of this topic can be conducted along the lines of information gathered from general experience. This, it is true, would not in itself bring forward any- thing very novel, but it would quite properly form part of the discussion of information contained on the work shects relating to heart disease.  : If it is desired to have data expressly for the group 30 to 60 years, mentioned by Dr- Armstrong in his memorandum of August 13th some additional computation would have to be made.  \"Alfred ’3.\"Lotka’*’  Sept, 25,1931", "Lotka, Alfred J. (Alfred James), 1880-1949", null, null, null, "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-webh-wntd.8w56", "00000000-0000-0000-D514-8AE03C9E605A", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Statistical Aspects of the Problem of Organic Heart Disease", "9918573882206676X139", null, "1925", "1 November 1925", "Louis Dublin, Statistical Aspects of the Problem of Organic Heart Disease, New York State Medical Journal, November 1, 1925. [item is a reprint]", "Articles", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "15", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, ")     +  SMB gs  Bes  Set RP RNR Reet oneal o. at. Dit Nee ett tn ey ine ‘ . a :  Reprinted from New York State JouRNAL oF MEDICINE, November 1, 1925.     STATISTICAL ASPECTS OF THE PROBLEM OF ORGANIC HEART DISEASE *  By LOUIS I. DUBLIN, Ph.D., NEW YORK CITY  EART disease in its various manifesta- | tions is first in the order of causes of death and, I am inclined to think, the first also in the amount of damage it does through disability and invalidism. The evidence for mor- tality is much more complete and decisive so that I shall give that first. The annual quota of deaths in the United States is now close to 200,000. If present conditions continue, one in every five of the population living at age of 10 will even- tually succumb to organic heart disease. The child at ten years of age is now three times as likely to die eventually from heart disease as from tuberculosis. At age 35, the probability of dying eventually from heart disease is, among males, nearly four times that for tuberculosis and, among females, the probability is almost six times that for tuberculosis. A revolutionary change has taken place in the general mortality picture during the last twenty-five years coinci- dent with the development of preventive medi- cine and the public health movement. It is also very likely that the pre-eminence of heart disease as a cause of death will increase  *Read at the Annual Meeting of the Medical Society of the State of New York, at Syracuse, May 13, 1925.  Otic De /  97 Su\" -    rather than decrease as time progresses. The gradual improvement in the death rate for such diseases as tuberculosis, pneumonia, and others that are coming under control, will transfer many additional persons to the later ages in life when heart disease is likely to strike them down. Under conditions of twenty or thirty years ago, many of them would have died in early life from the conditions referred to; today, they survive to middle life only to become victims of heart dis- ease, cancer, apoplexy, or Bright’s disease. This is an item which must not be lost sight of, especially in view of the fact that the medical profession is not so well organized to control heart disease as it has been for twenty years organized to combat tuberculosis. It is for this reason that I consider heart disease the out- standing problem: in contemporary preventive medicine. Nothing within the province of the physician today compares with it. I have in another place estimated that if heart disease could be eradicated as a cause of death, every one in the population would have about two years added to his expectation of life—not a small matter when the economic value of a year  ‘of life is considered.  The first question I would like to take up with you is whether the mortality from heart disease is beginning to show any signs of improvement. Between 1910 and 1918, the indications were that the mortality rate was rising slightly. The years 1917 and 1918 showed the maximum death rates of this period. The three following years, 1919, 1920, and 1921, were years of low rates, and there is every indication that this was largely due to the elimination of many persons through the influenza in 1918 who would ordinarily have died of heart disease in subsequent years. Be-  2  \\\\e ginning with 1922, the trend has been slightly upward, and there is really no saying what the picture in the immediate future will be like. In the first four months of 1925, the experience among the sixteen million Industrial policyhold- | ers of the Metropolitan, which is usually very sensitive as an index of what will be found later in the general population, showed an increase of four per cent over the same months of the year before. The facts for the fourteen years be- ginning with 1911 are shown in the following table: TABLE 1  Death rates per 100,000 for Organic Diseases of Heart.  Experience of Metropolitan Life Insurance Company,  Industrial Department (Ages one and over) and Expand- ing U. S. Registration Area (All ages).        Metropolitan Life - Expanding U. S.  Year 5 Industrial Dept. Rgistration Area Ages one and over (All ages)  1924 125.5 *  1923 128.7 157.3  1922 126.7: 148.4  1921 117.4 140.9  1920 117.0 141.9  1919 113.9 131.0  1918 141.7 153.3  1917 142.0 153.8  1916 140.2 150.6  1915 136.7 147.6  1914 138.1 142.2  1913 140.6 138.9  1912 143.8 142.8  1911 141.8 141.1           * Not available.  Heart disease is pre-eminently a condition of the older ages of life; but, it is by no means to be neglected as a cause of death in the early years. In 1924, for example, the Metropolitan Life Insurance Company recorded close to twenty thousand deaths from heart disease  3       among its Industrial policyholders. Of this num- ber, 1,600, or eight per cent were of persons under the age of twenty-five, and 3,400, or 17 per cent, were under the age of forty. In other words, one-sixth of the insured who died from heart disease were at their prime. At the younger ages of adult life, heart disease is responsible for as many deaths as are all forms of pneu- monia combined. After forty, the rate of mor- tality rises precipitously, and the priority of heart conditions is then unquestioned. But it should not be forgotten that these deaths of middle-aged men and women, in most instances, involve a loss of many years of productive life to which they would ordinarily have been en- titled, and often of years of diminished efficiency and even of complete invalidism prior to death. Deaths at these ages also produce in many cases broken families, widows, and orphaned children thrown upon the community for support. The curve of mortality from heart disease by age is is a very interesting one, and I present one here- with in contrast with the curve for tuberculosis. The latter disease now shows its maximum at or about twenty-five years of age and then de- clines with advancing years. But heart disease crosses the tuberculosis curve about the age of forty-five and then mounts to its huge maxi- mum at the oldest ages. The principal damage to the community has, however, been accom- plished long before old age is reached. Sixty- eight per cent of all heart disease deaths, in the experience of the Metropolitan Life Insurance Company, occur before the age of sixty-five. Among white persons, the death rates are very much the same for the two sexes up to the age of twenty-five. After the age of twenty-five, the death rate for whité males is higher than for  4    » © yo)        Deathrates per 100,000 for Organic Diseases of Heart  Metropolitan Life Insurance Co, Ind/. Dept. (Ages One & Over) end Expanding United States Registration Area, (All Ages)  ISIl to 1I924~  OEATHRATE PER 100,000 155            150 145 140 135 130 125 120  Laie)        110 Oo Po ee et eee eet ISh hee 13 14 15 16 17 18 19 ‘20 2I 22 ‘23 1924     STATISTICAL BUREAU ~ METROPOLITAN LIFE INSURANCE CQ.       females and the excess between the sexes be- comes greater with advancing age. The rate among colored people is at every age higher than for whites. In fact, during ine main age periods of life, the rates for colored people are  about twice that for whites at the correspond--  ing ages. It is also noteworthy that at some ages in adult life, the rates for colored females are higher than for colored males. It has been ‘suggested that the higher prevalence of such diseases as malaria, typhoid fever and especially syphilis in the colored race, plays an important part in creating this excess of heart disease among them. But possibly, also, the figures pre- sented are not all that they ought to be, because of the great difficulty in obtaining reliable state- ments of the causes of death on certificates. We shall always be troubled with our figures for heart disease until there are a larger number of  autopsies and the standards of medical practice -  are generally raised.  TABLE 2 -  Death Rates per 100,000 for Organic Diseases of the Heart. Metropolitan Life Insurance Company, Industrial Department, 1923.              Ree pees White Colored Males Females} Males Females oN Fee ONE) | ANSG 122.1 | 190.8 217.4 1 to 4 6.3 6.2 17.1 10.3 5 to 9: 10.3 10.8 17.5 10.8 10 to 14 19.8 23.2 28.9 16.0 15 to 19 27.6 23.9 28.4 19.3 20 to 24 23.9 . Ze 27.6 *) BB 25 to 34 39.6 32.4 68.0 58.7 35 to 44 86.6 70.7 180.3 184.7 45 to 54 253.3 184.9 424.6 470.4 55 to 64 681.3 535.6 | 831.9 948.8 65 to 74 1719.9 1545.4 |1595.6 1641.1              75 and over. 4060.8 3524.7 |2600.1 3016.5 :  - I have touched on the principal points in the mortality picture. I wish I could speak with as much assurance on the morbidity aspect of our subject. Unfortunately, that is impossible in the present state of our knowledge. We have made only the merest beginnings in collecting infor- mation on the incidence of heart disease in the community. Our very definitions are still vague. I should not have been able to make a presenta- tion today if I had been kept to a literal interpre- tation of the title of this symposium. For, what is really a cardiac cripple? When does the con- dition justify such a designation in that shadow zone between the functional and organic heart case? Primarily, the difficulty lies in the fact that organic heart impairments ordinarily are not discovered in the early stages, and even then, there is no provision for systematic records of the after-history of the cases. This most valuable information, therefore, lies hidden away either in the memories of the one hundred thousand or more practicing physicians, or in their uncompiled and unanalyzed records. It is only recently that we have made a beginning, through the work of the cardiac clinics, to gather such information as we need on the morbidity of heart disease. It will always be a source of satisfaction to me to have had an opportunity to co-operate with Dr. Alfred Cohn in the prepara- tion of the record forms used in these clinics. It is, indeed, fortunate that today we are able to tap this source of information from the un- published work of Dr. Wyckoff, who has com- piled and analyzed the records of a thousand patients in his service. These data are probably the only ones available at the present time, and I wish to acknowledge my great indebtedness to  ‘          Deathrates per 100,000 for Organic Diseases of Heart Metropolitan Life INSULENCE Co, Industria! Department, 1923                                                                                                                                                           DEATHRATE Pen 100000 WHITE PERSONS eR 1O0000 COLORED PERSONS 4,500 4,500 _ 4,000: 4,000 3,500|++ 3500}-+ : ——— FEMALES ——— FEMALES 3,000 i 3,000 : | 2 500F 2,500 i i \"2000 — 2,000 / 1,500 1,500 : i 5 7 // 4 : i} y 42517000 iH 1,000 4 4 / 5 500+ 5 4 500 p Y LA | ae ° 0 io Sssl0nl6 o0e5 5 46 55. Ge 7s. 55510 1500 (05s 9 35 45 555Gb. 75 to to to to to to to to to to to to to to to to to to to to 4 9 1419 24 34 Ad 54 64 74 OVER 4 9 14 19 24 34 44 54 64 74 OVER     STATISTICAL BUREAU ~ METROPOLITAN LIFE INSURANCE CO.  e \\\\ Ve  a~ -,       Dr. Wyckoff and his associates who placed this material at my disposal for this occasicn.  In a previous paper, I have indicated that the number of persons suffering from definite or- ganic heart disease approximated two per cent of the total population. This is a rough estimate. It is suggested by the findings of the life in- _ surance companies in their routine examination of applicants for insurance; by the findings of the Life Extension Institute in their examina- tions, and by the results of others who have made physical examinations of large numbers of school children, employees in shops and factories and of other groups. This figure should, of course, be considered only a first approximation and will give place to a more definite one as further work is done on the records. It will serve, however, as our starting point. On this basis, there are well in advance of two million men, women, and children with organic heart lesions, and if the number of deaths annually from this group of diseases is in effect 200,000, we may infer that the average duration of a case of heart disease is about ten years. This will, of course, vary with the age at which the lesion occurs, with the type of lesion, the care which the individual re- ceives and a host of other factors which bear on the condition. But, this figure of average duration will serve as a beginning to guide our discussion.  What are some of the outstanding findings in the tabulations of Dr. Wyckoff? The thousand cases were distributed etiologically as follows: About one-fourth presented rheumatic heart. dis- ease; about two-fifths, arterio-sclerotic; about one-tenth, syphilitic, and about another one-tenth, heart disease of unknown origin. The remaining 15 per cent are accounted for by a mixture of odds and ends which are not so easily classified.  9       The cases of rheumatic and unknown etiology are discovered, for the most part, at the earlier ages; the cases of arterio sclerotic etiology ap- pear in the later age groups. The great presump- tive importance of rheumatic fever as a causative factor is clearly indicated by the findings of Dr. Wyckoff and his associates. It would be very interesting to find out what the relative duration of these cases of rheumatic origin is in contrast with the duration of cases of degenerative heart disease. The seriousness of syphilis as a primary causative agent is no surprise. Probably, the figure of ten per cent is a minimal value in view of the difficulty of determining in every instance the presence of the spirochete and the incom- pleteness of case histories. It is important, how- ever, to observe that this type of heart disease is most prominent in the fifth and sixth decades of life. It is especially among the colored patients that syphilitic heart disease is prominent; in this group, it accounts for about a third of the cases.  An equally valuable classification prepared by Dr. Wyckoff and his associates is that showing the incidence of structural lesions in these 1,000 cases of organic heart disease. They found 88 per cent of the cases showing enlargements of  the heart, among whom one in five had enlarge-  ment of the heart only. Hypertrophy was, by all odds, the commonest impairment. Mitral insuf- ficiency was diagnosed in approximately half of all the cases. Mitral stenosis was present in 44 per cent of the cases; but, in nearly all of these, mitral regurgitation was also present. Aortic insufficiency was present in about 15 per cent of the cases; aortic stenosis in three per cent; aortitis in 17 per cent; and aneurism in a little over one per cent. The following table is taken from Dr. Wyckoff’s report, with his permission.  10  <a TABLE 3  Incidence of Structural Lesions in 1,000 Case of Organic Heart Disease.                 Males Females |Both Sexes Structural Lesion Per Per Per Now cent No. cent | No. | cent an Enlargement of Heart..}542 | 54.2 |341 | 34.1 | 884 |88.4 ¢ Enlargement of Heart, Only I ee ee 187 1013-7 |-00 |: 6:6: | 20321 20:3 Mitral Insufficiency ....}228 | 22.8 |207 | 26.7 | 495 |49.5 Mittal’ Stenosis. 3.1. .- Z16 | 21.6 |227 | 22.7 | 443 |44.3 Aortic Insufficiency ....1}178 | 17.8} 68 | 6.8 | 146 |14.6 PROGIC CStenosis 25342: 23: \\\\7- 2331.6 101298. 29 _ (NORUITIS 2 fee 142°1'14.2) 277), 2.7 | 169 | 16.9 ANeUnIsiINe ie Sle As it peed A122 Mitral Insufficiency and StenOSiSt.6 3). sess 202 | 20.2 219 | 21.9 | 421 |42.1 Mitral Insufficiency and| ._| Stenosis and Aortic] ||; ee Insuthiciencys a5: 82 | 82] 40 | 4.0 | 162 |16.2 Mitral Insufficiency and| - Stenosis and Aortic Insufficiency and Sten.| 6 O68 (Oo foe                          Deathrates per 100000. Tuberculosis and Heart Disease Compared in the Industrial Experience of the Metropolitan Life Insurance Company -!1923           CENTRAL AGES           11    This report when it is published should receive the careful attention of all physicians as it is, in fact, the first attempt, to my knowledge, to collect a large body of information of an authen- tic character in this field of heart disease preva- lence. We have, heretofore, had any number of sketchy and loose statements of diagnostic find- ings. But, this series stands by itself in the authority of the examiners and the fullness of the records. This report should stimulate others in the field to collect similar case records and to tabulate and analyze them along comparable lines. A very great service could be rendered to this branch of medicine by spreading the use of these forms not only in clinics, but also among physicians in private practice.  I wish now to turn to a third source of in- formation on the prevalence and significance of the heart diseases, namely, the records of the life insurance companies. A number of the larger companies have been for years liberal in their acceptance of certain types of heart cases for sub-standard insurance. The examinations for insurance are, of course, not. made with that same thoroughness which characterizes the work of the cardiac clinics. But, the insurance applica- tions do, nevertheless, make possible the classi- fication of the heart findings of large numbers of people with a fair degree of accuracy. The heart defect most frequently found is mitral regurgitation. Hypertrophy of the heart without other heart signs is next in importance. Mitral stenosis, aortic stenosis and aortic insufficiency follow in order of frequency. This order is very much like that of Dr. Wyckoff’s material; but, I doubt very much whether the examinations as conducted by insurance medical examiners in the field are of sufficient accuracy and refinement to  12 find all the cases with heart lesions or to diag- nose them correctly. Yet, the parallelism is very interesting. The medical directors of the life insurance companies are confronted with the practical problem of evaluating these lives and placing them in the several risk classes for which mortality rates and premiums have been com- puted. When insurance is granted, an excellent opportunity is afforded to the medical director of following the subsequent mortality experience on such persons.  A number of companies have, in this manner, collected considerable data on the after mortality of cases with mitral regurgitation which, I be- lieve, will interest you. The experience covers a period of close to twenty-five years and is based on many thousands of persons. Taken altogether, the rortality rate is about two and a half times as high as that which prevails among normal persons accepted for standard insurance, age period being considered. Those cases where the murmur is slight but not transmitted have only a slight excess over normal mortality. The presence of a well marked hypertrophy adds to the hazard, as does also the history of rheumatic fever or other acute inflammatory processes. You will all remember Dr. Mackenzie’s very favorable prognosis of these cases. He felt that the insurance companies were losing a great deal of good business in not accepting these cases. But, our experience has shown, I am afraid, that his impressions were not correct, although it may well be that these cases of mitral insuffi- ciency constitute the least impaired of the organic heart cases. In this connection, it is interesting to note that close to half of the deaths which occurred among the mitral insufficiency cases were from organic heart disease of one form or  13       another. Cases of aortic stenosis and of inter-  mittent heart disease gave an experience very much like that from mitral insufficiency, ice., double mortality. In a few instances, the insur- ance companies have accepted risks affected with mitral stenosis, but the experience is uniformly bad, and the present practice is to reject such cases. The available insurance experience is, however, insufficient to give reliable results on the precise effect on longevity of these very serious impairments.  I have mentioned the practice of the insurance companies in this connection with a special point in mind. The medical directors of the insurance companies are keenly interested in. the work of your clinics. As physicians they are, of course, very much concerned with the prevention of suffering and the postponement of death. But, as insurance men, they are also concerned with providing an equitable classification of the risks presented to their respective companies. They are all of them very anxious to provide protec- tion for the families of persons affected with impairments; but, it is always necessary that such insurance protection be granted with no unfairness to those who are already insured. For this reason, the medical directors have been com- pelled to proceed cautiously with the acceptance of risks showing heart impairments. It is quite possible that the new movement for the study of heart disease will provide the very informa- tion which will make it possible for the com- panies to extend their operations to such cases and to offer insurance to lives which are not now accepted because of the dearth of informa- tion on the after mortality of such risks. There are large opportunities for co-operation between your heart clinics on the one hand, and the in-  14  Nast  iH surance companies on the other. I believe it would be a very profitable procedure for both groups to get closer together if for no more than to explore the opportunities for further reseatch.", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, "New York State Journal of Medicine", "Medical Society of the State of New York (1807-)", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-x6fi.whkg~apfw", "00000000-0000-0000-197D-02404337E1C0", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Favorable Aspects of Heart Disease, with special reference to the Health Officer", "9918573882206676X140", null, "1933", "09 October 1933", "Presentation at the American Public Health Association Annual Meeting, 09 October 1933.", "Articles", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "36", "pages", "Text", "English", null, "Copyright may apply", null, null, "FAVORABLE ASPECTS OF HEART DISEASE  with Special Reference to the Health Officer  by  Louis I. Dustin, Pa.D.  THIRD VICE-PRESIDENT AND STATISTICIAN  and  Donatp B. Armstronc, M.D. THIRD VICE-PRESIDENT  of the  METROPOLITAN LIFE INSURANCE COMPANY  >  A paper presented before the Health Officers Section, + American Public Health Association, Indianapolis, Ind. October 9, 1933    FAVORABLE ASPECTS OF HEART DISEASE with Special Reference to the Health Officer*  BY  Louis I. Dusuin, Pu.D., Third Vice-President and Statistician, and Donatp B. Armstrone, M.D., Third Vice-President, Metropolitan Life Insurance Company, New York  I  ie ScopE and outlook of the public health movement have undergone great change in the last generation. In 1900, the deathrate from typhoid fever in the Registration Area was 31.3 per 100,000. Last year it was only 1.1 in the same ten states which originally constituted the Area. Today, typhoid fever is a serious problem in only a few of our cities and in certain rural areas. In 1900, the diphtheria deathrate was 40.4 per 100,000; last year it was 2.5 per 100,000 in the same states. So far as diphtheria is concerned, we are well on our way to eradicating the disease altogether. Tuberculosis, at the be- ginning of the century, was the leading cause of death in our popu- lation. Its deathrate then was 195.2 per 100,000, but last year it was only 58.7. The trend of mortality from this disease is still rapidly downward and by 1940 we are confident that it will have become a relatively minor item in our public health program. With the decline in the acute diseases of childhood and of early adult life, new problems have come to the fore which demand attention on the part of public health workers. The chronic diseases of later life have been constantly forging ahead and today occupy a position of outstanding importance. Over 60 per cent of all deaths now occur after age 45, and of this total nearly two- thirds are accounted for by chronic diseases of the heart and arteries, Bright’s disease, cerebral hemorrhage, cancer and diabetes. Of these, heart disease is responsible for far more deaths than any other cause. This new situation in heart disease is becoming more and more insistent, and for a number of reasons. It hasa just claim upon the time and thought of the health officer, because any  *A paper presented before the Health Officers Section, American Public Health Association, Indianapolis, October 9, 1933.  1       program of control will center about him. He can play his part well only if his attitude is based on the facts of the heart disease problem.  What are these essential facts? First, heart disease leads as a cause of death. In addition, it ranks very high as a cause of disability. Only rheumatism exceeds it in this respect. On the basis of their recent survey of chronic illness in Massachusetts, Bigelow and Lombard! estimate that there were approximately 84,000 persons ill with heart disease in that state alone. This incidence is probably higher than in most states because the population of Massachusetts has a higher average age than the country at large. In New York City, the study of chronic illness under the auspices of the Welfare Council? disclosed that at least one out of every eight of the chronically ill suffered from heart disease, and this figure did not include a large number of old persons, many of whom suffered from cardiovascular disease in some form. On a conservative estimate, the number of persons suffering from heart disease in the United States is probably close to two millions.  Another excellent measure of the current heart disease situation is the probability of dying from the disease. Under present con- ditions of mortality, one out of every five white males born will eventually die from the disease. The proportion is slightly larger for white females. Indeed, the chances of dying from it are not only greater than those from any other single cause, but exceed those for tuberculosis and cancer combined. These chances increase with advancing age so that one out of every four females at age 40 will, under present conditions, die ultimately from heart disease, and one out of every four males at age 50.  These figures present in bold outline the great importance of heart disease from the point of view of incidence and mortality. A great number of the cases, however, are old people who have lived out their life span. Their heart disease is, from one point of view, hardly more than a manifestation of senescent changes that are going on simultaneously throughout the body. Obviously, heart disease of this type is not a public health problem. What isa matter of concern to the health officer, however, is the frequency of heart disease that is preventable or postponable, and how much of it is premature and amenable to control through his efforts. To throw light on this problem we must consider whether or not this  2 disease is increasing, whether various types of the disease behave differently, and at which ages of life each type predominates.  Il  Crude figures on heart disease mortality show a very definite rise in rates. Thus, in the Registration Area, the deathrate from organic disease of the heart in 1932 was 184.3 per 100,000, whereas at the beginning of the century, the rate was only 111.2 per 100,000. An increase of over 60 per cent in the crude rates has been recorded in the space of a generation. These figures, however, do not take into account the changes in the age distribution of our population. In this interval, the population of the country has increased by 62 per cent, whereas the population at ages past 45 has more than doubled. Stated in another way, persons over 45 constituted only 17.7 per cent of our population at the beginning of the century, but in 1930 they made up 22.8 per cent. A large part of the increase in heart disease is necessarily a result of this increasing proportion of older persons in our midst.  We may correct for this factor by means of deathrates standard- ized in respect to age, sex and color. We have such a series of corrected deathrates available, covering the Expanding Regis- tration Area from 1911 to 1920, and the Registration States of 1920 from 1920 to 1930. The course of the standardized deathrate from organic heart disease in that twenty-year period is shown in Chart 1. The effect of the increasing proportion of older persons is here eliminated. In the early years of this series, heart disease was increasing at a rather moderate pace. The rate for the popu- lation as a whole did not show any appreciable increase during the influenza pandemic. The first important change in the picture was the sharp decline in heart disease mortality immediately follow- ing the pandemic. The deathrate in 1919 represented a new minimum and was approximately 15 per cent below the average for the eight years preceding. The most striking feature of this curve, however, is the rapid and almost uninterrupted increase since 1919. By 1922 the pre-influenza level was reached, and in the very next year exceeded. The extent of rise has been such as to carry the heart disease rate in recent years to a level approximately 25 per cent higher than that prevailing prior to 1918. It is clear, then, that even making allowance for changes in the age and sex  3       composition of our people, the recorded mortality from heart disease, compiled from official data, is higher than ever before.  STANDARDIZED DEATHRATES FROM ORGANIC HEART DISEASE IN THE UNITED STATES * 1911 Zo 1930  STANDARDIZED DEATHRATES R 000     190 180 /  170 7  160 A]  150 LZ.  140 oe VAY 130  120                                                                                      Ae eee ee hee eee SNH L~~A—~  eo lo ee 1911 1915 1920 1925 1930 * Expanding Registration Area, 1911-1919 aad Registration States of 1920, 1920-1930  CHART 1  iit  There are two reasons, however, why we are somewhat uncer- tain regarding the validity of the comparisons of present levels of heart disease mortality with those previous to 1920, as compiled from official data. In the first place, changes in heart disease classification introduced in 1921 tended to increase somewhat the rates from this disease, especially between 45 and 60 years of age. In the second place, the Registration Area grew very considerably between 1911 and 1920, and the level of heart disease rates in the states admitted to the Area from time to time was not the same as in the states comprised in it before. Consequently, we cannot be certain that the variations in the rates from 1911 to 1920, and the comparative levels before and since, reflect much more than these changes. Another item of some importance is that earlier official  4 data did not segregate the facts for white and colored persons, and consequently differential changes in the rates for the two races cannot be measured.  Fortunately, in the Metropolitan Life Insurance Company, we have kept mortality records of the many million Industrial policy- holders in such manner that the difficulties arising from classifi- cation changes have been avoided, and we have a separate record for the white and colored races from the very beginning of our series in 1911, In regard to geographical spread, the area covered  DEATHRATES FROM ORGANIC HEART DISEASE  IN THE GENERAL POPULATION  AND AMONG METROPOLITAN INDUSTRIAL POLICYHOLDERS I9ll - 1932  TOTAL PERSONS, AGES | to 74 STANDARDIZED  DEATHRATES PER 100,G00 160  ae 150 N  \"Taber AA NL mn                       * United an Loe » Lf) 110 4 A 7 me. [E Soh \\\\ on peas + mete, Sc e ee ae \\\\ Ls 0 ome a 7 \\\\ of  100 \\\\ :  \\\\  \\\\ ry  \\\\’  90                                                                          191] 1915 1920 1925 1930 # Expanding Registration Area, 1911-1919 and Resistration States of 1920, 1920-1930 CHART 2  by the Company has changed little and the growth in the number of Industrial policyholders is simply in the nature of an increasing proportion of persons living in the same communities. These policyholders reside, for the most part, in cities and towns of the United States and Canada, and the facts on heart disease relating  5       to them, must therefore, be construed as more typical of the urban population of the two countries than of the total population. They do constitute a sizeable fraction of this urban population, however, nearly one-fourth of the total—and the greatest single sample for which a record of heart disease mortality consistent in definition is available. This insured group, it should be pointed out, is deficient in its proportion of persons over age 75, and the rates for the insured group past this age are not reliable. Consequently, the experience is limited to ages 1 to 74. That the experience is dependable over this range of life is seen by comparing the general course of the heart disease rates, adjusted for age, sex and color, among these insured persons—the upper line in Chart 2—with the trend for ages 1 to 74 in the general population The latter is shown in the lower line of the chart. The main features of the two curves are similar, namely, a fairly stable level from 1911 to 1918, a sharp decline resulting in minimal rates between 1919 and 1921, and an unmistakable increase since that time. There is, however, one important difference: the recent level of mortality is still slightly below that of the pre-influenza years in the case of these Metro- politan policyholders, whereas in the population, rates of recent years have already far exceeded the level prevailing before 1918.  This general situation in heart disease mortality for the popu- lation as a whole does not hold true, however, for all the constituent parts. Analysis of the data reveals distinct differences between the trends for men as compared with women; for white persons, as compared with negroes; and for young persons as compared with older ones. For the reasons previously given we must use the insurance records for this part of our study.  Chart 3 shows for this group of policyholders the separate trends of the heart disease rate by color and sex. The rates are suitably adjusted to discount differences in age composition be- tween the various groups and the changes from year to year in the composition within each group. For white males, the trend has the characteristic detail of the insured population as a whole, namely, a level trend from 1911 to 1918, an upward trend since 1919 and a sharp break between the two periods. The rise since 1919 has been so great that the rates in some recent years have reached. or exceeded the level prevailing before the pandemic. Among white females, the course of the heart disease rate shows somewhat similar tendencies, but there are two important differ-  6 ences. First, heart disease rates among women began to fall after 1918, a year later than in the case of men; the decline was by no means so sharp, and the rebound has been very slight. As a result, heart disease deathrates among white females in recent years have remained appreciably lower than fifteen to twenty years ago. Among colored persons, pre-influenza rates fluctuated markedly, with no definite trend. This group likewise experienced a sharp fall in heart disease mortality, following upon the influenza pandemic, and a return towards pre-pandemic levels since. This is true for both sexes, but as among whites, the rebound has been sharper among colored males than.among colored females. There is, then, a distinct difference between males and females in the trends of heart disease mortality over the last twenty-two years.  TRENDS IN HEART DISEASE MORTALITY BY COLOR & SEX,AGES I-74  Metropolitan Life insurance Company , Industrial Department     1911-1932 STANDARDIZED STANDARDIZED Bap 00.000. WHITE PERSONS \"Figo 000 COLORED PERSONS 2  sliies YN NHLTTITISAVAN           200 soo HS uy [ y / sof NL i NN / Ti) 2 PTT | \\\\ TTTNUA NU 2 N Book “Al sy WA ZT SLL                                                                                                                                         i911 1915 1920\" 1925 1930 191 1915 1920 i925 1930 YEARS YEARS  CHART 3  Even more significant departures from the general trend are found at the various ages of life. Chart 4 shows the trends by color and sex at ages 1 to 24. The four curves are in general agreement, but distinctly different from the picture shown at all  7       ages. Among these young people, irrespective of color or sex, heart disease rates have declined and this is true both before and since the influenza outbreak. In young persons of both races, the minimal rates have been recorded in recent years. Among white persons, the rates have fallen nearly 50 per cent. The record among the colored is not so good as among whites, but they exhibit some improvement. In contrast to the generally unfavorable picture at all ages combined, therefore, a very healthy situation prevails at the younger ages.  TRENDS IN HEART DISEASE MORTALITY OF CHILDREN AND YOUNG ADULTS Metropolitan Life Insurance Company, Industrial Department                                                                                                                                                                             I911- 1932 BY COLOR & SEX, AGES 1to24 STANDARDIZED STANDARDIZED Be 100,060 WHITE PERSONS PER 1Gd000 COLORED PERSONS 45 45 40 40 35 35 ane ] emales,! | Yh 30 Fe | . 30 ta \\\\ males  - \\\\ J Ke N 25 NZ ic t 25 oe 4 ra  YS x J N \\\\ i\\\\  ANN ) LAIN A 20 \\\\ Be 20 \\\\/ AWA Ae =, v males] | SEAL UY TIM IM NN 10 10 5 5 0 OL! 1911 1915 1920 1925 1930 1911 1915 1920 1925 1930 YEARS YEARS  CHART 4  The changes in the heart disease picture in the main working ages of life—between 25 and 44—are brought out in Chart 5. Among white males at these ages, there has been a rather steady decline in heart disease mortality up to and including 1918; a precipitate fall during the next two years to a minimum level which was maintained through 1922; then a rebound back to the 1919 level around which the deathrate has fluctuated with very  8 moderate variations. Despite this sharp rebound, heart disease rates in these working ages have remained approximately 25 per cent below the pre-influenza level. Among white females, the heart disease deathrate prior to the pandemic also showed a down- ward tendency. The havoc which the epidemic played with pregnant women with heart disease is shown by an abrupt increase in heart disease rates in 1918. The next year, however, showed a fall in the rate to a lower level than in any previous year, and since that time the heart disease rate among women between ages 25 and 44 has shown, if anything, a downward tendency. Among the colored, the situation is somewhat more mixed. Colored males in this important period of life have suffered a sharp increase in mortality so that the rates in some recent years have approached  TRENDS IN HEART DISEASE MORTALITY IN THE MAIN WORKING PERIOD OF LIFE Metropolitan Life Insurance Company, Industrial Department                                                                                                                                                                 1911-1932 BY COLOR & SEX,AGES 25 to44 STANDARDI. PER ens WHITE PERSONS ren gate COLORED PERSONS | 140 140 A\\\\ / V ANALY Males J r g) i VY Females IN mls, 4 os N 120 120 NCU ‘ \\\\V \\\\L Hi \\\\ 100 100 = MN 80 NN 80 i Males tok j 60 \\\\ q SI = 60 N, ENA er | 1] f Females eggs ST NAN 40 chee 40 La | Rei eA tS LALIT PAC) Pt tO ASN Ol) ot er i911 915 1920 1925 i930 1911 915 1920 1925 1930 YEARS YEARS  CHART 5  the levels obtaining fifteen years ago. The trend has been slightly more favorable among young colored women. On the whole, then, the situation as regards heart disease in the important productive years is far better today than two decades ago and as in childhood  9       and adolescence, contrasts sharply with the unfavorable condition at all ages combined.  It is obvious from the foregoing that the increasing deathrate from heart disease must be a phenomenon of middle life and of old age. Chart 6 shows the deathrates in the broad age group, 45 to 74, by color and sex. Among insured white men at these ages, the deathrate from 1911 to 1918 is almost a straight line, except for a dip in the rate in 1915, and a moderate rise in 1917. Follow- ing 1918 there was a sharp fall, and the low level attained in 1919 was maintained for the next two years, since when the rate has risen sharply. In most recent years, the rate has exceeded the level of 1911 to 1918. Among white females at these ages, also,  TRENDS IN HEART DISEASE MORTALITY OF MIDDLE LIFE AND OLD AGE Metropolitan Life Insurance Company, Industrial Department                                                                                                                                                              1911-1932 BY COLOR & SEX, AGES 45 to74 STANDARDIZED STANDARDIZED Pe ioe obo WHITE PERSONS PER 193.308 COLORED PERSONS 00 N : /\\\\ 900 900 \\\\ /\\\\ / \\\\ y soe | aes | \\\\ 800 + 800 An ! 4 Wn A \\\\ K l LJ a, i NK Males 2 Vv 1 Vag 700 7 N / NL 700 f + fi! PTT / \\\\ / NV Females ~/ N\\\\ i MLA / 600;—- / N 600 iS Pade 7 rs \\\\ / - \\\\ ALAS WW an / Y Ne 500 , 7G WAY iy 500 ne | 400 aee8 +HHHH | | 400 Noe c ee eee ee OCC Cee 1911 1915 1920 1925 1930 1911 1915 1920 1925 1930 YEARS YEARS  CHART 6  the trend from 1911 to 1918 was practically level. After 1918, the rate fell to new low levels, but since 1921 has experienced a rebound; not so sharp, however, as among males. In this broad age group, the white female rate has remained slightly below the level of the early years of this experience. Among the colored, the situation  10  SF resembles that among white males. Indeed, the increases in their - deathrates from heart disease since 1921 have been so large that, within the past five years, the rates have attained a level higher than that of fifteen to twenty years ago.  If we dissect this broad age group further, we find that the heart disease situation grows steadily worse with advancing age. Thus, as Chart 7 shows, among white men between ages 45 and 4, the current rates are still below those prevailing prior to 1918. At all ages past 55, however, the rates have not only been increas- ing, but have attained a level appreciably in excess of those pre- vailing in the earlier years. Among white women between 45 and 54, the rate has been remarkably stable, and has displayed  DETAILED TRENDS IN ORGANIC HEART DISEASE MORTALITY AFTER AGE 45  Metropolitan Life Insurance Company , Industrial Department I9t} - 1932  WHITE MALES WHITE FEMALES  750  650  550  450  350  250     150 OR ee eee Oe oe ee ee 1911 915 1920 1925 1930 — 91 1915 (920 1925 1930 YEARS YEARS  CHART 7     little, if any, tendency to increase. Between 55 and 64, despite a perceptible upward trend since 1919, the mortality in recent years has not reached the pre-influenza level. Only at ages past 65 have recent deathrates among white women exceeded those of earlier years.  1l       This chart does not include curves for ages 75 and over because, as was stated earlier, the insurance experience contains too few persons at these older ages to give reliable deathrates. Official statistics indicate, however, that the rise in the deathrate at these advanced ages has gone on at a more rapid pace than for any other period of life. This is most significant because nearly one-third of all the deaths from heart disease occur after age 75.  Among older negroes, the situation is worse than among white persons. The detailed rates for them in these ten-year age groups fluctuate so widely from year to year, that it is difficult to make any clear-cut generalizations. Among colored men, however, even beginning with age 45, the deathrates in recent years have fre- quently attained the level of two decades ago, and in negroes of both sexes past 55, the rates in the past five years have been equal to, or even exceeded those of the pre-influenza years.  Summarized briefly, then, the heart disease mortality situation is amixed one. Certain aspects are decidedly favorable. In child- hood and in early adult life, heart disease mortality, in the aggre- gate, has been falling and the rates are now at minimal levels. In white women, this improvement extends well into middle life. Only in old age is the increase in heart disease mortality really serious. Among colored persons, the improvement in the situation has been much less substantial than among whites.  IV  The question has been raised by many as to whether the rise in heart disease, even in later life, is really authentic. They assert that the change in frequency of heart disease as a cause of death is very largely the result either of changes in the statistical methods of classifying deaths where two or more causes are stated by the physician on the official certificate; of the decline in the number of deaths assigned to senility and of the increased propensity of physicians to specify heart disease as the cause of death where it is accompanied by so-called degenerative changes in the kidneys and arteries. An example of this attitude toward the situation is the attempt of the Bolduans? to show that there has been no increase whatever in heart disease in New York City. To demon- strate this thesis, they bring together all the diseases of the heart— including the acute bacterial types—apoplexy, arterial disease,  12 nephritis and half the deaths assigned to senility. At all ages combined, the mortality from this aggregate of conditions has not increased for New York City, but some rise is recorded at ages 65 and over, and, in the last decade, between ages 45 and 65. There is, in some degree, a medical basis for the assumptions underlying this reasoning, but the case for it is not proved by the purely arithmetical procedure of adding up the deaths from this varied list of causes. The extent of the changes in heart disease mortality is too great to be explained away in this manner. Moreover, the trend from this composite of diseases has certainly less validity than the carefully compiled statistics on heart disease alone. It is not convincing for the additional reason that, among men past 45, there has been an increase in the deathrate from all causes some- what parallel to that in heart disease. In older females, where the increase in heart disease occurs only at advanced ages, the total deathrate during old age has maintained a fairly constant level. During this period there has been a decline of considerable pro- portions in the mortality of old people from infections, notably the pneumonias and tuberculosis. These declines would have resulted in an appreciable fall in the total deathrate, but were counteracted by an increase in the deathrate from some other cause or causes. It is logical then to assume that at least part of the recorded rise in heart disease is authentic.  Vv  What do these varying and contrasting trends mean? Why should deathrates from heart disease in early life go down and the rates in later life increase? The reasons for this diversity in trend must be sought in a consideration of the causes and types of heart disease which prevail in the main age periods of life. For heart disease is not a single entity. It is a complex of many factors, etiological and pathological. Not only are there many recognized types of heart defects, but some of them have the same origins, others are of different origins. Moreover, in the same diseased heart there may coexist two or more types of structural change which may or may not be of the same etiological origin.  It will clarify our discussion considerably if we review rapidly the causes of heart disease and their relation to structural changes in the heart. Unfortunately, careful study on a statistical basis  13       of the frequency of the various etiological types of heart disease has been long in getting started, and only in recent years have really reliable facts been collected. Even this limited material must be handled with caution because often the groups studied are selected from one point of view or another, and due regard is not given to the differences in age composition between various groups studied. The facts given in the literature must be taken as tentative, and not as final. For them we are indebted to the investigations of Cohn and his co-workers, 56 in New York, White,’ in Boston, and others.8-6 Acute rheumatic fever is found to be responsible for about 30 per cent of all cases of heart disease in and around New York. Under age 20, however, four out of five cases of heart disease have a rheumatic origin, and indeed, if we exclude cases of unknown etiology, nine out of ten cases. In the next decade of life, two-thirds of all cases have a rheumatic back- ground; in the fourth decade more than half, and in the fifth, one- fourth of male heart cases and one-third of female cases. After age 50, rheumatic infection plays a relatively small part in the causation of heart diseases.  Syphilis is responsible for about one out of twenty cases of heart disease. But this cause shows very sharp differentials in the various sex and age groups. Under age 30, it is relatively uncommon as a factor in deaths except for a negligible proportion of children with congenital syphilis. Among females, on the whole, it is also relatively uncommon except between ages 40 and 50, when it reaches a maximum of 10 per cent of all heart disease cases. The problem of syphilitic heart disease is largely concentrated in men. Between ages 30 and 40, about 10 per cent of the cases of heart disease have this origin; between 40 and 60, about 20 per cent; and after 60, about 10 per cent.  By far the largest proportion of heart disease is of the senescent or arteriosclerotic type. Such cases constitute about one-half of the total at all ages combined. They include primarily those suffering arterial changes in the heart, and the rarer cases in which the degenerative change in the heart muscle is not associated with the arteries. Arteriosclerotic heart disease is found very infre- quently before age 40, but after that age constitutes a large and increasing proportion. Between 40 and 50, about one-sixth of the cases are of this type; between 50 and 60, about one-half; but after age 60 about three-fourths.  14 Other causes, in the aggregate, account for a sizeable proportion of heart disease, but no single one of them stands out, except hyperthyroidism, which, in women between 30 and 50, is a rather frequent cause of heart symptoms.  The proportions just cited relate to conditions in the north- eastern section of the country. It will be realized without going into detail that they vary from place to place. For example, in the south, the frequency of the rheumatic type of heart disease is much less than in the north. On the other hand, there, and in many communities with large negro populations, the syphilitic types are more common. It should be noted also that the proportion of cases of undetermined etiology differs so widely from place to place and from study to study that they tend to make exact comparison difficult.  Next let us review briefly the diseases of the heart structures arising from these various causes. Endocarditis or inflammation of the internal serous membrane of the heart is caused chiefly by diseases of the infectious type; rheumatic, streptococcicor syphilitic. Disease of the heart valves is frequently caused by these infections but often it is the result of atheromatous or sclerotic changes. Mitral valve disease is chiefly but not exclusively of rheumatic origin. This type of heart disease is most common in youth and middle age, and particularly affects females, among whom the incidence is probably 50 per cent greater than in males. Aortic valve disease is also of infectious origin, but the offending organism is more frequently the spirochete of syphilis. Aortic disease is most common in middle life and affects males more frequently than females, the ratio being roughly three to one.  Myocardial disease is by far the most important type of heart trouble. Its principal manifestation is an increase in the size of the heart. The cause of this cardiac enlargement, whether by hypertrophy or dilatation or both, is heart strain, the chief factors of which are ascribed, on the one hand, to hypertension and, on the other, to valvular disease. The latter is, as we have seen, chiefly of rheumatic origin. But hypertensive cases greatly out-number those with a valvular background. Degeneration and fibrosis, the other i", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cajv.fs4d-nqqt", "00000000-0000-0000-B02B-8C9AF468CA67", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "The Problem of Heart Disease", "9918573882206676X141", null, "1927", "1927", "Louis Dublin, The Problem of Heart Disease. Harper's Monthly Magazine, January 1927.", "Articles", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "11", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "(ste  The Problem of Heart Disease  Reprinted from Harper’s Monthly Magazine Janoary, 1927  . BY LOUIS I. DUBLIN, Pu. D., Statistician Metropolitan Life Insurance Company, New York 1926    eae eile’,  Th       VEVOS  THE PROBLEM OF HEART DISEASE  BY LOUIS I. DUBLIN  time as we count human affairs;  yet, in this brief space, a revolu- tion has occurred in the hazards to life and health. The change has passed almost unnoticed. The discoveries of modern medicine and the development of the public-health movement have brought one infectious disease after an- other under control. The ravages of typhoid fever, yellow fever, cholera, diphtheria, scarlet fever, and infantile  [Mine 28 we co years is a short  enteritis have been checked and are now, ©  in the most advanced countries at least, well on the road to extinction. Tuber- culosis, which a quarter of a century ago was the leader among the destroyers of life, has been reduced more than fifty per cent. It is now relegated to fifth place in the list of causes of death. Asa result of this progress, more than ten years have been added, in this generation, to the life expectation of the whole people. A new order of vigor prevails to-day in the civilized world. Men are able to live and work more freely, unhampered by the fear of premature death. Life is safer and easier and, for that very reason, is economically more productive. In the struggle to overcome the bacterial ene- mies of mankind the outlook has never been more promising.  These victories have brought into relief hazards of another sort. The con- trol over the external infections has re- sulted in a greater interest in the defects of our internal organism. To-day the emphasis is shifting from the infectious diseases of youth to the degenerative conditions of middle life, such as heart disease, the hardening of the arteries,  Bright’s disease, the nervous disorders, and cancer. There is good evidence that these conditions, taken together, are increasing. It is entirely possible that the way we now live has a good deal  to do with the situation. The crowding  of immense populations into the cities, their intense and noisy activity, the drive for money: and for the excite- ments which money supplies are certainly not conducive to orderly and repose- ful living. Leading psychiatrists have stressed the possibility that the new ways are injurious to our nervous organization, and some of them believe that they may predispose to the degenerative diseases. But we really know very little in regard to this relationship. Much remains to be discovered in this field of medicine. In any case, it is time that the public in- terest should be directed to what is hap- pening. The present trend of the more important of this group of diseases should be more generally appreciated. This is a first requisite to any steps that may be taken to check their effects. Heart disease is now the first in the list of causes of death. It is also first in the amount of damage it does through producing disability and invalidism. In the United States the deaths of nearly two hundred thousand persons are as- cribed to it each year. If the present situation should continue unchecked, one in every five of the population now living will eventually succumb to this disease. At age 10 one is three times as likely to die eventually from it as from tuberculosis. A man at 30 is four times as likely to die of heart disease as from tuberculosis, and for a woman the prob-  Copyright, 1926, by Harper & Brothers        ability at that age is more than five times as great. If these deaths from heart disease were limited to old people, one could hardly complain; for this mode of exit is as good as any for those who have rounded out their life work. But, as we shall see more fully later, the deaths from heart disease are not limited to the aged. There are all too many young and middle-aged victims whose premature death is often preceded by long periods of disability. For every death from heart disease that occurs annually, there are probably ten persons living impaired and deficient lives because of the break- down of heart function. Altogether, two million people in the United States,  or about two per cent of the entire popu- |  lation, suffer from some form of heart trouble, many of whom cannot engage in productive activities and are, to a greater or less degree, a drain on the resources of other people. Proper medical and nursing care of such patients is expen- sive. There is to-day no more fruitful source of impoverishment and family breakdown. The annual reports of our charitable organizations show strikingly the prominence of heart disease as a primary cause of dependence. It has more than replaced tuberculosis, which is being relegated to a position of decreasing importance. The economic loss to the community from heart disease is enormous. I consider the problem involved an outstanding one in contem- porary medicine. ,° Il  In discussing heart diseases we must remember that they are not the result of one, but. of a combination of many fac- tors and related disorders. The heart is a complicated organ. Essentially, it is a muscular pump composed of four cham- bers and their incoming and outgoing blood vessels. The action of these cham- bers is co-ordinated and controlled by an intricate nervous mechanism. The chambers are paired into a right half and a left half. The upper chamber on each side is called the auricle; the lower, the  \\\\.. HARPER’S MONTHLY ‘MAGAZINE  ventricle. Each auricle is separated from its ventricle by a muscular valve which permits the flow of blood down- ward but prevents the leakage of blood backward. Venous blood arriving from all parts of the body in the right auricle passes from the auricle through the valve into the right ventricle. It is then pumped through the pulmonary arteries to the lungs where it is aerated. The blood then returns to the left auricle, passes down through the valve on that side into the left ventricle, whence it is pumped out through the aorta to be dis- tributed to all parts of the body.  This complicated pumping mechan- ism may get out of order in many ways. A group of very common heart disorders arise from difficulties outside of the heart itself. These are the so-called functional cases. The nervous mechanism which regulates the heart action may be tem- porarily and slightly deranged, producing heart symptoms without causing struc- tural changes in the heart. Emotional states and severe bodily exercise will increase the demands on the circulation and overstrain the heart. The symp- toms in such cases are sometimes very distressing but usually are of no very great importance. If they are allowed to continue for long periods of time, how- ever, these functional disorders may result in permanent heart impairment. The cases of so-called organic heart dis- ease, on the other hand, reflect condi- tions within the heart itself, and are much more serious in character. Dis- ease of the heart valves is lamentably common, usually the aftermath or an ac- companiment of disease elsewhere in the body, such as scarlet fever, serious dental infection, tonsillitis, or acute rheumatic fever, more rarely syphilis. It is esti- mated that acute rheumatic fever alone is responsible for twenty-five per cent of all cases of heart disease, and that syphi- lis accounts for ten per cent more. Rheumatic heart disease is usually acute, occurs in early life, and more often affects the valve between the auricle and ventri- cle on the left side. Syphilitic heart dis-          THE PROBLEM  ease, on the other hand, is much Anore chronic in character, occurs amofig per- sons between 40 and 60 years ofage and affects the aorta or the aoftic valve, which is on the right side gf the heart. The affected valves in such cases become scarred and deformed and no longer func- tion perfectly. The inflow of blood from one chamber to another may be impeded, or an incompletely closed valve may permit blood once through to leak back into the chamber whence it came. Obviously, to meet the body’s needs, the heart must increase its labors under such conditions and, ultimately, the muscle will become exhausted. Frequently, val- vular disease is accompanied by certain sounds called “heart murmurs.”  The most common form of heart trouble is associated with hardening of the arteries or with elevated blood pres- sure. Forty per cent of all heart cases are of this type. The arteries are nor- mally elastic tubes, expanding and con- tracting, and thus aid in the distribution of the blood. As they become hardened, there is an increased resistance to blood flow and, as a consequence, more work for the heart. The hardening of the arteries may take place in the muscular walls of the heart itself, thus impairing its nutrition. Bright’s disease, in much the same manner as arteriosclerosis— perhaps together with it—may increase the load upon the heart and predispose to ultimate disaster. The enlargement and the degeneration of the heart muscle are the physical signs of this type of disease. The valves are usually not involved at first. The disease is chronic in character and occurs, for the most part, in persons after age 50. A varia- tion of this class of case.is that found in old age. The senescent heart, even without any local disease process in the muscle or valves, is not able to function properly. The origin of about ten per cent of the cases of heart disease is still unknown. In the remaining fifteen per cent of the cases the cause may be one of a number not so easily classified. They include such cases as result from certain  F HEART DISEASE 3  excesses, such as alcoholism, or from an attack of goiter, and from a number of other primary disorders.  These are some of the commoner ways which lead to cardiac failure. The trained physician can usually determine  whether there is any considerable en-  largement of the heart muscle by the physical examination of the chest, supplemented by means of fluoroscopic or x-ray pictures, and can determine the particular lesion by the unusual sounds or murmurs heard in the heart as the blood flows through from chamber to chamber. The subjective symptoms which the patient brings to the attention  of the physician are of the greatest im-  portance in leading to a diagnosis. By means of an instrument of precision, the electrocardiograph, the heart specialist is able to diagnose more accurately still the particular defect which exists, even in its very early stages. Finally, by the actual test of the heart’s ability to tolerate standard types of exercise, the skilled physician is able to determine the amount of the heart’s disability. The first question for the vital statisti- cian to answer is whether heart disease as a cause of disability and death is now actually increasing or decreasing. We  must examine the crude death rates over —  a period of years. In 1900, when the earliest reliable figures for the U. 5S. Death Registration Area became avail- able, the rate was 111.2 deaths per 100,000 of population. This means that one-tenth of one per cent of the total population died from this cause during the course of a year. Between 1900 and 1910 there was a continuous rise in the rates, the figure for 1910 being 141.5 per 100,000. In the decade beginning with 1910 the rates were first fairly stationary and then rose regularly up to the war and influenza years, 1917 and 1918, when figures above 153 per 100,000 were reached. Following the influenza epi- demic, the three years 1919, 1920, and 1921 were years of comparatively low rates. There is every indication that this decline was, in large measure, the  ws,    4 HARPER’S  MONTHLY MAGAZINE     Deathrate per 100000  Deathrates per 100,000 population. Organic Diseases of the Heart United States Registration Area, 1900 to 1924     160     Lat \\\\\\\\/     I AA                                   / Ye 1Z 10 7 \"= eens UD a . a1 4. 4. 4 i at 4 1 4. | i 1 t 1 | 4 1 1 1 1 1 1 | 00 03 #30 1938. 1920 1924              result of the epidemic itself. Physicians  everywhere noted that the influenza was very often fatal to those who had been suffering from serious heart affections. Many died of influenza in 1918 and 1919 who, in the ordinary course of events, would have died two or three years later from their heart disease. Since 1922, the rate has been rising slightly each year, bringing the figures back to what they were immediately before the in- fluenza period and even beyond.  The general impression given by these figures and which is confirmed by the graph, is one of increasing heart disease incidence during the last twenty-five years. But this rough method of anal- ysis is subject to very decided limita- tions. Heart disease is pre-eminently a condition of the older ages of life, and it may well be that such increase in the number of deaths as has occurred has resulted not so much from an increase in the hazard from heart disease as from the shifting of the age distribution of the population. Inrecent years more people have reached the older ages when heart disease is more likely to occur. The  gradual reduction in the death rate from such diseases as tuberculosis, pneumonia, and the other infections has transferred many additional persons to the later age groups. It is then that heart disease is most frequent. Under conditions of twenty or thirty years ago, many of these people would have died in early life. To-day they survive to middle life and old age and then are much more likely to develop a heart defect and to die of it. Our first question, accordingly, resolves itself into a second, namely: “Is the hazard of dying from heart disease actu- ally increasing at the various ages of life as well as during the aggregate life span?” This question can be answered only by reference to certain figures which we call age-specific death rates. These tell us the probability of dying from heart disease during the specific age periods in question.  A comparison of the age-specific death rates of 1920 with those for 1900 shows at once a number of interesting items. Among young children under 5, the rates declined considerably during this twenty- year interval. Between ages 5 and 45 THE PROBLEM OF HEART DISEASE                                                                                Deathrates per 100000. Organic Diseases of the Heart In States Comprising Original Registration States in 1900. 1920 ond 1900. Oeathrate . qo per 100,000 4000 3500 ommmmmn 19 20 3000 awe |D00 | 2500 / 2000 / Z §§00 o aes 1900 vA 7 Y a“ Pot 500 7 goon\" nok 1 510 toro = 151 2010 2510 3510 45 10 $510 6510 75 AND 4 ) 4 9 24 34 “4 34 oA cc) OVER        the specific death rates remained un-  changed. Conditions, to be sure, varied somewhat from year to year, but taken altogether, the disease was in statu quo over this period of life. One in every seven deaths from heart disease still  takes place among persons under age 45. »  It may be interesting at this point to con- trast the contour of the age curve for heart disease with that for tuberculosis. The latter disease under present condi- tions shows its maximum at or about the age of 25 and then declines with ad- vancing years. The curve for heart dis- ease crosses that for tuberculosis at about age 45 and then mounts to its huge maxi- mum at the oldest ages. After age 45 the rates were uniformly higher in 1920 than in 1900 and the excess of 1920 over 1900 is greater with each advancing age period. After age 75 the 1920 fig- ures were 114.8 per cent higher than in 1900. Among both men and women the increase in the total rate in recent years has been the result of the marked changes in the death rates at the older ages, be- ginning at forty-five or fifty years. Not only do more people die from heart dis-  ease because more arrive at the older ages, but also because there is an in- creasing hazard of dying from heart disease in the later ages of life.  Til  It should be of some interest, then, to determine under conditions as they now exist what the probability of dying from heart disease actually is. The real ques- tion is, “What is the chance that an ordi-— nary man or woman will die eventually from heart disease?”’—and this question cannot be answered as easily as it seems. However, calculations have been made, and the reader may be interested in the underlying principle of the method used. We begin with a group of 100,000 per- sons at a fixed age, let us say age 10. We keep in touch with these people, recording their deaths as they occur until all have passed away. We note also in each case what was the particular cause of the death. We thus learn how many persons in the group die at each year of age after 10. If we sum up the number of deaths from heart disease             SSS a                                                                                   6 HARPER’S MONTHLY MAGAZINE Deathtates per 100,000. Tuberculosis 6 Heart Disease Compdred, in the Industrial Department of the come;  “Ctropolitan Life Insurance Company ~ 1923 ce PER 300TH » : a OO”  i : i 250 } 250 u : i 200 200 / = 150 ra me 150 z, / J y | & st 100 / 3 100, y 50 rai 50 ee Pa ; a cs ‘ 2k, ik lea 21h chk 274. 323 57k 428 479 _S2s 573 G2 678 723 Woe CENTRAL AGES =        Bee Pathe + ae re     from age 10 onward to the end of the life table and divide by the total number of persons in the group, we obtain the measure of the probability of dying from heart disease. For example, if we began with 100,000 boys at age 10, we should find that ultimately there would be a total of 19,218 deaths from heart disease before the entire group had passed out of life. This means that for a boy 10 years of age there are 19 chances of dying eventually from heart disease as against 81 chances of dying from some other cause. In the same way, the probability of dying from heart disease can be calculated at any other age. We find, for example, that for this particular disease the probability of dying eventu- ally from it increases slightly from age 10 onward. At fifty the probability is 22 per cent; at 70 it is 24 per cent. In other words, virtually one out of every four persons at age 70 will die from this cause. The figures for girls are approxi- mately the same. The probability of dying eventually from heart disease is one in five at age 10, and a little less than one in four at age 70. It will now be possible to see what has actually hap- pened in the last fifteen years; for in 1910  the probability at age 10 of dying even- tually from heart disease was only 15 ina hundred. In other words, there has been an increase in the probability of 31 per cent during this short interval. At age 50 the probability of dying ulti- mately of heart disease in 1924 was 26 per cent higher than in 1910, and at 70 the increase was 26 per cent. These figures better than any at our disposal indicate how great is the budget of heart- disease mortality at present. No other disease or condition shares its primary position when studied from this angle. Another interesting statistical com- parison relates to the incidence of heart disease in the several races that compose the American population. The most striking difference is shown by the white and colored’ races. During the main age-periods of life the rates for colored people are about twice those for whites at the corresponding ages. It has been suggested that the higher prevalence of such diseases as malaria, typhoid fever, and especially of syphilis, plays an im- portant part in creating this excess of heart disease among the colored people. The lower standards of personal hygiene and the more difficult conditions of life of THE PROBLEM OF HEART DISEASE 7  this race are clearly important items. There are also marked differences in the amount of heart disease prevailing among the various nationalities that make up the white population in our country. Those of foreign birth divide into two groups: those with a low and those with a high incidence of heart dis- ease. Those of native birth are inter- mediate between the two foreign groups. The favorable foreign stocks include those born in the former Austro-Hun- garian Empire, the Russians (mostly Jews), and the Italians. These are the newer immigrants of eastern and south- ern Europe. The stocks with high heart disease rates include those born in Eng- land, Scotland, and Wales, Germany and Ireland. The figures for the Irish are particularly high, recalling those for the colored people rather than those for any white group. Why this should be so it is difficult to say. No such unfavorable conditions occur among the Irish people in the old country. But they are uni- formly bad among the Irish in America. It will be an interesting study in future years to determine whether the descend- ants of Irish immigrants are able in their own generation to overcome this handi- cap of their parents and grandparents and to approximate the better condi- tions prevailing among Americans of other racial origins.  But, irrespective of race or group, the figures for heart disease are as engrossing as any we can present to-day. No other _ disease in the entire field of medicine, with the possible exception of cancer, offers so large an opportunity for life- saving service. It is too true, however, that the medical profession and their allies, the public-health workers, have only recently become concerned over the hazards of heart disease and have taken the public into their confidence in devel- oping a program of relief. What can, in fact, be done in the present state of our knowledge to bring this condition under some control? The answer must in the nature of the case be somewhat cautious and conservative. The campaign is at  its very inception. We shall be better able to define the issues and to move more quickly as the program is tried out. The experience that the public-health workers have gathered in their attack on other conditions, especially tuberculosis, will prove of great service in the cam- paign against heart disease. At the very outset two lines of attack present them- selves. The first affects the individual; the second concerns the community as a whole. Each has its proper place. No great headway will be made until the two efforts are thoroughly co-ordinated and put into operation with a full measure of public support.  The campaign as it concerns the in- dividual redutes itself largely to one of personal hygiene. We must, all of us, so live as to avoid heart impairments. More definitely, this means that children must be protected against the infections which often bring heart disease in their train. Unfortunately, we do not yet know the true cause of rheumatic fever, an arch enemy of childhood. But we can prevent and give adequate con- valescent care to many cases of tonsil- litis and sore throat which appear often to bring on rheumatic complications. The prevention of cases of typhoid fever, of diphtheria, and of scarlet fever through specific inoculation of children against them will cut down a proportion of heart sequele. Among young adults the reduction of syphilis will have its effect in fewer heart cases later in life. Saner and more temperate living on the part of all, with due regard to rest and exercise, will result in healthier bodies better prepared to avoid heart impair- ments. But probably the most potent of all personal efforts will be the devel- opment of the habit of the annual medi- cal examination. The periodic health examination is the key to our problem so far as the individual is concerned. This rule applies equally to children as to adults. It is a striking fact that most persons who have heart disease have dis- covered this in the most accidental and casual fashion. Usually the cases are       8 HARPER’S  found as the result of a life-insurance examination or through the health examination of children at school, or by industrial physicians in examining ap- plicants for employment. Usually be- tween one and two per cent of all such examinations result in the discovery of unmistakable heart disease. The great majority of those who are found to have a heart lesion of one kind or another have not been aware of it. On the other hand, those who believe they have heart dis- ease and go to doctors to have their suspicions confirmed are usually in error. I have no doubt at all that if periodic health examinations became the rule in the United States tens of thousands of persons would be discovered every year with early cardiac defects when it would still be possible to remedy the situation. Changes of habit or of occupation are often sufficient to keep an early lesion under control, certainly for long periods of time.  The contribution the community can make is likewise many sided. The first step is to spread the knowledge of right living; to inculcate good habits of life through the agency of the schools and the other channels of public education. The public schools are in a particularly strategic position. They can discover the large group of children who are pre- disposed to heart disease and can provide for them a special regimen of instruction and of care which will serve to protect them from further deterioration of their heart structure. As in the successful campaign against tuberculosis, the or- ganized health agencies of states and cities must provide heart clinics with skilled examiners and adequate equip- ment to help discover and treat cases of this disease, especially among adults. New York City, stimulated by the wise counsel of the Heart Committee of the New York Tuberculosis and Health Association, has taken the lead in such activities. There are now forty-seven heart clinics distributed over the city. It is not at all surprising that these are crowded, with an ever-increasing num-  MONTHLY MAGAZINE  ber of patients. In the year 1925 there were about twelve thousand active patients under care in these clinics of New York City, fully as many as there - were receiving care in the much older tuberculosis clinics of the city. Young people for whose relief there is much hope constitute a large number of the patients, as might be expected. The clinics serve as centers of supervision and instruction and as exchanges for the dis- tribution of patients to those agencies that are best adapted to keep them.  Sanatoria and convalescent homes are especially necessary in the attack on heart disease. For in this disease there are periods of acute outbreaks when the patient must receive intensive care and complete rest before he can be released. to his family or his work. And, what is equally important, suitable work must be found for such patients if they are not to slip and become helpless cripples. Only the organized agencies of the com- munity can provide such service. For- tunately, most American cities are find- ing themselves in a position where they can provide such care without involving themselves in great initial expense. General hospitals with their out-patient departments and their staff of trained social workers are ordinarily well adapted for this type of service. There is also the possibility of utilizing vacant beds or even whole wings of tuberculosis sana- toria now more or less unoccupied. Actual experience has shown in several well-managed tuberculosis hospitals that the type of care and the equipment necessary for the conduct of tubercu- losis sanatoria are adapted also for the care of heart patients. There is nothing inherently difficult or dangerous in the undertaking, and it should be entirely possible as the tuberculosis death-rate declines to shift the facilities of organized health agencies, both public and private, to the care of incipient and moderately advanced cases of heart disease.  The life insurance companies occupy a particularly favorable position in the campaign we are sketching. The large THE PROBLEM OF HEART DISEASE 9  number of examinations made annually by insurance medical examiners discover many new cases. A number of com- panies offer free annual examinations to their policyholders and these, too, result in bringing many cases to light. The value of this service can hardly be over- estimated. A few of the larger and more powerful companies, because they pay out millions of dollars in death claims each year on account of heart disease, are especially active in spreading hygi- enic information broadcast among their policyholders and the general public. But what is the attitude of the com- panies toward those who on examina- tion are found to have a heart defect? Do they insure such persons and, if they do, what has been their experience? On the whole, the companies have been rather conservative. Lacking informa- tion as to the gravity of most heart de- fects, they have permitted themselves to insure limited numbers and those only who had the least dangerous lesions. The experience has been concentrated in afew companies. Taken altogether, the mortality rate has been found to be about two and a half times as high as that which prevails among normal people ac- cepted for standard insurance. The cases have been almost altogether lim- ited to those having the hypertensive or arteriosclerotic type of the disease, cases with a slight enlargement, and in which the mitral murmur is not pronounced. These are the cases which Doctor Mackenzie, the great English heart specialist, looked upon very favorably. He felt that the insurance companies could take a great many such people without loss, but experience has shown that he was not correct. Only the least impaired cases of organic heart disease are insurable; the rest require premi- ums so high as to put insurance out of reach of most persons. As infor- mation and experience increase, protec- tion will undoubtedly be offered to larger numbers.  IV  What shall we say as to the future of our problem? The prognosis is neither very clear nor very encouraging. ‘There is good reason to believe that the exten- sion of the program of prevention along the lines we have outlined will save many lives and prevent much suffermg among young people especially. With more effort it should be possible to keep larger numbers of persons with impaired hearts active for longer periods with greater comfort for themselves and less financial strain on their families. There are many people who suffer from chronic heart affections and who break down at or before age sixty. Nothing could be more desirable than to postpone their premature collapse. The possibilities for such service are good. There is, however, much disappointment in store for those in the heart movement who are sanguine of quick results. This enemy of mankind will not be easily vanquished. Wherever we suffer from the results of man’s habits, relief will come slowly. There will always remain a heavy tax payable by way of heart disease. The future will probably show more heart\\", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3wqt~tsa2-atjm", "00000000-0000-0000-FEE8-48632160DA4E", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Heart Disease and Public Health", "9918573882206676X142", null, "1942", "1942", "Citation: Louis Dublin, Heart Disease and Public Health, American Heart Journal 23 (1942): 16-21.", "Articles", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "8", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "HEART DISEASE AND PUBLIC HEALTH  LOUIS I. DUBLIN, PH.D. New York, N. Y.  Reprinted from AMERICAN HEART JOURNAL St. Louis  Vol. 28, No. 1, Pages 16-21, January, 1942 (Printed in the U. S. A.)          HEART DISEASE AND PUBLIC HEALTH CURRENT TRENDS AND PROSPECTS  Louis I. Dusuin,* PH.D. New York, N. Y.  S I am to discuss heart disease and public health, it will avoid mis-  “understanding if at the outset I attempt to define what we mean by public health. It is that branch of medicine which concerns itself with the prevention and control of disease from the point of view of the community as a whole. In contrast with the practicing physician, the health officer has the people of the city, county, state, or nation as his patients, and he utilizes the resources of the community in his ef- fort. We all know that, under modern conditions, the individual patient and the individual doctor are often helpless in combatting the spread of infection, as, for example, in controlling yellow fever, typhoid fever, or malaria. It takes the resources of the state to do that. Even in relation to such diseases as tuberculosis and syphilis, it has become a well-established principle, learned through sad experience, that not only prevention, but treatment, is properly a public health function. The individual patient, in many instances, is unable to provide himself with the care which he needs, and the community is to that extent endangered. The public interest, therefore, demands that the necessary treatment be given, even if at public expense. In all this, there is no real conflict between the public health officer and the individual practicing physician. There are broad areas of mutual interest. The wise health officer will attempt to make all practicing physicians his associates, and all good doctors will look to their health officer for guidance and support in help- ing them with their patients. These general principles are gradually being evolved, although they have not yet been completely crystallized. The functions of the health officer, on the one hand, and of the prac- ticing physician, on the other, are being worked out in the light of ex- perience and of changing conditions, and the public interest, in the last analysis, determines their mutual responsibilities.  What are the most important problems in heart disease, and what is or should be the relationship of the public health officer to them? Heart disease is the outstanding feature of the medical picture, and will be increasingly so in the future. Clearly, the health officer cannot ignore a field so wide in extent and affecting the public health so vitally. Essen-     Presented before the Annual Meeting of the American Heart Association, Cleveland, Ohio, May 30, 1941.  Received for publication June 9, 1941.  *Third Vice-President and Statistician, Metropolitan Life Insurance Company, New York. :       tially, his problems in this field are the same as those of the practicing physician, although his approach and functions are necessarily different. As I have already indicated, the health officer must concern himself with those phases of heart disease in which large-scale prevention or measures of control are attainable. The well-recognized public health procedures are clearly applicable to the control of rheumatic fever and syphilis. The control of premature hypertensive and arteriosclerotie heart disease has to date received scarcely any attention from health officers. Im- perceptibly shading off from the last category is the numerically most important group, which comprises cardiac disease in old people. Toa greater or lesser degree, the health officer, as we shall see, can function to advantage in all of these four phases.  Rheumatic fever is today one of the foremost health problems of child- hood. Between the ages of 5 and 9, deaths from it are outnumbered only by those from the four principal communicable diseases of ehild- hood, as a group, and by pneumonia.* At the ages of 10 to 14, it is the leading cause of death. Between the ages of 15 and 25, it is second only to tuberculosis. Although it is true that the mortality from rheumatic fever has declined, the rate of fall is less than that from other diseases, so that the proportion of deaths from rheumatic fever to the total num- ber of deaths among persons under the age of 25 has increased.  By its very nature, rheumatic fever is a disease in every phase of which the health officer can be of great help, but he has done compara- tively little. He may actively participate in a case-finding program. He can take over or amplify facilities for the treatment and eare of children with the disease. Practically nowhere are these facilities com- mensurate with the needs. His laboratory can function in research in rheumatic fever, the cause of which has thus far eluded scientific in- vestigation, and his records may help to unravel the complex epidemi- ology of the disease. He can provide convalescent and sanatorium care in suitable types of institutions. In certain parts of the country he can make use of facilities built for other purposes, but which are not fully used, particularly the tuberculosis sanatoriums. Furthermore, since non- specific factors seem to be responsible for most of the decline in rheu- matic fever, public health officials can actively promote improvement in certain matters which come under their aegis or in which they have some influence, as, for example, in housing and in popular education in health and nutrition.  In the control of syphilis, the health officer has already contributed a great deal. In fact, the progress that has been made reflects the de- gree to which health officers have been willing and able to take respon- sibility, not only in finding cases but also in treatment. In the Sean- dinavian countries, where syphilis has been largely controlled, it has     *Since this paper was written, mortality from pneumonia has fallen below that for rheumatic fever.  4 been the public health service which has carried the brunt of the work. If in our country some progress in this direction has been made, there is no denying the fact that in the present national emergency, with huge numbers of young men concentrated in camps and in defense industries, and with no correspondingly increased facilities for handling the case load, we may lose the gains of the last ten years. The task of the health officer is manyfold—finding the victims of the disease, and providing adequate facilities for treatment and seeing that they are used. He needs a broad concept of his job and resourcefulness in dealing with it.  The most difficult aspect of the problem in heart disease, both for medicine and for public health, is constituted by the relatively large numbers of persons in the prime of life who fall prey to early and some- times preventable heart and coronary artery diseases of other etiologic types. They affect men chiefly. The root of much of this problem is probably related to the process of aging of the human organism, the study of which has been scarcely begun. Fortunately, there is increas- ing awareness of the necessity for research on the fundamental prob- lems involved, and a number of medical men and other scientists and public agencies are already engaging in this work. Notable is the work being done in one public institution, the Research Unit of New York City’s Hospital for Chronic Diseases, and in several other places. But a greater part of our public medical resources can and should be de- voted to this field.  Apart from this, however—and the wide difference between the death rate of the two sexes is, in my judgment, a good indication—a large number of early deaths from heart disease can be avoided or postponed. Many are probably due to faulty living habits with respect to the routine of daily life, or to lack of attention to infections. Intensive research and popular health education, both of which are important functions of modern public health organizations, can do a great deal in this regard.  For one thing, the health officer can help in the early detection of incipient heart disease by popularizing the periodic medical examina- tion. In this matter a more receptive attitude on the part of the medi- cal profession generally is much to be desired. It is admittedly true that symptoms of early heart disease are often hard, sometimes impos- sible, to detect, but too many patients receive little or no medical super- vision until long after the disease has progressed. It may not be too early for the two professions to consider the desirability of providing public clinies for the periodic medical examination of certain groups of the population that cannot themselves afford such service.  The public health officer should consider in what ways, efficient and economical, he can utilize available medical resources in cases of early heart disease, or in acute attacks of chronic disease. For example, do we not all know of working men and women with cardiac disease who  5    would benefit from relatively short periods of sanatorium care, where prescribed rest and other measures would be carried out under suitable conditions, and where re-education of the patient in his way of life could be carried out better than in the atmosphere of worry and strain of the home and work place? In a few places, the overbuilt sanatorium facilities for tuberculosis, or other hospitals now unutilized, could be devoted to this purpose.  The health officer can make increasing use of the visiting nurse serv- ices under his supervision for chronic cardiac disease and, when such services are lacking, can develop or amplify them. Visiting nurses now operating very widely all over the country are of value not merely in actual bedside care, but in the broader fields of psychologic adjustment of the patient or his family, and in interpreting the doctor’s instruc- tions to them. These phases are often neglected because they take more time than the busy physician can give them. But they can be handled well by visiting nurses, particularly if nurses are given special training.  Old people with cardiovascular disease present a somewhat different set of problems which are much more difficult in character and limited in scope. The chief needs are for additional clinic facilities, for more home care, and for institutional care of the semihospital type. The numbers of aged cardiac patients are already so great and so rapidly increasing that there is urgent need for a long range program for their care. The very nature of the situation is such that a major part of the financial burden must be borne by public funds. Public health officers must recognize their responsibility here and seek the help and guidance of the whole medical profession in developing programs that are con- sonant with the welfare of the patients, the public, and the doctors. We cannot safely add to the burdens of voluntary hospital clinies if they are to continue to do good work. A larger number of public clinics, run by full-time men, is the only desirable solution for impoverished ambulant patients. When such patients are confined at home, public medical service may be necessary, but the work of physicians may be lightened and rendered more efficient by the proper use of visiting nurses and, in selected cases, of medical social workers. Increasing numbers may best be handled in institutions, but these cannot and need not be elaborate and costly hospitals.  Heart disease, then, presents many different tasks, responsibilities, and opportunities for public health officers. But before any large-scale advance can be made, both they and the medical profession as a whole must accept the view that heart disease is a proper field of work for the health officer. They need also to agree on the respective roles of the private and public physician.  In this respect, the problem of heart disease reminds one of the sit- uation which prevailed in tuberculosis about thirty years ago. At that time health officers were just beginning to recognize that the problem  6 of the tuberculous patients was largely one for them to solve. There was considerable hesitation in entering the field, and uncertainty in developing the necessary technique and procedures. They lacked diag- nostic classifications and standards, staffs trained in the special treat- ment of the disease, and sufficient diagnostic and sanatorium facilities for the care of patients.  What a change thirty years has brought about, thanks to the increas- ingly better understanding of the basic factors of the situation by both practicing physicians and the growing profession of health officers. Once it was realized that a disease so widespread, yet everywhere so concen- trated among poor people, was the province of the public authorities, constructive steps, one after another, were taken by health officers and physicians in developing new procedures, with the result that, within a single generation, prodigious advances were made, not only in cut- ting down the ravages of the disease, and in protecting persons against it, but also in developing an understanding of the nature of the disease itself.  I am bold enough today to stress this analogy with reference to heart disease. I appreciate, of course, inherent and fundamental differences between the one disease which stems from an invasion of the body by a bacillus, and the other disease or group of diseases, the bulk of which represents sequelae of many factors, both exogenous and endogenous in nature, in the aging patient. Prevention of tuberculosis did not prove a hard nut to crack once the nature of the problem was understood and properly organized efforts were brought to bear. Prevention of heart disease will be a very different proposition because so large a part of it is the end result of accumulated insults incidental to the functioning of the body. On the other hand, there are enough phases of heart disease in which prevention is possible to excite the health officer with the real job he can do in this field.  But my major interest is the idea that the field of heart disease is fundamentally one in which the health officer can function and bear a major responsibility. There is every reason why the organized official and voluntary public health agencies should participate in the work to be done. Health officers have a tremendous contribution to make in supplementing and strengthening the efforts of the medical profession and in developing appropriate administrative procedures for the prac- tical care of patients. For this disease, the most common in adult life, involves such long periods of disability that the medical costs are far beyond the resources of most of its victims. The very economics of the situation make it impossible for the ordinary relationships of physi- cian and private patient to be fully effective. Thus far this situation has been met by the establishment of free or low-cost outpatient clinics in public and private hospitals, in which much of the service of the physician is rendered without compensation. Patients either cannot be  7       seen sufficiently often or are treated too superficially because of the crowded conditions of most of the clinics. This situation will get worse, rather than better. Obviously, we must think in terms of modifying our present types of organization and medical care for these people.  Leaders in medicine and public health must face frankly a situation as patent as this, and take the necessary steps for integrating the care of those suffering from heart disease into a general scheme, in which the proper share is borne by public funds, under the administration of the health officer, just as has been done in the case of tuberculosis. I have no illusions that this scheme can be developed overnight. The tubereu- losis problem, which was simpler, took decades to get under way toward solution. But the important and first step is the acceptance by physi- cians and health officers of the concept that heart disease is a suitable field for similar exploitation. Then, in good will, all interested parties can get together in planning their respective parts of the job, on ways of effective cooperation, and on the sequence of steps which would be most likely to assure progress and win the support of the community.", "Dublin, Louis I. (Louis Israel), 1882-1969. ; American Heart Association", null, "The American Heart Journal", "Mosby, Inc.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yk33_kuz3-45kv", "00000000-0000-0000-C8D1-BD68335DF94F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "The Incidence of Heart Disease in the Community", "9918573882206676X143", null, "1922", "August 1922", "Louis Dublin, The Incidence of Heart Disease in the Community, The Nation's Health 4 (1922).", "Articles", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "4", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Reprinted from the NATION’s HEALTH, August, 1922, issue, Vol. IV, No. 8  Incidence of Heart Disease in the Community’  Out of Systematic Study and Full Records in Every Case Will Grow Adequate Preventive Methods  By LOUIS I. DUBLIN, PuH.D., STATIsTICAN, METROPOLITAN LIFE INSURANCE COMPANY, NEW York CITY.  DISCUSSION of the incidence a of heart disease in the commu- nity is especially pertinent at  this time in view of the high death rate from this disease that has prevailed during the present year. We in the Metropolitan Life Insurance Company have an advantageous position watch- ing the experience among the many millions of our policyholders. We can see month by month and, in fact, week by week, just what conditions of health prevail in the country at large. Since November, 1921, and continu- ing each month to date, the death rate from heart disease has been ap- preciably higher than it was during the corresponding months of the pre- vious year. The same has been true for the associated organic diseases, such as, Bright’s disease, cerebral hemorrhage, and apoplexy. It must be remembered that the last three years were favorable ones for heart disease  mortality... [he sates fer-4919,-1920,-        The fundamental importance of collecting all pertinent facts in regard to the incidence of heart disease in the community is urged. Uniformity of record, with such details of history and physical findings as will enable comparison, would bring out a mass of data which would just- ify definite prognosis which is now impossible. Effective pre- ventive work on a wide scale must await such a mode of pro- cedure.  Nor should such data, to be ef- fective, be drawn exclusively from mortality records. Morbid- ity records need to be so ex- tended as to point out the in- ciptent case, and the. predispos- ing environmental or constitu- tional condition which led to disabling heart lesions. Our  portant as these two conditions are in affecting the heart disease rate ad- versely. We shall have to watch the developments for the rest of the year very carefully.  These facts are disturbing to all engaged in public health work because the figures for recent years had given some encouragement that a definite  downward tendency had come for  heart disease. It must be remem- bered that the great improvement in the rate for tuberculosis has left heart disease in the first place as the leading cause of death. During the first eight years of the decade, there was hardly any decline in the figures among insured lives. In the general population of the Registration Area, there was an almost continuous in- crease in the mortality from heart disease. But, in the years 1919, 1920, and 1921, the great improvement in the figures already referred to gave     and 1921 were the lowest recorded during the last decade. When the in- crease began in the winter months of 1921, the change attracted little at- tention. The possibility that it ushered in a definite check in the fa- vorable downward tendency for heart disease mortality was not seriously considered. But, by the end of Jan- uary, 1922, there could be no longer any question that a definite upward tendency was in progress. As _ will be seen from the graph, the heart disease death rate increased sharply month by month until in March the rate reached the maximum of 168.2 deaths per 100,000 living, one of the highest figures recorded in recent years among Metropolitan Industrial policyholders. (Chart I)  Obviously, some of this increase for heart disease is the direct result of the influenza epidemic which broke out in the early months of this year. But this cannot be the whole cause because heart disease death rates that were higher than normal prevailed for several months before the influ- enza epidemic and have continued for several months after its close. The experience among insured lives would seem to indicate that we are experi-  *Read before the Boston Association of Cardiac Clinics, Boston, Mass., May 18, 1922.  present statistical studies are sertously inadequate in this re-              gard.        encing in the current year a serious increase in the incidence of heart dis- ease deaths quite apart from the ef- fect of influenza and pneumonia, im-     Chart 1 Death Retes per 100,000 from ORGANIC DISKASES OF HEART In Specified Menthe of 1921 and 1922.  hetropolitan Life Insurance Company, Industrial Departnent,                 Chart I.—The heart disease rate increased sharply month by month until in March the rate reached the maximum of 168.2 deaths per 100,000 living, one of the high- est figures recorded in recent years among Metropolitan industrial policy holders.  cause tor—hepefulness—that-—a new—-sit--——  uation had been inaugurated. Condi- tions at the present time are not very encouraging in this respect and would seem to indicate that the favorable heart disease mortality experience of the last few years was possibly only a temporary one, reflecting in part the result of the influenza epidemic of 1918 and 1919, when a large num- ber of advanced cardiac patients were very likely eliminated, and also the effect of improved economic conditions for the wage working population dur- ing the war. It is a good deal of a question in view of all the facts, as shown in the _ following graph, whether there has been any lasting improvement in heart disease mor- tality during the last decade. This disease is today the chief cause of death. It is likewise a condition of the greatest importance from a public health standpoint. In spite of its leading position in mortality and mor- bidity, there has been virtually no gain in its control; the campaign against heart disease is very much in the same position as that against tuberculosis fifteen or twenty years ago (Chart II).  The incidence of any disease may be studied from two angles: (1) as a cause of sickness and, (2) as a cause of death. In respect to sick-          Chart If     Death Rates per 100,200 from ORGANIC DISSASES OF HRAPT. Experience of the Metropolitan Life Insurance Co., Industrial Tep't., and of the U. 8. Registration Aren (ages 1 tc /4) Compared.  Death Rates 1911 to 1921.  per 100,000                       (ees er  ‘ peabe ssl 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921  , Trend line, 1911 to 1920.           Chart II.—Conditions at present would seem to indicate that the favorable heart disease mortality experience was possibly only a temporary one. All facts considered, it is a question whether there has been any lasting improvement in heart disease mor- tality during the last decade.  ness or morbidity, we have very few facts indeed. There is very little au- thentic information on the preval- ence of various types of heart affec- tions. More recently, some informa- tion has been accumulated on the prev- alence of heart lesions among school children as a result of the work of school medical inspectors. The exam- inations are, however, often made and recorded in a perfunctory manner. The greatest variations appear in the tabulations. It is not always possible to determine whether the figures in- clude functional as well as organic disturbances. The result is the great- est variability, some cities showing an incidence of two per cent of the children affected and others less than one-half of one per cent. On the other hand, careful examinations have been made among certain industrial groups which show a considerable prevalence of heart affections. The findings of Dr. Schereschewsky of the United States Public Health Service among gar- ment workers in New York; of Rob- inson and Wilson among employees in various industries in Cincinnati; and of Harris and Dublin among food handlers in New York, indicate that about two per cent of working adults have organic heart disease of one kind or another. The army medical ex- aminers in connection with the draft and camp examinations rejected about four and a half per cent of those ex- amined because of heart defects. The figures will, of course, vary consid- erably with the group of persons ex- amined, the severity of their occu- pations and the age of the men ex-  amined. But, altogether, the evidence would seem to indicate that at least two per cent of the population show cardiac defects on examination. This means that in the population of the United States, more than two million persons of all ages suffer from serious heart impairments. This fact alone indicates the magnitude of this par-  ticular health problem, and what pro-.  vision we must expect to make in the next decade to provide for the discov- ery and the care of these persons. The statistics of heart disease mor- tality are more satisfactory, both in point of areas covered, of detail as  ' to color, sex and age, and of diagnos-  tic accuracy. Deaths are reported, even if the cause is not always stated absolutely clearly, especially in con- ditions where heart disease is asso- ciated with disturbances of the vas- cular and renal systems. Valuable use may be made of the figures, how- ever, both in the reports of the Reg- istration Area, and more especially for persons insured in the larger com- panies. ° 1. am- in ~a - ‘position. to present the figures for the ten-year period 1911 to 1920 among the many millions of persons insured in the In- dustrial Department of the Metro- politan and to show the incidence of heart disease as a cause of death at the various age periods of life, in each one of the main classes of this group, that is, among the white males, the white females, the colored males, and colored females.  Before we proceed, it is well to  explain just how we measure the mor- tality from the several diseases. The index, or death rate, is the number of deaths from any disease during a calendar year for each 100,000 per- sons exposed during the year. The figures in the following table are ob- tained in this way. The only point to be remembered is that the rates are specific and refer to the particular class of persons designated. Thus, the rate 7.1 in the column “white males” at the ages one to four years means that seven deaths from heart disease occurred among white male children ages one to four for every 100,000 such white male children who were insured in that year. The other figures are obtained in a similar man- ner—(Table I and Chart IIT).  The first point that comes to view from an examination of the tables and of the graphic illustration is that the incidence of heart disease as a cause of death increases consistently with age. At the age period 35 to 44 when persons should be at the height of their productivity, one white person dies from heart disease in every thousand living and two colored per- sons out of each thousand. At the age period 65 to 74, the number of deaths from heart disease has in- creased to about 15 in each one thou- sand living, or to put the facts in another way, deaths from heart- dis- ease constitute 9.8 per cent of all deaths at ages 35 to 44, but, at the older period, 65 to 74, they are re- sponsible for 21.9 per cent of the     Death Rates per 100,000  2614.  2000 1600  1600 | White Males  1400 colored: Males *-357—\"* |  1200  1000  254034  Age Periods     Chart Ill Death Rates per 100,000 from ORGANIC DISHASES OF HHART. white and Colored Groups Compsred by Sex and by Age Periods.  Metropolitan Life Insurance Company, Industrial Department, 1911 to 1920. Death Rates                               100,000 2ebat  2000  1800 whee} Fenales): —.:- .  Colored Females  1600 De 1400 1200 1000  800 |  600  Age Periods        Chart III.—Statistics of heart disease distributed according to color, «ge and sex, and diagnosis jndieate increased incidence with age, greater incidence among colored persons than white. Tho rates are higher for females in the age groups below thirty, but higher for males from that age onward, the difference being regularly greater for males with advancing years.  2  eT)    TABLE I.—Death Rates per 100,000 from Organic Diseases of the Heart.  Metropolitan Life  Insurance Company, Industrial Department, 1911 to 1920.                                   White Colored Age Periods | Male Female Male Female Miiaes ison ee ee isa 199.8 189.1 200.4 DA ls ee aa tee te | it 5.9 13.4 | 17.5 Gch a eee cee | 14.0 16.7 i | 16.5 OSI eo ee | 21.8 29.1 20.2 28.9 USO a, ies 28.9 30.0 PAS ee 36.7 ONG et ee | O76 30.8 BOA 40.2 Deen eke ca, | 47.5 45.3 83.2 15.7 BE res ewe ee ieee cal 108.6 89.3 199.0 199.8 Ab EOE i ne eae eet | 245.1 195.2 403.1 414.5 DDIOA oe cout ae 622.2 516.0 BE3.0-72) 785.6 ObTAe eee ate oe 6000 1,445.9 1,717.0 1,570.0 Gore ee ee | 3,158.5 2,910.4 2,674.1 2,662.1 } deaths. There is no exception to this Some relations have been discovered rule. The rates are also very much to exist between the prevalence of  higher for colored persons than for whites. The sex ratios of heart dis- ease mortality are also rather inter- esting. The rates are usually higher for females than for males up to age 30. From that age onward, the rates for males are higher, the dif- ference becoming regularly greater with advancing years.  But, if heart disease is particularly important in middle life and at the older ages, it is already an impor- tant condition in childhood and early adult life. Thus, the number of deaths between the ages of 5 to 9 are as many as from two such impor-     tant infectious diseases of childhood ~  as measles and whooping cough. Be- tween 15 and 24 years, the deaths from heart disease are more numer- ous than from typhoid fever. Be- tween the ages 25 and 34, heart dis- ease caused each year almost as many deaths as lobar pneumonia. So heart disease is not to be overlooked as a factor in the mortality of young peo- ple. It takes a heavy toll through- out life.  It is difficult to understand just why the rates for young girls after age 5 should be much higher than for boys at the corresponding ages. Perhaps the same causes are at work which make the death rates from tuberculosis higher among grow- ing girls than among boys. The greater prevalence of heart disease among colored people is notorious. Colored males show rates from heart disease during the main period of lite trom 65. to. 80 per cent higher than for white males at the same ages; those for colored women are twice as high as for white women at a number of age periods of life. Possibly, the higher prevalence of such diseases as syphilis, malaria, and typhoid fever in the colored race plays an important part in creating the excess of heart disease mortality.  heart disease and occupation. While the figures are not entirely trust- worthy, it would appear that of all occupations, those which are carried on upon the water have the highest heart disease rates. Thus, sailors, fishermen, and to a less degree, barge- men, show a very high prevalence of heart affection. It is possible that this relationship is in some way re- lated to exposure to greater dampness and cold. Next to these occupations are those exposed to alcoholism, in- cluding brewers, and those exposed to lead poisoning. There are high rates for metal workers, blacksmiths, and     All sedentary occupations have favor- able death rates from heart disease.  Summary  To summarize the facts then, we may say that, according to our best knowledge, there are about two and one-half million persons in the United States who, on examination, would show some type of organic heart le- sion. These persons are not all sick. Many of them are engaged in their ordinary pursuits and have no idea of their impairment. Yet, they are seriously impaired lives who, unless they take note of their condition and adapt themselves to their lesser ca- pacity to labor, will break down at a premature age. Insurance studies have shown that persons impaired with such minor defects as mitral re- gurgitation have, as a group, double mortality for their age. It is the business of American physicians, and especially of those in the cardiac clinics, to discover for each commun- ity those who are in any way suffer- ing from one type or another of heart defect. No one knows the amount of loss sustained annually through the disability for work which results from the varying incapacity among these two and one-half million people.  3  In addition, there are each year in the United States about one hundred and fifty thousand deaths from heart disease and the number is not de- clining. Even under age 45, there are each year over 22,000 deaths. Each one of these deaths represents a distinct loss to the community since these persons are presumably at an age where production may be ex- pected to be at its highest. They leave good sized families of minor children who suffer from the loss of a parent and, more usually, the father. This is the extent of the com- munity problem which is brought about each year by heart disease.  It is very obvious, however, that our information with reference to heart disease both as a cause of sickness and of death is very frag- mentary. At the present time, there is really no agency or machinery for collecting the facts by means of which the campaign against heart dis- ease can be properly guided. It is just this sort of machinery that is called for at the present stage of the movement. This information will provide checks against misdirected enterprise, and will suggest which of many possible lines of activity are really worthwhile.  What promises to fill this need is _  the plan of the Association for the Prevention and Relief of Heart Dis- ease in the City of New York. This Association, which conducts a consid- erable number of cardiac clinics, pro- poses to keep systematic records in connection with its work. The great- est emphasis has been placed upon a full history in each case, and on a complete record of the findings on each examination. A series of fol- low-up visits by social workers is planned, and the findings from this source are likewise provided for in the record. A system of tabulation and of analysis is being considered, so as to answer the outstanding ques- tions which the directors of this As- sociation have in mind. It is hoped that out of these records it will be possible to throw light on the prog- nosis in various types of cases, on the duration of the various heart lesions from inception to death and on the value of certain types of treatment in relation to the restoration of work- ing capacity. A goodly number of other factual items will naturally sug- gest themselves at the outset of a campaign against a disease of such magnitude as heart disease. We ap- pend to this article a reproduction of the first page of the record form (Fig: 1). _ What is being planned for the city of New York should, of course, be seriously considered by associations of heart disease clinics in other cities of the country. It is certainly to be hoped that the clinics of such cities as Boston, Chicago, and Philadelphia, whether carried out under municipal or private auspices, will develop a similar system of records and plan similar tabulations and analyses. The greatest value of this effort will come from the multiplication of sources of information and out of the exchange of experience from place to place. :  What will probably remain a most fertile field for the development of knowledge is the practice of individ- ual physicians who must, for a long time, continue to be the chief instru- ments in the campaign against heart disease. The individual doctor should appreciate, more and more, the neces- sity of keeping a full history and cur- rent record of his cardiac patients. The record form recommended for use in New York City should be indorsed by the leaders of the profession, and its use encouraged by the great body of physicians. It will then be pos- sible at regular intervals to send the material to some central agency where the records might be tabulated and the conclusions made available to the whole profession. On a foun- dation of established fact, the cam- paign against heart disease will make rapid and substantial progress.  ASSOCIATION FOR THE PREVENTION AND RELIEF OF HEART DISEASE--First Record              Hospital Out-patient dept. or clinic | Date Serial No. (A. P-R.H.) Issuing Clinic No. Name of patient Address Sex Color or race Age Single Reisgion Married Widowed PLACE OF BIRTH: Patient attending Country SCHOOL?  Town or city | Country of birth of patient's mother        Number and addressof SC oo Days lost from SCHOQL during past five years: 4 5     OCCUPATION OF PATIENT:     [Time lost acct. sickness each  History Type of Work No. of war hours per week  Teed ough Wage Date entered  General nature of industry or business per week  Trade or particular kind of work     Present occu pation     Past occupation (1)     Past occupation (2)     Past occupation (3)                    Past occupation (4)           FAMILY HISTORY:                            Ttem  age;       from  If of death. disease     PRESENT HABITS AND HYGIENE: Sleep—hours Sleep—{Good—fair—poor—very bad)  Sleep—How many pillows  Habits. (State “Heavy “—\" moderate” —“‘none™ Alcohol | Tea Coffee Sweets | Drugs | Tobacco  Appetite (Good—fair—poor—very bad)              ‘How many per day? Bowels regular? pes ‘igars                 Pi | Cigarettes | Ghew (Yes or no) PATIENT'S PAST HISTORY: HEART HISTORY: (Congenital Giotal(derscnnyothllnere Noi times confined toibed” | Days cut of | How many days and in what year was paticnt disabled for work beeen or acquired? Years ‘Months A 2nd attack | 3rd at -[¥ eteackalusthte taskn|icikis tase javier aca :  tack Yr | Ye  Yr  t Home In Hospital Days     work past year] Ist attack | Yr. | Days     tac! | Days} Yr. Ee     ac Yr ie        Yr eee  HISTORY OF OTHER DISEASE: (If patient has had the disease, indicate by “ Y” and give year of occurrence. If not, write\"-N\" Indicate severity: S\"—Severe, “M\"\"-Moderate, “L\"’-Lighz}                                                    Yes or No               Yes or No        Year Yes or No        Disease Year Dieease Disease Year Disease Yes or No Yenr                     Ist attack onsillitis 2nd  Side  Rheumatic fever Ist pr and: 2: ordi                            fever         Purpura ooth and gum                Rheumatic nodules     orticollis cough           Erythema nodosum or growing pains      PREVIOUS MEDICAL TREATMENT Treated                 » Treatment  Time in Bed                      ! Private Physician  { Clinic  | Hospital Sanatorium Osteopath  Chirepractor       SYMPTOMS—PAST AND PRESENT: (Answer yes (Y) or no (N), and give dates if possible). _If any symptoms are brought about by exertion or excitement, apecify aymptom by writing (E).                          First symptom Fatigue | Dyspnoesa Orthopnoea Palpitation Cough Swelling—lege or abdomen | Headache Pallor Nausea | Vomiting “Pain under right costa! margin Loss of weight’ | Giddiness Faintness Flushing Sweating t Tremor Nose-Bleed Pain—site Pain—rediation | Pain—character           Fig. 1.—The greatest emphasis is placed upon systematic records in each case of heart disease. This record form, adopted by the New York Association for the Prevention and Relief of Heart Disease provides for full history and complete record of physical findings on each examination. Such records will develop possibilities of prognosis in given conditions.  THE MODERN HOSPITAL PUBLISHING CO., CHICAGO cublishers, The Modern Hospital ;  The Modern Hospital Year Book  The Nation’s Health", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, "Nation's Health (Washington, DC)", "American Public Health Association", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ipbk_fuf8-fb2b", "00000000-0000-0000-575D-CB43FB0BAD5F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "The Incidence of Heart Disease in Adults", "9918573882206676X144", null, "1920", "10 April 1920", "Louis Dublin, The Incidence of Heart Disease in Adults, New York Medical Journal, 4/10/1921. Read at the New York Academy of Medicine, February 5, 1920 before the Association for Prevention and Releif of Heart Disease.", "Articles", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "5", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Reprinted from the New York Medical Journal for April 10, 1920.  ILE INCI DENCH OF HEART DISEASE IN ADULIS.  By Louis. > DUBLIN; Phew. New York,  Statistician, Metropolitan Life Insurance Company, New York.  There are today two outstanding public health problems in America, namely, tuberculosis and heart disease.. Both are responsible for about the same amount of sickness and death. There are about two million persons in the United States who are seri- ously impaired from heart disease and also from pulmonary tuberculosis and approximately one hundred and fifty thousand of them die each year from each of these two causes. These two fields of public health work have been very differ- ently exploited. That of tuberculosis has for over a period of thirty years received the greatest public attention. Many millions- of dollars have been spent annually in the campaign against this condi- tion. Thousands of doctors have been trained in the special diagnostic and therapeutic work for the tuberculous. Large numbers of sanatoria and of hospitals for the care of the tuberculous have been established. ‘In fact, a well defined, nationwide program has been in operation against this disease. The results obtained have been commensurate with the effort expended, for during the same period the death rate from tuberculosis has been reduced about a third. On the other hand, in the campaign against heart disease, virtually nothing has been done. The Association for the Prevention and Relief of Heart Disease is only a few years old and is the pioneer in the field of preventing heart disease. The surface has hardly been scratched, but we have come to     *Read at the New York Academy of Medicine, February 5, 1920, before the Association for Prevention and Relief of Heart Disease.  Copyright, 1920, by A. R. Elliott Publishing Company.    Dublin: Heart Disease in Adults.  realize how large_a field of public health work is presented by the incidence of heart disease.  The evidence upon which these statements are based is derived from various sources. In the first place, about two per cent. of persons examined by the insurance companies are rejected each year because of various organic heart defects. This agrees with the findings in the examination of in- dustrial workers, such as those of Dr. J. W. Schereschewsky, of the United States Public Health Service, among garment workers in New York; of Robinson and Wilson: among employees in various industries in Cincinnati; and of Harris and Dublin among food handlers in New York. About the same results were obtained by the army medical ex- aminers in connection with the draft and camp examinations, 2.6 per cent. of the men examined having been rejected on account of heart defects. Likewise children in the schools of New York show on examination an incidence between one and one half and two per cent. of important cardiac defects. We may, therefore, summarize with the statement that about two per cent. of the population or, in continental United States, over two million persons of all ages, suffer from heart impairments.  Medical research has also shown that the presence © of heart disease seriously curtails the longevity of persons thus afflicted. Studies of statistics of the New York and the Metropolitan life insurance companies indicate that insured persons suffering from well compensated mitral regurgitations at the time of their insurance have a subsequent mortality varying from fifty to 100 per cent. in excess of the normal for persons at their respective ages. It should be remembered that these cases of mitral regurgitation were carefully selected as to every other physical character and that this form of heart disease is probably the least serious of the more important cardiac impairments. Similar results were shown for females in these insurance studies.  2 Dublin: Heart Disease in Adults.  For other heart impairments, such as aortic regurgitation and aortic stenosis, the excess mortal- ity is much higher than for mitral regurgitation.  MORTALITY FROM HEART DISEASE.  It should not surprise us, therefore, in view of the heavy incidence of heart impairments and of their serious effect on longevity, to find high death rates from heart disease. In 1917, the figures for the registration area showed organic heart disease to be the first cause of death, with a rate of 153.1 in 100,000. In 1918, the year of the epidemic, the rate for heart disease was 152.3, second only to pneumonia and influenza. Among the insured lives in the industrial population, organic heart disease is second only to tuberculosis.  These are very high death rates and if they have risen to first place in the list of causes of death, it is because the mortality from heart disease has re- mained high for a number of years, while that from tuberculosis has steadily declined. A remarkable condition as to the death rate from heart disease in 1919 is disclosed by the experience of the Metro- politan Life Insurance Company. The rate for the first time in years dropped about twenty-one per cent., compared with the preceding year. But this drop is probably only a temporary one and may only reflect the result of the influenza epidemic of the preceding fall and winter. It should be remembered that during the last three months of 1918 and the first few months of 1919 the influenza pneumonia epidemic carried off a large number of persons who had impaired hearts who would have lived for some time longer had not the influenza prevailed. The heart disease death rate went up sixteen per cent. in the last three months of 1918 coincident with the epidemic. Many deaths from heart disease, which would normally have occurred in 1919, took place in 1918 and the 1919 rate accordingly dropped. It is interesting also to find that during 1919 the im- provement in the death rate from heart disease was felt at every age period. .       Dublin: Heart Disease in Adults.  The death rate from heart disease varies con- siderably with the age period of life, being lowest at the early ages and highest at the advanced ages. But it must not be supposed that deaths from heart disease are of rare occurrence even in early life, for the death rate under twenty-five years is as high as that from typhoid fever. For ages between twenty-five and thirty-four years, the death rate from heart disease is as high as for lobar pneu- monia; between thirty-five and forty-four years, it is higher than for Bright’s disease, and after forty- five years the death rate is higher than for any other cause of death. The accompanying table presents the rates in 100,000 for persons insured in the In- dustrial Department of the Metropolitan Life for the period of 1911 to 1916 inclusive.     MortaLity FRoM OrGANIC DISEASES OF THE HEART CLASSIFIED BY Cotor, SEx anp By AGE PERIOD. Death Rates in Joo,000 Persons Exposed, 1911 to 1916.  ——— _ W hite—, ———Colored-——  Age Period. Persons. Males. Females. Males. Females. All ages—one and over 140.1 125.9 137.0 191.0 202.0 BECO so aysra io oye, olen oes 7.2 7.0 6.4 14,1 14.6 GrtO 00 cco = 6.2 14.9 18.2 11.4 I4.1 TOs COA asa as do ss 26.7 2237, 3154 19.9 28.3 Teh tQ; SO} ae ce oss 0b 30.2 28.7 31.2 20.4 34.9 2010! 24 ee 30:02 20.5 30.4 22,2 42.8 2h S10 84. oes de 53.5 51.6 45.6 89.6 72.8 95% 10 AAs ae is es T2185 120.1 92.6 201.2 2IT.7 4540 54 SES Ae 253.6 259.0 201.1 416.0 433-1 Shs €0.04 neu as cue 604.8 641.2 526.8 SOSA 2787.8 65 10-74-35... 1523.1 1624.2 1443.0 1702.2 1530.3 75 and Sovers i447 2808.1 3033.1 2703.1 2647.8 2613.2     ‘The death rates, it will be observed, are very dif- ferent for the two races and the two sexes. Colored persons suffer much more seriously than do white persons. This is especially marked during the first years of life and during the period of thirty-five to fifty-four, when the rates for the colored are  almost double those for the whites. Almost without -  exception the rates for females are higher than for males up to and including the age period of twenty to twenty-four years. After this age period, how- ever, the situation is reversed, the death rates for  4 Dublin: Heart Disease in Adults.  males being much higher than for females. This is uniformly true for white persons but there are a few exceptions among the colored. It would appear, therefore, that these organic heart diseases in their higher incidence among adult males strike heavily at the chief or only income producers of families, often after long periods of sickness in which the wage earner has been unable to work actively all or part of the time. These diseases thus bring about hardship and distress which cannot be shown in figures. In fact, if it were possible to calculate the money loss to the country through deaths from the heart affections and the long periods of sickness which precede them, the importance of cardiac disease economically would be much more impres- sively demonstrated than is possible by the publica- tion of mere numbers of deaths and the corresponding death rates.  I believe I have presented sufficient evidence to justify the statement with which I began this paper, namely, that heart disease is as important as tuber- culosis as a public health problem. A _ splendid opportunity awaits the medical profession in its attack on this disease. A carefully planned program must be developed by Federal, State, municipal and private agencies against this disease. The older organization of public health activities, which was directed toward sanitation of the environment, must be augmented by activities aimed at improvement of the physical state of the individual. The con- quest of heart disease and the related disorders of the vascular and renal. systems will depend in a measure upon the continuation of time tried en- deavors in public hygiene, but more so upon the knowledge of personal hygiene disseminated by the educational arm of health services. Especially promising is the development of the routine annual physical examinations of children and young adults. In many instances these will disclose incipient heart lesions which instruction and treatment may either  abort or control. |", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, null, "New York Medical Journal", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yzxb_ga48_kr6a", "00000000-0000-0000-8E06-12246FD83C85", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Incidence of Tuberculosis in the Industrial Population", "9918573882206676X145", null, "1932", "1932", "Louis Dublin, Incidence of Tuberculosis in the Industrial Population, American Journal of Public Health 22 (1932): 281-291.", "Articles", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "11", "pages", "Text", "English", "This item may be under copyright protection; contact the copyright owner for permission before re-use.", "Copyright may apply", null, null, "Reprinted from American JourNAL or Pusric Heart, Vol. XXII, No. 3, March, 1932  Incidence of Tuberculosis in the  Industrial Population’ LOUIS I. DUBLIN, Pu.D., F. A. P. H. A. (Life Member)  Third Vice-President and Statistician, Metropolitan Life Insurance Company, New York, N. Y.  fo8 a long time, it has been realized by those who have studied the tuberculosis problem that the disease is not uniformly distributed in the population as a cause of sickness and of death, but that it is con- centrated in certain stratums and groups. In fact, the campaign to control the disease is more and more directing its attention to these classes and groups. Common observation and everyday experience have shown that tuberculosis is much more prevalent in the industrial population of the country than in the agricultural, the commercial, or in the professional trades. It is the purpose of this paper to point out certain variations in the tuberculosis death rate in the various elements which compose the industrial population. This should prove to be an important aid to the movement against tuberculosis, as it will show where the disease takes the heaviest toll of life, and where it is less prevalent. Such knowledge should make it possible to economize effort and to concentrate resources where they will do the most good.  By the industrial population I mean that large section of the people who live, for the most part, in the cities and towns, and who draw their support from employment in the industries of the country. They are, almost altogether, wage earners and their dependents, in- cluding women and children. I use this term “ wage earners,” know- ing well its limitations, to identify the group I shall discuss with that large number of people who are insured in the industrial insurance companies of the country. I might have entitled this paper with more propriety, “The Incidence of Tuberculosis among Those Insured in the Industrial Department of the Metropolitan Life Insurance Com- pany.” The advantages gained by using this material are many. It covers a period of 20 years; relates to a population extending over the states and provinces of the United States and Canada; and includes  millions of negroes as well as white persons—virtually all city dwellers who live on wages. |  * Read at a Joint Session of the Vital Statistics and Epidemiology Sections of the American Public Health Association at the Sixtieth Annual Meeting at Montreal, Canada, September 16, 1931.  [281]       282 AMERICAN JOURNAL OF PuBLic HEALTH  Tuberculosis is primarily a disease of the working classes of the country. In 1930, for example, the death rate from all forms of tu- berculous disease, among the industrial policy holders of the Metro- politan, was 81.3 per 100,000. In contrast, the death rate for the ordinary policy holders for that year was only 48.7 per 100,000.  These ordinary policy holders are persons who carry insurance ranging from a minimum of $1,000 up to hundreds of thousands of dollars. In the Metropolitan, a great many of the ordinary policy  holders carry industrial insurance also. The ordinary department |  death rate for tuberculosis would be lower still if these were excluded. For example, among those who carry insurance in policies for $5,000 or more (among whom very few carry industrial insurance), the rate was only 17 per 100,000; and among the many employees of the com- pany the rate was 40 per 100,000. It is clear, therefore, that the two main classes of the population are sufficiently far apart in their tu- berculosis death rate to show how important is the industrial factor.  We may still further drive home this contrast by recording the fact that the death rate from tuberculosis in the general population of the U. S. Registration Area for the last year available, 1929, was 76 per 100,000 of population, as compared with 87 for wage earners and their dependents. But this figure (76) includes deaths from tuberculosis in the industrial population, as well as in all of the rest. If it were possible (which unfortunately it is not) to obtain the death rate from tuberculosis for the entire population, outside of the industrial class, the rate would be considerably lower than 76. A closer approxima- tion, perhaps, to the tuberculosis death rate of the non-industrial popu- lation is the rate for 1929 for the rural area of the United States. That death rate was 74.7 per 100,000,* but even this figure is much higher than the true rate for actual, permanent residents of the rural districts, because it includes that large number of deaths of city resi- dents who went to rural communities in search of health, and died there either in sanatoriums, hospitals, or in private homes. a  There can be no question, in view of these figures, of the heavy concentration of tuberculosis as a cause of death in the urban industrial group of the population. Every refinement we could make, which would separate out those who work for salary, or on farms, from those who work for wages in industrial establishments, would make the dis- tinction all the greater. I do not for a moment suggest that tubercu- losis is not an important factor in the mortality of the farming popu- lation, or that it does not strike down others than the industrial work- ers in cities; but it does this more infrequently. |  * When general population death rates for 1930 become available a still lower rate will be shown, and a greater contrast with the 1930 rate for the industrial policy holders, 81.3. DeEaTH RATES PER 100,000 FRomM TUBERCULOSIS—ALL FORMS.  TUBERCULOSIS  TABLE I  1911 To 1930  283  By Racsg,.SEx AND AGE PERIODS. METROPOLITAN LIFE INSURANCE COMPANY, WEEKLY PREMIUM-PAYING INDUSTRIAL BUSINESS.        Tuberculosis—AIl Forms                                                                          One 10 to | 15 to | 20 to }| 25 to | 35 to | 45 to | 55 to | 65 to Year and |ito4)Sto9! 14 | 19 | a4:| 34 | 44 | sal 6a | 74 Over  White Males 1930........... 68.0 | 21.7 8.6 7.9 | 34.3 | 80.2 | 100.6 | 141.0 | 177.2 | 172.2 | 151.2 1929. .......... Sel 25.6 9.0 7.2 38.3 81.4 | 120.8 | 153.9 | 183.6 | 167.8 | 159.1 1) 77.9 29.7 10.0 9.2 44.2 85.4 | 129.9 | 167.4 | 191.0 | 174.9 | 153.1 1925........... 84.3 | 29.1 9.5} 11.4] 53.7 | 103.0 | 135.6 | 187.1 | 204.0 | 181.8 | 179.9 1923........... 97.9 37.2 13.6 11.1 52.9 | 130.3 | 169.3 | 202.5 | 225.5 | 206.7 | 201.7 1921........... 99.5 35.8 15.2] 14.6 62.5 | 142.8 | 160.2 | 207.4 | 215.5 | 203.9 | 192.9 1919... 0.0.0... 145.2 57.8 19.0 17.4 90.0 | 153.9 | 240.4 | 352.4 | 353.1 | 271.2 | 242.5 1917. 194.3 71.2 23.7 20.1 | 106.7 | 211.5 | 344.1 | 488.7 | 461.6 | 360.9 | 282.6 191 SP eee 201.1 68.7 28.0 15.8 91.7 | 218.8 | 359.4 | 517.6 | 456.7 | 356.4 | 262.9 1913. 218.2 80.5 23.8 19.9 | 113.0 | 252.9 | 404.3 | 542.5 | 473.5 | 366.1 | 256.2 1911 230.8 75.6 27.8 21.4 | 115.9 | 288.6 | 424.1 | 606.1 | 455.5 | 392.8 | 303.6  White Females 1930. S7oi 23.2 8.7 14.1 69.5 | 108.6 90.7 66.3 57.1 66.7 72.4 1929........... 63.0 | 28.0 8.1] 14.4] 70.8 | 130.9} 105.3] 71.6] 61.0 | 66.3 | 81.2 1927........... 70.9} 27.8) 10.4] 15.8] 93.3 | 141.7] 118.5] 79.6] 67.7] 69.2 | 82.8 . 1925........... 76.7 30.3 12.5 18.1 92.9 | 155.4 | 130.3 92.7 70.9 74.7 81.1 1923........... 88.2 31.3 11.8 24.4 | 107.4 | 167.4 | 144.7 | 111.3 91.9 81.2 96.3 1921........... 95.2 | 36.8] 14.8] 28.4 | 118.9 | 186.9 | 151.8 | 118.6} 90.9 | -87.9 | 106.2 1919........... 125.2 54.3 22.2 35.6 | 145.8 | 228.4 | 214.3 | 162.7 | 121.1 | 115.4 | 116.2 Oar ee 135.0 | 69.4 | 24.5] 38.8 | 160.0 | 238.0 | 220.0 | 180.6 | 134.9 | 128.7 | 117.9 1915... ........ 141.5 70:7. 26.0 37.3 | 146.4 | 224.5 | 234.8 | 206.5 | 141.8 | 140.9 | 133.9 1913........... 147.7 88.1 29.8 39.7 (144.5 | 230.0 | 247.7 | 214.0 | 151.0 | 136.4 | 154.6 1910. 2 165.4 81.6 35.8 39.1 | 155.2 | 263.3 | 293.6 | 260.4 | 161.7 | 150.7 | 149.4  Colored Males 1930........... 223.8 97.2 51.1 73.3 | 245.4 | 351.5 | 295.1 | 258.7 | 275.8 | 204.4 | 229.8 1929. 0. eke: 226.4 | 125.0 55.4 65.8 | 272.1 | 377.7 | 288.2 | 266.5 | 245.6 | 200.4 | 174.0 1927. .1:227.6 | 129.6 55.7 65.4 | 273.0 | 341.5 | 273.2 | 290.7 | 251.5 | 212.7 | 191.7 1925........... 224.9 | 137.2 43.7 85.3 | 300.6 | 334.7 | 276.5 | 264.7 | 242.7 | 211.4 166.0 1923........0.. 242.9 | 139.2 86.0 76.2 | 285.3 | 362.3 | 314.7 | 286.3 | 261.3 | 204.2 206.1 1921........... 249.1 | 139.3 77.0 | 101.3 | 293.4 | 353.2 | 309.9 | 295.1 | 264.3 | 238.0 272.2 1919........... 319.7 | 203.8 | 102.7 | 177.7 | 339.2 | 435.4 | 388.9 | 392.5 | 351.9 | 289.4 | 317.1 1917.....0..... 414.9 | 197.4 | 133.4 | 136.1 | 450.0 | 547.5 | 527.8 | 562.8 | 482.3 | 350.5 381.3 1915........... 432.8 | 217.8 | 134.0 | 140.1 389.6 | 532.8 | 583.6 | 608.4 | 436.2 | 486.6 | 357.0 1913505 455--- 428.6 | 282.2 | 134.8 | 147.1 | 412.7 | 592.5 | 545.7 | 575.8 | 494.7 | 446.7 350.6 1911........... 422.2 | 345.5 175.4 151.8 | 416.2 | 610.6 | 548.8 | 488.6 | 478.2 | 465.6 | 364.4  Colored Females  1930... 0.00... 213.0 97.7 53.9 | 123.1 | 368.7 | 398.1 | 301.9 | 182.4 | 139.2 | 113.6 | 113.9 1929........... 220.1 | 126.0 66.2 | 125.6 | 383.7 | 432.0 | 293.1 | 194.9 | 149.0 100.8 84.0 1927 22 ee: 228.4 | 118.4 49.6 | 126.5 | 421.6 | 433.8 | 339.6 | 177.3 | 124.0 97.2 | 106.9 1925 0 oe ne 228.0 | 116.0 | 56.2 | 154.4 | 431.4 | 421.2 | 301.8 | 184.5 | 134.1 | 116.0 99.5 1923........... 245.7 | 107.6 59.3. | 153.6 | 444.2 | 489.7 | 317.1 | 214.2 | 161.6 | 125.4 | 105.0 1921.05.00... 285.8 | 133.4 74.2 | 205.1 | 474.4 | 622.4 | 374.6 | 214.7 | 178.1 | 137.0 | 123.1 1919. ..0 0000.05 328.9 | 157.6 | 122.3 |-240.4 | 642.2 | 551.6 | 404.1 | 283:9 | 191.1 |°173.6 |-196.5 1917........... 371.7 | 231.1 | 163.7 | 291.3 | 660.7 | 587.5 | 479.4 | 332.0 | 213.8 176.7 | 189.7 1915...0....... 394.2 | 315.2 | 145.9 | 285.4 | 665.1 | 638.7 | 497.3 | 345.5 | 271.1 | 220.1 184.7 1913 084 8 tes 363.1 | 313.0 | 132.7 | 243.2 | 601.5 | 612.1 | 443.0 |'366.2 | 187.8 | 159:8 | 194.3 LOD. eee 415.1 | 269.7 | 178.9 | 315.7 | 643.1 | 735.8 | 511.3 | 379.1 | 234.4 225.4 | 173.2                         284 AMERICAN JOURNAL OF PuBLIC HEALTH  It is safe to say at this time that, by and large, tuberculosis is rapidly becoming a minor cause of death in all but the industrial classes of the population. We shall later call attention to a few apparent ex- ceptions to this statement. But, to illustrate the essential truth of what I am saying, it is only necessary to note the tuberculosis death rates in 1929 of 27 for the State of Utah; 32 for Nebraska; 33 for Iowa; 34 for Wyoming; 35 for North Dakota; and 37 for Idaho and Kansas. In these states, there is a total population of 7,589,190, of whom about two-thirds (63.4 per cent) are rural dwellers, and only a small percentage may be classified as in the industrial population. In these states certainly it may be said that tuberculosis has already be- come a minor cause of death.  It would be misleading, however, if we did not call attention to the tremendous improvement that has occurred in the incidence of tu- berculosis in the industrial population during the last 20 years. In fact, the improvement has been greater in the industrial classes than in the nation as a whole, or in any other major group of the population; for we find, as between 1911 and 1930, a decline in the mortality from tuberculosis of 64 per cent. In other words, for every 10 lives lost on account of tuberculosis in 1911, only 4 were lost in 1930. In the gen- eral population, the reduction in 1929, as compared with 1911, was only 52 per cent. Again, I wish it were possible to indicate the de- cline in other specific population groups, but the only other one for which data are available is a constantly increasing rural area, which between 1910 and 1929 shows a decline of 41 per cent. Obviously, the disease we are discussing was a much more important factor in the life of all elements of the population 20 years ago than it is now; but this applies particularly to the industrial classes. Yet tuberculosis is still the third cause of death in the industrial and the seventh in the general as well as in the rural population.  Within the industrial classification, the disease will be found con- centrated primarily among males and among colored persons. Tak- ing the figures for 1930 for insured white persons as our guide, we find no significant difference between the sexes under age 10. From ages 10 to 15, however, the mortality among females is higher by more than 75 per cent; and between 15 and 20 it is twice as high among white females as among males. Between 20 and 25, it is still 35 per cent higher among white females; but after age 25, the rate is uniformly lower among females; and at the age period 45 to 54 it is only one- third as high. Figure I* shows the relative importance of the disease  * The three charts used in this paper are similar in form to those prepared by Miss Jessamine S. Whitney,  Statistician, National Tuberculosis Association, for her ‘‘ Facts and Figures about Tuberculosis.” The charts have been brought up to date.  4 TUBERCULOSIS 285.  FIGURE I -     DS °o oO  DEATHRATES PER 100.000 FROM TUBERCULOSIS -A// forms WHITE MALES Y FEMALES By Age Periods  METROPOLITAN LIFE INSURANCE COMPANY eeRly Premium Paying Industrial Business 1930  a o  a o  rs °  © o  KEY  GAME White Males White Females  @ ° So o  Deathrates per 100,000 Males and Females o o  40  20  WDB Ky’”iW  “4  49-54 9-  20-24 25-34 35% Age Periods  .WWWWO16'*o—6 FOR           as a cause of death, in 1930, in the two sexes of the white classification.  Uniformly, the colored people show higher rates than the whites. At some of the younger ages, the rates for the colored are from 5 to 9 times as high as those for the whites, although after age 35 they are less than twice as high for males. Toward old age, the differences be- come less marked. Table I shows the contrast between white and colored persons on the basis of the 1930 mortality experience in the Metropolitan’s industrial department.  With the colored, as with the white, there is substantially no differ- ence under 10 years in the mortality rates of the sexes. Between 10 and 20 the rates for females run from 50 to nearly 70 per cent higher than for males, instead of 100 per cent higher, as they do in the case of © the whites. With the colored, it is not until age 35 is reached that the death rate for males exceeds that of females, whereas this condition obtains from age 25 with the whites. |  Occupation plays an important part in the incidence of tuberculosis and in its mortality. The very highest rates for the disease are found among those exposed to silica dust. Included here are a large num- ber of workers in ore mills and mines (except coal mines), in the       286 AMERICAN JOURNAL OF PuBLIC HEALTH  building industries, and among grinders and buffers, stone workers, pottery workers, employees of foundries and other metal industries, and of the clay and glass industries. Another group having a high death rate from tuberculosis includes those employed in low grade oc- cupations, more particularly common laborers in a variety of industries, longshoremen, porters, freight handlers, watchmen, hucksters, and peddlers. —  Certain types of factory work have long been associated with high death rates from tuberculosis, although the mortality among these em- ployees does not reach the figures for those exposed to silica dust. In- cluded among these are the cigar and tobacco workers, shoe factory operatives, those engaged in certain textile occupations, and to a lesser extent workers in the printing trade.  Again, there are a number of occupations, in which there is more or less exposure to organic dust, and which show high mortality from tu- berculosis. Among these are barbers and hairdressers, furniture and other wood workers, bakers, textile mill workers, hatters and hat workers (both wool and fur felt), and cigar makers and tobacco workers. The last named occupation has long been recognized as one with an especially high incidence of tuberculosis. In addition to the dust hazard, there is exposure to nicotine and often to general insani- tary conditions in the workshops. But it is necessary to point out that in this industry wages are low, and the work attracts many men of poor physique and low resistance. .  Another group in which tuberculosis takes a large death toll is laundry workers. This may be due, in part, to the fact that these workers are sometimes exposed to infected material; but the chief fac- tor in their high mortality is probably the conditions of heat and humidity in which they work. |  There are other employments in which there is a relatively high incidence of tuberculosis, although they are not associated with an definite industrial risk. Among these are tailors and other clothing workers, cobblers, and slaughter and packing house employees. Cleri- cal workers and bookkeepers also have a higher than average incidence. Here the factors of confinement, poor ventilation, and fatigue, un- doubtedly play their part in reducing resistance to infection.  This paper should not close without a brief reference to the geo- graphical aspects of the mortality from tuberculosis. For this pur- pose I shall have to use data for the general population, even though this paper’s title would seem to restrict my discussion to the industrial population. I have already referred to the extremely low mortality in the general population for certain states with predominantly rural, TUBERCULOSIS | 287  and non-industrial populations, in order to contrast their low tubercu- losis death rates with those of states where the people are more or less concentrated in the cities—and are largely wage earners.  First of all, it will be necessary to disregard the figures for certain states, like California, Colorado, New Mexico, and Arizona, which have very high tuberculosis death rates, solely because thousands of tu- berculous people go there every year in the hope that favorable cli- matic conditions will arrest the disease. Many of these people die each year and thus swell the mortality rates for these states to a point many times in excess of the figure for the permanent residents. There are, however, 4 states where the ravages of tuberculosis are worse than in any others, namely, Tennessee, Kentucky, Maryland, and Virginia. This applies, with the exception of Virginia, to both the white and colored populations. Aside from these states, the following (in addi- tion to the “‘cure seeker” states) have tuberculosis death rates in ex- cess of the average: Alabama, Arkansas, Delaware, Louisiana, Missis- sippi, Nevada, North Carolina, and South Carolina.  FIGURE II DEATHRATES FROM TUBERCULOSIS ~ ALL FORMS              YY Y         REGISTRATION AREA ~ 1929  LESS THAN 50 DEATHS PER 100,000 POPULATION 50 TO 75 DEATHS  BRBBA 75 TO 100 DEATHS  GEES /00 DEATHS AND OVER  CJ NON-REGISTRATION AREA DEATH RATE UNITED STATES REG. AREA ~ 76 PER 100,000    AMERICAN JOURNAL OF PuBLIC HEALTH           288 FIGURE III RANK OF REGISTRATION STATES In Death Rates from Tuberculosis ~ All forms and FerCent Decrease ~ 1920 and 1929 Percent Decrease Deathrate per 100,000 population 1929 as compared with 1920 . . ARIZONA * Con NEW MEXICO * 707] COLORADO a ~d22 TENNESSEE (22M Cd G4 CALIFORNIA Os? KENTUCKY er MARYLAND VOfMm «4d; C47 VIRGINIA (Com) 43 NEVADA * LOUISIANA ce Cd ALABAMA* EG MISSISSIPPI [oad 27 NORTH CAROLINA cy DELAWARE sa CC~<“C~SSC*SdS‘I 4G SOUTH CAROLINA Comm S~«di 20 ARKANSAS * 3] ean net on fon ee GEORGIA* 75) MISSOURI (cy Cid 07 NEW YORK i J 120 INDIANA 7 109 NEW JERSEY ee 4 FLORIDA i) OHIO Ome jios VERMONT [7ORE ] 82 ILLINOIS ORE id i00 RHODE ISLAND ) es MASSACHUSETTS Pd 4 WEST VIRGINIA* ro5] WASHINGTON [ory J 09 MICHIGAN a WS PENNSYLVANIA (COM «das MONTANA T] 76 CONNECTICUT J 120 MAINE comm SS=«*d‘ ‘COD MINNESOTA Ec] 20 WISCONSIN eS OKLAHOMA* Sa) NEW HAMPSHIRE E97 OREGON Om] 89 KANSAS [37m | 43 IDAHO * 7] NORTH DAKOTA* [iia WYOMING * GA 1920 IOWA * oa 1929 NEBRASKA Sa) +3 UTAH 27m j 29 * Not included in Registration Area in 1920          TUBERCULOSIS .« 289  Figures II and III show graphically the tuberculosis mortality rates in the several states, in 1929, together with the per cent decline in that year, as compared with 1920 for each state. The map shows the 8 “black spots,” states in which the tuberculosis death rate in 1929 ranged from 100 per 100,000 up. As noted previously, the high mortality in Arizona, New Mexico, Colorado, and California is due to deaths of non-residents, and the most serious situation prevails in Tennessee, Kentucky, Maryland, and Virginia. In the last named, the tuberculosis death rate among the white population is not far from the average figure for whites in the United States. Negroes, however, constitute 27 per cent of Virginia’s population; and their excessive mortality from tuberculosis raises the death rate for the state to one of the highest figures in the entire country. In Maryland, too, the prob-  _ lem is largely with the negroes, although the mortality from tubercu-  losis among the whites is also above the average.  If it were possible to obtain resident death rates for tuberculosis for each of the states the shadings in the map would be changed in several instances. I strongly suspect, for example, that the figures for Nevada and North Carolina would be materially lower if deaths of non-residents were excluded.  In Figure III, attention is directed in particular to the per cent declines for the several states. It is encouraging to note that no state fails to show a lower tuberculosis death rate in 1929 than in 1920; but one is immediately impressed with the fact that for each of the 4 states where the situation is the gravest, the drop in the death rate has been less than the average decrease in the United States (expanding reg- istration area).  _ Large cities whose tuberculosis mortality in 1929 was in excess of the average for all cities in the registration states, considered as a group, are: Albany, Atlanta, Baltimore, Birmingham, Boston, Buffalo, Chattanooga, Cincinnati (very high), Cleveland, Columbus, Denver  -(due entirely to tubercular transients from other states), Detroit,  Duluth, El Paso (for the same reason as Denver), Fall River, Houston, Indianapolis, Jacksonville, Kansas City, Kansas, Kansas City, Mis- souri, Knoxville, Los Angeles (for the same reason as Denver), Mem- phis (very high), Nashville (very high), Newark, New Bedford, New Orleans (very high), Manhattan and Richmond Boroughs of New York City, Norfolk, Philadelphia, Richmond, San Diego, St. Louis, San Antonio (very high—probably from non-resident deaths), San Francisco, Scranton, Toledo, Trenton, Washington (due to an ex- tremely high rate among negroes—the mortality is not high among whites), and Worcester.    290 _ AMERICAN JOURNAL oF PusLic HEALTH  I have, also, comparable data to show the relative mortality from tuberculosis among the insured Canadian wage earning population, as compared with the death rate in the same group in the United States. The figures for Canada are based upon the mortality experience of about 174 millions of industrial policy holders of the Metropolitan Life Insurance Company. They relate to a period of 6 years.  TABLE II  DEATH RATES PER 100,000 FRoM TUBERCULOSIS (ALL ForMS) AMONG INDUSTRIAL PoLicy HOLDERS IN THE UNITED STATES AND CANADA COMPARED                 Death Rates per 100,000 Per cent Excess  . Canada over  Year United States Canada United States (Total Persons) (White) Total Persons White  1930 79.7 59.0 97.3 64.9 1929 86.2 : 64.9 96.8 49.2 1928 89.8 68.4 94.3 37.9 1927 93.0 71.7 100.9 40.7 1926 98.8 77.6 102.5 32.1 1925 97.7 78.4 104.1 32.8                 It will be seen, first of all, that the tuberculosis death rate runs uniformly higher in Canada. Furthermore, the decline has been small in recent years, by no means matching the improvement in the United States. Even when we include the mortality among almost 2/2 millions of negro policy holders (as we do in the first column of this table), the Canadian death rate exceeds that for the United States; and it must be borne in mind that the tuberculosis mortality rate for negroes runs about three and one-half times that for the whites. As the Metropolitan has comparatively no negroes at risk in Canada, the really significant comparison is that with the insured whites in the United States. There is a wide gap between the Ca-  nadian and United States figures. As a matter of fact, with the slow:  decline in Canada and the abrupt drop in the United States the excess in the Canadian tuberculosis death rate is becoming greater every year. These figures, it must be remembered, relate almost exclusively to city dwellers. Canada’s relatively unfavorable record is due en- tirely to the very high death rates in the industrial populations of the cities in Quebec, Nova Scotia, and New Brunswick. For the remain- ing five provinces, the mortality from this disease is well below the average for white wage earners in the United States.  On what elements of the population, then, should the attack on tu- berculosis be concentrated from now on? First of all, upon the wage  a    pane 7 ~~  ‘TUBERCULOSIS 291  earners in American and Canadian industries, and their dependents. In this group, despite the splendid progress that has been made, tu- berculosis still ranks third among the causes of death. Thus, where the greatest progress has been made lies a great opportunity for further progress. Within this wage earning element, the concentration should be primarily on males, except between the ages of 10 and 25, where the mortality among females runs from 14 per cent to more than 100 per cent higher. Second, upon the negroes, both in the industrial and general population, where the reduction in the mortality rate has by no means kept pace with that among the whites—more especially within the last 10 years. Here probably lies the greatest opportunity of all, especially in the age groups under 25, where the negro death rate exceeds the white by from four- to almost ten-fold. Third (and this lies within the field of the industrial hygienist), among those engaged in the several occupations I have mentioned—more particularly among those exposed to silica dust. Fourth, in the general population, both white and colored, of Tennessee and Kentucky, and to a lesser extent in other states, mostly in the South, where the tuberculosis death rate runs above the average; also among the populations of over 30 cities. Fifth, among industrial wage workers and their dependents in the cities of the Canadian provinces of Quebec, Nova Scotia, and New Bruns- wick.  There is every reason to believe that if the health officers and health agencies of the United States and Canada would concentrate their efforts on these groups of the population, a very definite reduc- tion in both cases and deaths would result. Up to the present time, even lacking such particular concentration of effort, the death rate has declined each year among the industrial population. It should be pos- sible, without undue expenditures of money, to increase the rate of de- cline. The important thing to do is to concentrate the energy and in- telligence of the medical and public health services of the communities along the lines I have indicated. The immediate goal of the tubercu- losis movement should be to accomplish for the industrial population what has apparently been brought to pass for those who are somewhat  more advantageously placed socially and economically. This can and will be done.", "Dublin, Louis I. (Louis Israel), 1882-1969.", null, "American Journal of Public Health", "American Public Health Association", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fcwg.rfui_nx25", "00000000-0000-0000-4881-879C9209759A", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "The Causes for the Recent Decline In Tuberculosis and the Outlook for the Future", "9918573882206676X146", null, "1923", "1923", "An address delivered before the nineteenth annual meeting of the National Tuberculosis Association at Santa Barbara, California, June 20, 1923.", "Speeches", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "32", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "os An Address De 1B  . the National | a oe Tp. Association Se _ “at Si nta Barbara, Cal., ee 20; 19 Sp  - LOUIS ie ‘DUBLL De Statistician | = | Murreoro1.t ITAN v Lae INSURANCE Company, New \\\\ York ae    THE CAUSES FOR THE RECENT DECLINE IN » TUBERCULOSIS AND THE OUTLOOK | FOR THE FUTURE  By Louis I. Dusiin, Ph.D., STATISTICIAN METROPOLITAN Lir—E INSURANCE COMPANY.  New York  I. THE STATEMENT OF THE PROBLEM  Tue decline in the tuberculosis death rate during the last twenty years is, in my judgment, the most outstanding fact in the tuberculosis prob- lem. This decline has been large in amount and continuous in character. There has been no backsliding; there has never been any doubt as to the tendency of the tuberculosis death rate during the last twenty years. In 1900, (and that, by the way, was the first year for reliable statistics on tuberculosis for any large part of the United States) the death rate was 195.2 per hundred thousand of population. In 1910, the rate in the same geographical area, namely, the Original Registration States and the District of Columbia, had dropped to 164.7, or 15.6 per cent., in the ten year period. In 1920, the rate in the same states was 112.0. This is 42.6 per cent. less than the figure for 1900. In the second decade, that is, from 1910 to 1920, the rate fell 32 per cent., or a little better than twice as fast as in the first decade. In 1921, the rate went down to the low figure of 94.2 per 100,000, which is less than one-half the figure for 1900, only twenty-one years before. The general character of the de- cline is clear enough and is shown in Chart 1 and Table 1 on page 2. The straight line expresses the tendency during the period and is the most convenient expression, mathematically, of what has happened in the two decades.  Let me leave with you another picture, and that is of a group of some fifteen million people insured in the Industrial Department of the Metro- politan Life Insurance Company. Since 1911, accurate records of their mortality have been kept. Every death is recorded and the number of persons insured is also.known. ‘There is little room for error in the calculations. Now, in the year rgit, the rate of mortality for tuberculosis was 224.6 per 100,000 insured persons. These are working people, mind you,—men, women and children,—constituted, as to age and sex, about the same as is the general population. By 1921, the tuberculosis rate among these people had dropped to 117.4, and in 1922, it declined to 114.2. The decline between 1911 and 1922 was 49.2 per cent. The im-  , 1    CHART I AND TABLE I        DEATH RATE per 100,000-TUBERCULOSIS-ALL FORMS  ORIGINAL REGISTRATION STATES AND DISTRICT OF COLUMBIA,  DEATHRATE PER 100,000                 190 180 170 160 150 140 130 120 0 100  90  OLt to} jy to} poe pt pep ttt pt | tt | 1900 190! 1902 1903 1904 1905 1906 1907 1908 1909 i910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921 YEARS  Year 1900  ISO!  1902 1903 1904 1905 1906 1907 1908  1909  I910  I900 TO 192I DATA  Rete Yeer Rate 195.2 (911 159.0 1898 012 49.8 174.1 1913 148.7 77.1 IN14 1486 188.5 I9I5 146.7 180.9 N16 143.8 17.8 17147. 156. = 1918 «151.0 694. = 191s 1249 163.3 1920 112.0 1647 921 942        SISOTNOWAGNAL NI ANIIONG LNAOAY AHL UOT SasaAVvo    LOUIS I, DUBLIN, PH.D. 3  provement is continuing this year and, in the first six months, we have recorded+a decline of 5.3 per cent. among white policyholders and of 2.4 per cent. among colored from the corresponding figures for 1922. Chart 2 and Table 2 on page 4 show what happened in this group of insured wage earners.  I have shown you that with reference to the general population, the death rate from all forms of tuberculosis has declined fifty per cent. in twenty years, and that in a large group of insured persons, the same amount of decline has occurred in a little over ten years. The improve- ment in the figures means that a hundred thousand fewer persons are dying each year in the United States from tuberculosis than would have died if the tuberculosis death rate of twenty years ago were still pre- vailing. That is an amazing fact, and, I believe, justifies my first re- mark that the decline in the death rate is the most outstanding item in the tuberculosis problem. -  My purpose, however, is to discuss the causes underlying the decline. As you know, there is much difference of opinion as to what has caused the decline in tuberculosis mortality. The first step in a scientific dis- cussion is to set for yourself a theory or a probable explanation on the basis of such evidence as is available and then to see how that theory squares with all the facts of the case. In connection with the decline of the tuberculosis death rate, there are two out-standing explanations. I shall consider these two opposing views and attempt to show you whether or not they square with the facts.  The first is espoused by most of the workers in the field of public health and ascribes the fall in the tuberculosis death rate during this period of twenty years and for a long period before to the great im- provement in the general wellbeing of the population. The improvement is due, they say, primarily to activities within human control. Those who hold this view say that the great mass of the population is pretty generally exposed to infection, and that those who break down and become active cases of tuberculosis, and later die, are those who have not had the resistance to localize and control the foci of infection within them. With that thought in mind, the proponents of this view early developed a broad campaign of general education of the population. A knowledge of personal hygiene was widely disseminated in order that individuals might learn how to strengthen their resistance against the development of active symptoms of the disease. This was the keynote of the general campaign against tuberculosis as outlined by this Asso- ciation when it was founded in 1904.  The campaign against tuberculosis was accordingly directed toward finding the large numbers of individuals who had broken down with the disease, to strengthen their resistance and to make them well again if possible and, second, to prevent their infecting others. That meant, of course, the establishment of such institutions as the tuberculosis clinic,       4 CAUSES FOR THE RECENT DECLINE IN TUBERCULOSIS  CHART 2 AND TABLE 2        Death Rate per 100,000-Tuberculosis-All Forms  Metropolitan Life Insurance Co. Industrial Dept, 1911101922 & Expanding U.S. Registration Area (aes 1 to74 years) 1911 to 1921 .  DEATHRATE PER 100,000 225  DATA Deathrate per 100,000  M.L.I.Co. U.S.Reg. Area Yeer Indl. — Ages 1°to 74 years 191! 224.6 IS7.6  200  2 2129 14@.G: 13 206.7 «146.5 115 94 2045 14623 915 197.8 145.4 2 16 = 190.2) «141 jo” = 188.9 «145.8 125 18 ~=—«18D9.0—s: 148.6 9 1565 125.2  1920 IS7.9 12.5        100 192] 7.4 97.7 90 ee eae ee ered ar = i 12 113 “4 15 16 17 “18 19 *20 ‘ZI i922 ee 14.2 YEARS        the training of many physicians in the technique of diagnosis, the build- ing of sanatoria for the care of early cases and of other institutions for the more advanced cases, and the segregation of those individuals, who having broken down, might become a source of danger to others. T need not enumerate all the activities and agencies which have grown out of this conception of the origin and control of tuberculosis. It is LOUIS I. DUBLIN, PH.D. o  sufficient to say that all over the world a definite technique for the con- trol of tuberculosis has been developed on the basis of this theory. This probably has received its best and most acceptable expression in the writings and practical works of Dr. Philip of Edinburgh.  The underlying principle of his campaign and of his followers has been that tuberculosis was subject to human..eontrol. The disease de- veloped because of infection and the weakened defenses of the individual. This might be due either to undesirable conditions of the organic and physical environment or to weakened personal constitution, or to both. This school did not lose sight of the importance of the constitutional factor. It realized that there are differences in the capacity of individuals to resist the development of infection and breakdown. But, it saw that with respect to the constitutional factor, man could do very little except to make the most of what constitutional equipment he had. The emphasis of this group has, therefore, been placed where it could be effective, namely, in the constant improvement of the status of human beings. The campaign against tuberculosis has attempted to make the lot of men and women happier and easier, to improve their personal hygiene, to remove possibilities of gross infection and to give many individuals a fighting chance to throw off disease which they would not otherwise have in their usual methods of life. Acting on this theory, a flourishing and world-wide activity has developed and those associated with it have very naturally. claimed that a measure of the decline which has appeared in this period may be credited to their and allied efforts.  The second explanation or theory minimizes the importance of the environmental factors in the control of tuberculosis. It places great emphasis on the fact that the decline in tuberculosis antedates by many years the development of the tuberculosis campaign as at present un- derstood, going back, in fact, to the early decades of the nineteenth cen-’ tury. Those who support this second view emphasize the all importance of the genetic, that is, the constitutional factors. They grant the uni- versality of infection, but they insist that those who break down are a special group whose constitutions, in advance, have doomed them to tuberculous disease. They insist, moreover, that the tendency to have tuberculosis is inherited like other physical characteristics, and that ob- servation will discover a distinctively tuberculous stock or phthisical con- stitution, quite irrespective of the environment, mode of life, or any effort that man might make either to avoid infection or to build up indi- vidual resistance.  A corollary of this explanation has been that hardly any of the decline to which we have called attention could have resulted from the tuber- culosis campaign. It is not in the nature of the disease, this group says, to be amenable to the kind of treatment that has been given to environ- mental factors.There is, as you know, a school of biological writers, who, for many years have labored to show that the tuberculosis campaign as       6 CAUSES FOR THE RECENT DECLINE IN TUBERCULOSIS  it has crystallized in official and voluntary agencies, was doomed to fail- ure. In its extreme form as stated by Dr. Given and quoted by Professor Pearl of Johns Hopkins University, the view is expressed that “there is no evidence that anything that man has done has affected in either one way or the other the decline in the mortality of tuberculosis which has been continuous for nearly three-quarters of a century.” In fact, it has been urged that such effort as has been made is likely to have done more harm than good by encouraging the reproduction of phthisical stocks and thus thwarting the influence of natural selection. The decline in tuberculosis, on this view, is ascribed to the influence of the disease itself as a selective agency in eliminating less fit stocks and thus preventing the reproduction of such individuals. Possibly, it is best to quote Professor Pearson’s own statement.*  “Has or has not the selection due to many years of heavy phthisis mortality left us with a more immune and resistant population? If it has—and that I believe will be found to be the ultimate explanation of the fall, especially the retarded fall in the phthisis death rate—then in- fection is not the only factor worth investigating. There is the question of hereditary immunity. It may be a bitter pill for mankind to swallow, when we suggest that natural selection may have done more for racial health in this matter than medical science, but it may have its compen- sations from the economic standpoint. Above all, it may suggest that Evolution helps man better than he at present knows how to help him- self, and that possibly he would learn to help himself better if he studied her processes of racial selection a little more closely.”  II. A STATEMENT OF THE FACTS  These then are the two views which, I hope, I have stated clearly and without bias. The first emphasizes the importance of the environmental factors in the causation of tuberculosis and credits the improvement in the death rate to those human activities which have centered around the amelioration of the environment of the great body of people. The second emphasizes the importance of stock and ascribes the decline to secular changes in the germ plasm of nations and of races which follow from the full interplay of natural forces rather than from the con- scious interference of man with nature. After all, these are only at- tempts at an explanation. They have no assured validity unless they square with the mass of facts. I will, therefore, rehearse some of the commoner characteristics of the disease, tuberculosis, and shall try to see how often each of the two views appears to be consistent with or in opposition to the facts. The factual data are so numerous and often so clear-cut that we should have little or no difficulty in determining which of the hypotheses we have outlined has the better of the argument.  *“The Fight against Tuberculosis and the Death Rate from Phthisis.”—pp. 34-35. LOUIS I. DUBLIN, PH.D. r 7  TABLE 3 Death rates per 100,000 Population in U. S. Registration States . Tuberculosis—all forms 1917  Urban and Rural Areas Compared*                    States Urban Rural  Total Reg. States of 1917.......... 0... cece eee 159.2 130.3 California... 0.0.0 c cece eee tect e eee 179.9 190.1 Colorado... 1. eee ec cence eee eens 250.3 ~ 129.9 Connecticut. ..... 0.0... c ec cece eee eee eens 171.0 155.7 Indiana... .. lee cece cee cece cence eee enee 155.2 133.8 Kansas... cece ccc cee ee eee cece eee ee te eee 82.2 49.6 Kentucky... cece cece eee cece ete e ene ees 227.1 191.1 White... oc cc cece ee ee eee eee ens 168.9 164.7 Colored...........00000ee ee 526.5 462.4 Maine. ...... ccc cee ce tee eee e eee eee ences 144.5 105.5 METH EINE. Soo 000000600000008b500000000000009009 225.5 178.4 White... ccc ce ccc cee cece eee eben eee e eee ence 164.4 144.1 Colored. ....... 0c cece ce ete eee eee eens 596.2 322.9 Massachusetts.....-.......0.eeeee eee “se Poi eee 141.5 152.4 Michigan... 2.1.2... ce cece cece ee eee e rece eens 131.1 90.5 Minnesota... ... 2. cect eee cee ete eee eee eens isle 86.7 MiSSOULL. .. 0. cee ec ce eee eee eens 166.8 134.7 Montana... .. cece eee ccc ce eee eee eee eee es 149.3 105.3 New Hampshire. ........ 0.00. cc ees ee ect eeeee 119.1 107.9 New Jersey... 0. fs eee bee cet eet s essence ote. 138.6 180.7 Ne WaV/on een ne ene Choe cirrnciiiciar: 168.3 131.4 North Carolina. . 00.0.0... cece cece eee eens 271.2 128.1 White eco es cies site aie oes esi edeiele lclelineolrenete enero 196.5 88.0 Colored. ..... cece ec ete ete ee eee eens 402.1 218.0 Ohio ner 167.5 117.6 Pennsylvania............ 00sec e eee cece ete eee 152.9 118.8 Rhode Island........... 0. cece eee eee eens 173.6 113.3 South Carolina... 2.0.0... cece eee eens 249.3 134.6 7 aris cn ee tre 139.2 63.3 Colored... 1... ce ee eee eee eae 384.7 195.3 Tennessee... 1... ec cee tee rete tees 234.2 190.8 White... oc cece cc eee cece eee ee neces 148.6 157.1 Colored. ...... cee eee eee tees 413.5 347.2 ITA; a a re Sr ed atid 52.0 38.6 Vermont........... Be Oe ee ETL 132.0 83.6 Virgitiia. ccc ee eee oe cae ee ee ec eee 194.4 (105.2 AE ee ee ee 128.4 106.3 (GOOG Ce ee 328.3 302.3  > Washington. ..:....c ccc reece eet eer ee ee cee tees 72.4 90.0 ie 3     Wisconsin. ......- cece ce eee eet eee eee tenets 102. 96.     *The term “urban” indicates municipalities of 10,000 inhabitants or more in 1910, smaller places being included under the term “rural.”       8 CAUSES FOR THE RECENT DECLINE IN- TUBERCULOSIS  _ Geographical Variation in the Tuberculosis Death Rate—The first fact is the great variation in the amount of fatal tuberculosis in various parts of the United States. Tuberculosis is preeminently an urban dis- ease. It is, of course, present in rural sections of the country, but to a lesser degree. The latest figures that we have showing the contrast are for the Registration States in 1917. In that year, the rate in the cities was 159.2 and in the rural area 130.3 per 100,000. The Census Bureau has not published any figures distinguishing between rural and urban areas since then. We have also figures for each of the states of the | country showing the distinction between rural and urban groups of the population. In almost every instance, the rate for the urban areas is materially higher than for the rural areas. Please remember, also, in this connection, that many of the sanatoria and hospitals for the tuber- culous are located in rural areas, and that these civil divisions are usually charged with all the deaths that occur there, although many of the patients are city residents. If the necessary corrections were made for original residence, the difference between the two rates would become all the more marked in favor of the country. Table 3 on page 7 presents the data for the urban and rural parts of the Registration States in IQI7,  Then, too, you are familiar with the fact that the death rate from tu- berculosis is very much lower in some states than in others. The lowest rate in 1921 is found in Nebraska, namely, 37.1 per 100,000. Utah had a rate of 39.9; Kansas, a rate of 43.3. Beginning with these minimum rates, the figures increase gradually until we get to New York with a rate of 102.4, Rhode Island with a rate of 108.0 and Delaware with a rate of 140.6. There are even higher rates as, for example, for California and Colorado, but we realize that these high rates are due in large measure to the migration of tuberculous persons to those areas. It is, therefore, unfair to use the last figures in interstate comparisons. But, excluding these states, the figures, varying from 37 in Nebraska to 140 in Delaware, present a fair range of variation in the states of the country. (See Table 4, page 9).  Finally, we may note the diverse rates in the large cities of the country. There is first Akron with a rate of 46.9 in 1921; Salt Lake City, 64.7, and Grand Rapids, 63.7. Further down the list, we find such cities as New York with a rate of 104.3, Cambridge, 125.0 and Toledo, 125.3. At the end of the table, we find cities like Cincinnati, 152.7, and Denver and San Antonio, with rates above 200 per 100,000. But, the high rates in the last two cities reflect the concentration of migrant tuberculosis within their limits. (Table 5, page 11).  Here, then, are our first facts. Let us try to square them with the two explanations. The environmentalists, although I use that term ad- visedly, approach this phenomenon of extreme variation in the tubercu- losis death rate without hesitation. They are familiar with it and have, in fact, based much of their assurance on it. Those who live in the country,—the rural population—they say, might be expected to have a  “4 LOUIS I. DUBLIN, PH.D.  TABLE 4  Death rates per 100,000 Population in U, S. Registration States and Area Tuberculosis—all forms.           1921 Area and States Death rate Area and States Death rate  U. S. Reg. Area.......... 99 .4 iNonthg@arolinasts 9c. ee 104.8 \\\\inite resi ee 69.3 Orig. Reg. States of 1900.. 94.2 @Colotedh a= 187.4 States ranked according Rhode Island Erna iar ire 108.0 Penne rer neOode Mississippi We a eee 111.1 White.................. 49.5 Nebraska 37.1 Colored................. 167.2 Utah. 21s eee 39.9 South Carolina............ 111-2 IKAWIEES . cobb 6h doudone ce 43.3 Vite 52.4 Vortec tc ane 61.8 Goled ee ee 168.0 Vermo eS es 2 cae 69.5 Louisiana.:............... 122.4 Nae Je White .........0..0. 00. 69.4 INIEOB PEER. 5 ooo uscec aoe: 75.2 Colored. ................ 207.2 pie BO a Ba 76.0 District of Columbia ....... 126.6 MN BESOLA fe aaig i 77.0 Une eee 75.2 New Hampshire. ....... 78.9 (Colonedinnne iar ree 278.2 oe oes eee ee 82.1 Kentuckye reer a a 131.2 oa ee 82.7 Whitel. 9.7... 2 110.0 Illinois... 2.1... eee. 84.9 Coles 333.1 a line 89.1 Wineries ee nee 133.4 Pennsylvania Sas ee sane 92.3 Te 86.1 Witte 83.8 Colored. ................ 246.0 + ee es 333.0 Maryland................. 135.5 MGI a ge ee a White.............0..-. 102.1 NS CUS ae ae Cac 301.8 New So 93.6 Tennessee................. 139.9 BlOn Gay ea ager at he 106.6 Se 62.4 (olonedin sas ea 282.0 ee eee ee 140.6 Tenareene, meres s : California............6. 00. 151.1 Nese 102.4 Colorado.................. 184.6  White.............. 97.7  Coloredhere tae Blea              lower rate. They are engaged in more healthful occupations; they live in less congested places ; they live more normal, happier lives; they enjoy a larger and better supply of food and lead altogether a more whole-  some existence.  Those who live in cities, on the other hand, are in  more congested quarters and, therefore, more subject to infection; they are often engaged in industries which are more distinctly hazardous to general health; they are on the whole poorer and less well supplied with       10 CAUSES FOR THE RECENT DECLINE IN TUBERCULOSIS  good food and fresh air. Life is harder and more hazardous in the cities and there are more chances for the natural resistance of the in- dividual to break down.  They explain the differences in the rates in the various states and cities in much the same way. They stress the fact that often those places which have the lowest rates excel in their efforts to make life better. Some are the states and cities where the people are most intelli- gent and prosperous, least crowded and driven in their efforts to make a living. Often they are the centers which have deliberately made the greatest effort to meet their tuberculosis situation by providing adequate facilities for the care of the sick and for the education and protection of the well. The geographical differences in the death rates reflect for the most part, the environmentalists say, the differences in the arrange- ments which the communities have made to make life in them happier and better. |  The geneticists or the constitutionalists, on the other hand, have not made much of the facts of geographic variation, and it is difficult to summarize the views that they hold in explanation of these facts. Obviously, it would be necessary for them to assume that the differences in the death rates represents differences in the innate stocks with various degrees of immunity or resistance to tuberculosis in the several parts of the country. They are compelled to posit a hierarchy of constitutional vigors corresponding to the low rates and the high rates. They must assume that the population in the rural areas is constitutionally better selected than that in the cities; that the population living in Nebraska, for example, is a fitter population than that living in Delaware or in New York; that the people living in Akron are innately more vigorous than those living in Providence. Without any desire to prejudge the case from this single line of evidence, it must be clear, I think, that on the score of variation of the death rate, the environmentalists have very much the better of the argument on the first count. Their assumptions are consistent with everyday observations of the differences in the modes _ of life and in the general level of wellbeing in the various parts of the country. Their explanation does not add difficulties which in their turn need expounding. But, the explanation of the second group has simply substituted one difficulty for another. There is no warrant in fact for any such degree of difference in our races and populations as their theory would demand. The people of the various states and cities are, with a few outstanding exceptions, altogether too much alike in their inherited qualities and their race histories to explain the enormous differences we have described.  Sex and Age Variations.—But, of course, geographical variation is only one and the first test. Another fact about the tuberculosis death rate which is almost universal, is the marked difference in the death rate in the two sexes. Tuberculosis mortality is much higher among males than  ‘ Death rates per 100,000 Population.  LOUIS I. DUBLIN, PH.D.  TABLE 5  Tuberculosis—all forms.  11  U. S,. Registration Cities in 1921              City Deathrate City Deathrate Akron................. 46.9 Wilmington (Del.) ......... 106.7 Salt Lake City ......... 61.7 Buffalo................... 110.7 Grand Rapids.......... 63.7 Houston.................. 114.4 Portland (Oregon) ...... 64.2 White.................. 93.3 Spokane............... 64.2 Colored................. 180.5 Rochester.............. 67.8 Philadelphia............... 115.7 Syracuse............... 69.9 Boston................... 116.4 Milwaukee............. 70.0 Albany................... 117.4 Seattle..............5. 70.7 Atlanta. ..........5.22.... 119.0 Reading............... 72.4 White.................. 65.0 Oakland............... 73.3 Colored. ................ 239.0 Hartford.............. 73.9 Kansas City (Kan.)........ 119.4 Springfield (Mass.)...... 76.5 VAMIDIEBs S 5 S'o Gav oae ec ound 76.7 Scranton.............. 77.0 Colored i saan 367.3 Omaha................ 77.1 New Bedford.............. 124.0 Youngstown........... 80.1 San Francisco ............. 124.5 New Haven............ 80.9 Indianapolis............... 124.7 Camden............... 81.1 White.................. 101.1 Newark............... 81.1 Colored. .............0.. 310.0 Lowell................. 82.7 Cambridge................ 125.0 Minneapolis............ 84.7 Toledo... -........5-5.42. 125.3 Ghicasoue an 85.0 Washington, D. C.......... 126.6 | Dallas................. 86.5 White.................. 75.2  White............... 54.8 Colored................. 278.2 Colored............. 270.4 Louisville... ............. 131.7 Jersey City............ 86.9 IME SO oS SS bs 9 0 aco 98.9 St. Louis.............. 87.4 Colored. ................ 292.7 Dayton................ 89.8 Baltimore................. 139.5 Detroit................ 89.9 White.................. 96.3 Trenton............... 90.5 Colored. ................ 388.1 Columbus.............. 93.2 Birmingham............... 145.1 Fall River............. 96.9 White.................. 52.2 Cleveland.............. 97.9 Colored. ................ 288.6 Providence............. 98.0 Richmond................. 145.2 St. Paul............... 98.8 White, ..........5...... 91.5 Bridgeport............. 99.0 Colored. ................ 264.9 Kansas City (Mo.)...... 99.7 Cincinnati................ 152, 0 Paterson.......... ae 101.9 Nashville................. 159.0 Pittsburgh............. 102.5 White.................. 95.6 Yonkers............... 102.6 Colored. ................ 309.0 Worcester.............. 102.7 Memphis................. 168.9 New York. ............ 104.3 White...............0.. 80.8 Queens.............. 77.5 Colored. ................ 317.8 Bronx............... 86.6 Los Angeles............... 173.4 Brooklyn............ 88.3 New Orleans.............. 189.8 Manhattan .......... 128.0 White.................. 136.2 Richmond........... 154.2 (Colored aan nar 341.4 Norfolk. ............... 105.6 Denver................... 219.2 White............... 51.5 San Antonio............... PIV) Colored. ............. 194.8                .12 CAUSES FOR THE RECENT DECLINE IN -TUBERCULOSIS  among females. Among the policyholders of the Metropolitan, (and similar conditions are found in the general population of the United States,) the death rate among white males during the period 1911-1920 was 36 per cent. higher than among white females. That is for all ages combined. Among the colored, the excess of the male over female mor- tality in the same period was 8 per cent. But, let us analyze these figures a little with reference to age. In Chart 3, page 13, the solid line shows the death rate from tuberculosis among males in the various age periods of life beginning with early childhood, increasing by five-year periods so old age. The broken line illustrates the facts for females. The aggregate, that is, taking the whole of life together, shows, as I have said, an excess of tuberculosis deaths among males. But, if we keep in mind the age element, the picture is somewhat different. In the first two age periods of life, up to the age of ten, the rates are so nearly the same that there is no point in making a distinction between the sexes. Beginning with age ten, the rate for females is higher than for males, and that continues for the next fifteen years of life up to age twenty-five for both the white and the colored groups. The excess of female over male mortality is, moreover, considerable. Beyond age thirty the rate for females drops rapidly and continues throughout the rest of life below that of males. The white male rate reaches its maximum at about age forty-two years and is then 477.2 per 100,000: the white female rate is highest at about twenty-seven, when it is 240.2 per 100,000. The curves for the two sexes are clearly very different. The excess rate of males is limited to the ages beyond thirty, while the females show an excess at the younger ages. ;  How do the two explanations square with this fact? The environ- mentalists would say that the boys and girls in our country are pretty much the same as to exposure and as to resistance. The higher rate for females at the ages of adolescence and early adult life they would explain on the assumption that at this period, developmental changes in the organism involve greater hazards than among males. The intense in- ternal changes that accompany puberty and the mechanism of develop- ment to maturity in the female are sufficient, they say, to explain their lower resistance. The hazard of childbearing must also be considered. Pregnancy is often fatal among those who have incipient tuberculosis, and many breakdowns are traceable to the first childbirth. Moreover, in the last twenty years, a large and ever increasing number of young women and girls have gone into gainful occupations, remaining at work for greater or lesser periods and sharing with men the hazards of work, though possibly less well equipped for such strains.  In any case, the change in the sex-ratio of tuberculosis mortality comes at about the twenty-fifth year of life, and thereafter females enjoy decided advantages. Fewer of them are engaged in industry with the hardships of factory routine; they lead’a more regular and sheltered 13  LOUIS I, DUBLIN, PH.D.  Cuart 3     Death Rates from Tuberculosis-All Forms Males and Females Compared by Color  METROPOLITAN LIFE INSURANCE CO.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-pzbw_dbp3_29as", "00000000-0000-0000-DCDC-0AE13B5B0D8E", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "A War on Consumption", "9918573882206676X147", null, "1912", "1912", null, "Pamphlets", null, "Collecting and Applying Data to Reduce Risks: Life Insurance Companies and Public Health Agencies, 1900-1950", "17", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "A WAR ON CONSUMPTION  THE NATURE OF THE DISEASE ITS EXTENT, GROWTH AND SPREAD  ITS CURE AND PREVENTION  Including Friendly Advice to Persons having Diseases of the Lungs  “Take care of the consumptive at the right time, in the right place and in the right way until he is well, and not at the wrong time, in the wrong place and in the wrong way until he is dead.’>—PRYOR     PRINTED AND DISTRIBUTED BY THE  Metropolitan Life Insurance Company OF NEW YORK  FOR THE USE OF ITS POLICY-HOLDERS  1912       IMPORTANT ANNOUNCEMENT  TO OUR POLICY-HOLDERS: .  This pamphlet has been prepared by the Company for your benefit. Read it carefully. It may help to save your life and the lives of your dear ones. Study the pictures, which show     clearly the awful ravages of tuberculosis and indicate at a glance the wide range of this dread disease. When you have made yourself thoroughly acquainted with the contents of this little volume, we believe you will be in a position to protect yourself more thoroughly against the so-called White Plague, if you will follow the instructions given. |  In addition to this pamphlet we have prepared a list of the sanatoria, tuberculosis hospitals, dispensaries, anti-tuberculosis associations and classes: in the United States and Canada. As yet there are not enough of these for all patients. In some of the smaller cities and towns there are none at all. If you wish such a list and will write to us, we shall be glad to send you one for your city, State and adjoining States.     METROPOLITAN LIFE INSURANCE COMPANY OF NEW YORK TUBERCULOSIS OR CONSUMPTION.     ITS NATURE. This disease, known also as ““phthisis,” is caused by a living plant or germ, called the “bacillus tuberculosis,’ which  multiplies with great rapidity in the living tissues of animals. The germ is called “bacillus” because it resembles a little rod,  TUBERCULOSIS GERMS.     TUBERCULOSIS GERMS IN SPUTUM, MAGNIFIED ABOUT 650 TIMES.  this being the meaning of the word “bacillus.” A picture of these germs, magnified about 650 times, is shown above. Outside of the human body, the germ may live in warm,    4 METROPOLITAN LIFE INSURANCE COMPANY.     moist, dark places for a longtime. By direct sunlight the germ is killed in a few hours, in a few days by ordinary daylight, and immediately by boiling water. If the germ finds its way into the       WHERE CONSUMPTION BREEDS.  lungs, it becomes attached and rapidly increases in number. A strong, healthy person will resist the germs, but an individual who is weak will not ordinarily repel them, and the germs will rapidly multiply until the lungs are consumed and the person dies. The germ generally obtains entrance to the body through the mouth and nose, and most frequently lodges in the air passages of the lungs. It may however, get into the glands of the neck, attack the throat, the bowels, the kidneys, the brain, or any other organ of the body, as well as the bones or the joints. Fortunately, strong, healthy people possess the power of resisting these germs, otherwise it is likely that the disease would kill off whole communities: NEARLY EVERYBODY at some period of his life BREATHES IN the living GERMS of the disease, but owing to the power of resistance of a HEALTHY body, the germs are not able to multiply. If they do not imme- diately die they produce little lumps about an eighth of an A WAR ON CONSUMPTION. > 5     inch in diameter called ‘‘tubercles,’’ from which comes the name ‘“‘tuberculosis.” If these form in the lungs, . they continue to grow, soften, break open, and are eventually expelled by coughing or otherwise. For this reason, the sputum, or spit, of an individual who has consumption is filled with the germs of tuberculosis.  In the early stages of the disease the germ is found in small numbers in the sputum, in larger numbers as the disease progresses, and in countless millions in the later stages. Unless this sputum is destroyed by burning or by disinfectants, it may become the most common method of carrying tuberculosis to _ other individuals.  It is now generally believed that CONSUMPTION IS NOT INHERITED. It is true that the children of con- sumptive parents are frequently of low vitality and generally of poor physique. This does not mean, however, that they are bound to become consumptives. They will get consumption only if the germ enters | their body at a time when it is in poor condition. Being weak, and being unable to re- sist the action of the bacillus, they are more susceptible than indi- viduals who are phys- ically well and strong. There is no reason whatever why such children, if properly cared for, should not grow to be healthy and well-developed men and women who will never get Consumption.     By courtesy of the Metropolitan Magazine.       6 METROPOLITAN LIFE INSURANCE COMPANY.     It is possible to HAVE THE DISEASE for some time AND NOT KNOW IT or suspect it. Anexamination of the patient’s chest by a good doctor and a microscopical examination of the sputum may discover it, but if both of these tests fail, it does not definitely mean that tuberculosis is not present. Repeated EXAMINATIONS SHOULD BE MADE from timeto time IF THE EARLIER SIGNS OF TUBERCULOSIS ARE PRESENT. Among these are the following: Slight cough, lasting a month or longer; loss of weight; slight fever each after- noon; bleeding-from the lungs; tired feeling. If these symptoms are present, do not delay, but consult a physician AT ONCE.  From what has been said above, it will be seen that as consumption isa GERM DISEASE, it isa COMMUNICABLE DISEASE, and as such a PREVENTABLE DISEASE.  - IF IT IS TREATED PROPERLY IN ITS EARLY STAGES, IT IS-A CURABLE DISEASE.  THE EXTENT OF THE DISEASE.  Tuberculosis is the chief cause of death in middle life. About one-third of all the deaths that occur between the ages of twenty and forty-five years are from tuberculosis. IT IS ESTIMATED THAT ONE HUNDRED AND SIXTY THOUSAND PEOPLE DIE EACH YEAR IN THE UNITED STATES FROM TUBERCULOSIS. — Between the ages of twenty and thirty-five, one-half of all deaths are from tuberculosis. During the four years of the Civil War the total loss of life was two hundred and five thousand and seventy. In the period of four years, the tubercle bacillus now destroys in the United States alone over six hundred thousand people.  When we hear of yellow fever, we make every possible effort to stamp out the disease at once. The same is true of smallpox and other so-called contagious diseases, and yet it is estimated that the total number of deaths from yellow fever in the United States during one hundred years was only ‘one A WAR ON CONSUMPTION. _ i     hundred thousand. The annual loss in money from consump- tion in the United States is estimated very conservatively at $330,000,000. IS IT WORTH WHILE FOR YOU TO HELP us and all others who are engaged in this war against tuberculosis?  DEATHS ett Prot.  a! ie beat Sus  ait bade) aoe tas THE UNITED. SU  Pane YELLOW Aa  By courtesy of the Metropolitan Magazine  a: Ma i: BL) l     _ Take the experience of our own Company. At the end of 1911 we had 11,100,387 Industrial policies in force. During 1911, 18.67 per cent. of our deaths of males was from tubercu-       8 METROPOLITAN LIFE INSURANCE COMPANY.     losis, and 15.98 per cent. of our deaths of females; the total per- centage of deaths from tuberculosis, both sexes, was 17.3 per cent.  You will understand the awfulness of this scourge best, perhaps, when we tell you that one of our policy-holders died from tuberculosis every thirty-two minutes from the lst of January, day and night, to the 31st of December, 1911. During this period one policy-holder of the Metropolitan died every six minutes, of whom, as you can see, more than every sixth one died of tuberculosis.  THE SPREAD OF THE DISEASE.  THE GREAT MEDIUM FOR THE SPREAD OF THE DISEASE IS THE CONSUMPTIVE’S SPIT. When the consumptive coughs or sneezes, he fills the air in front of him with particles of moisture almost too small to be seen, which are filled with germs. When he spits upon the floor or the sidewalk, millions of germs are deposited, and are ready to find their way upon the clothes or hands and thus into the mouths and into the lungs, stomach and intestines of children who play upon the floor or walk. The careless consumptive’s handker- chief, the pocket in which he carries his handkerchief, the bedding, and especially the pillow cover, and the towel used by him, are apt to be laden with germs.  When a member of the family has consumption and the spit is not carefully collected and destroyed, the house is apt to become infected and other members of the family take the disease.  When a consumptive removes or dies, and other persons move into the house, some of them are very apt to take the disease unless the house is thoroughly cleaned and disinfected, particularly the floors and walls.  Impure air and deficient sunlight favor the development of the germ. For this reason, a consumptive is more frequently met with in the crowded parts of cities, where houses are built closely together and in which air cannot circulate freely, and    A WAR ON CONSUMPT ION. 9  where sunlight does not enter. Over-crowded, poorly-ventilated houses, offices and workshops, all help to spread the disease. | A consumptive is much less common in the suburbs where people live in separate houses.  HOW THE GERMS OF CONSUMPTION ARE CARRIED FROM THE SICK TO THE WELL.        The spit dries and care- less sweeping, dusting or draughts cause the germs to float in the air.  Consumptive spitting on floor. Flies feeding on it, carry the germs of the disease to food.  The germs may enter the bodies of children play- ing on the floor, through sores or wounds.              GIVE US A BITE     Others may get the disease by breathing or swallowing the germs.  Spray given off in sneezing or coughing, contains germs in a moist and active state.  into the mouth after a consumptive has poisoned them with his spit.     Dirt, dampness and darkness are three of the most active | friends of the tuberculosis germ. On the other hand, SUN- | SHINE, PURE AIR AND CLEANLINESS ARE ITS GREATEST ENEMIES. It is highly desirable for this    10 METROPOLITAN LIFE INSURANCE COMPANY.     reason that you keep your home perfectly clean and constantly remove from it dust and dirt. Every room should have a thorough spring and fall house cleaning each year. Rooms which have been occupied by consumptives frequently become _ infected with the germs. Such rooms should never be used without having been previously disinfected. Hot water and soap should be used freely.  CONSUMPTION’S ALLIES—AVOID THEM AND YOU ARE SAFEGUARDING AGAINST | THE DISEASE.                   Intemperance and The closed window. other excesses. .        Sondet sfeeping, living ’ Mouth breathing,  and working rooms. often due to adenoids.  REMEMBER THAT THE MOST ACTIVE AGENT FOR SPREADING TUBERCULOSIS IS THE SPIT OF THE CONSUMPTIVE. If this is thoroughly burned or destroyed at once there is little danger of infection.  If the body is weakened by overwork, or by dissipation or  by excesses of any kind, the individual is more apt to contract tuberculosis than if he keeps himself strong and well. In    A WAR ON CONSUMPTION. 11     fact, healthy persons living a proper life when infected frequently » ~ get over the disease so quickly and so readily that they do not even know that they have had it.  PEOPLE WHO ARE ADDICTED TO THE USE OF ALCOHOL IN ANY FORM ARE MORE LIKELY TO GET TUBERCULOSIS THAN OTHERS.  THE CURE OF THE DISEASE.  Consumption is no longer the hopeless disease of the past—IT IS CURABLE.  The earlier it is detected in an individual case, the greater are the possibilities for a cure. Therefore, HEI.P YOUR FRIEND, YOUR NEIGHBOR, YOUR RELATIVE, TO REC- OGNIZE AND TREAT THIS DISEASE AT THE START.  If you should be unfortunate enough to be afflicted with tuberculosis or consumption, FIRST OF ALL GET THE ADVICE OF A RELIABLE PHYSICIAN and follow his instructions conscientiously and religiously. There is no anti- toxin for treating tuberculosis such as is used in diphtheria. The ONLY CURE we know FOR TUBERCULOSIS IS TO INCREASE THE BODILY STRENGTH, so that the body will resist and gradually destroy the germ. This is a slow process. The best means are plenty of fresh air all the time, plenty of good food, rest, freedom from worry, and out- of-door life. Medicines are of very little use in the cure of consumption. PATENT MEDICINES DO NOT CURE CONSUMPTION. MOST OF THEM ARE ALCOHOLIC DRINKS IN DISGUISE, WHICH ARE DANGEROUS TO THE CONSUMPTIVE.  For the best treatment of tuberculosis, so as to afford the patient outdoor treatment as much as possible, special hospitals, called sanatoria, have been erected in all parts of the United    12 METROPOLITAN LIFE INSURANCE COMPANY.     States and Europe. It is highly desirable, in order to cure the consumptive as rapidly as possible, that he be treated in such a sanatorium. ‘There are, however, as yet not sufficient of these to accommodate everybody, and for this and for other reasons it is frequently necessary for the patient to be treated at home. If the latter method be resorted to, it should be done under the advice of a physician.  IN CASE OF CONSUMPTION, LOOK TO THESE FOR CURE.     THE DOCTOR. SUNLIGHT. _ OUT-DOOR AIR      REST  The physician will tell you how to carry on this HOME TREATMENT in the best manner. A person who has pul- monary tuberculosis, or consumption, is not dangerous to have in the house if he is careful and clean, and if he follows the usual rules laid down to prevent infection of other members of the family. The patient’s window should be open day and night, and he should occupy the room alone. Preferably there should be no carpet or rug on the floor. The sheets and the body linen should be frequently washed and well boiled. The    A WAR ON CONSUMPTION. 13     roca should be dusted with a damp cloth or a damp broom. The dishes from which he eats should be used by him exclusively and should be well boiled.  A CAREFUL CONSUMPTIVE—NOT DANGEROUS TO LIVE WITH.      Coughs, spits and  sneezes into paper ee . .  or cloth,— Burns or boils it ‘or puts it into a before it dries,— disinfectant,— —                         <—e fi ron eee ame a S SS a Washes her hands Always uses the same And sleeps before and after dishes and boils them alone. uh eating,— in water before wash- J, t ¥  ing with other dishes,—°  THE PREVENTION OF THE DISEASE  To prevent consumption, two things are required: (1) to collect and destroy the germs in the consumptive’s spit, and (2) to: keep the body in good general health, so that it will be able to resist the germs.  ' ‘The consumptive, by careless spitting, almost certainly will give the disease to his family, friends or fellow workmen, but if he carefully destroys all his spit, he is harmless. He should preferably use paper napkins, which can be burned immediately. They should not be carried loose in the pocket after using. When coughing or sneezing he should hold one of these before    14 METROPOLITAN LIFE INSURANCE COMPANY.     his mouth. If the handkerchief is ever used for this purpose it should be immediately disinfected, by being placed either in boiling water or in a three per cent. solution of carbolic acid.  He should spit into a pasteboard sputum cup, which at the end of each day can be burned, or into a vessel which can be easily and completely cleaned daily. The ordinary spittoon is most difficult to clean, and should never be used by a con- sumptive. When the consumptive is at work or riding on the street-car, or traveling, he should use a pocket sputum cup or flask which can be kept tightly closed until he can empty it at night.  Paper napkins and sputum cups are cheap. You can learn where to get them through your physician.  THE CAREFUL CONSUMPTIVE IS NOT DAN- GEROUS. Tuberculosis is not contagious by the breath (except when the consumptive coughs or sneezes), or in the Same way as small-pox, or diphtheria, or scarlet fever, but through the sputum. hess  Even though every effort is made to collect and destroy the germs, it is probable that EVERY ONE OF US on account of the prevalence of the disease and the large number of con- sumptives who are careless or do not understand the importance of destroying their spit, WILI, RECEIVE AT SOME TIME OR OTHER THE GERMS IN OUR LUNGS. It is most important, therefore, that the lungs be in proper condition and that the general health be good.  THOROUGH VENTILATION OF BEDROOMS is one of the most important means to this end. ‘Too often the bed- room is small, dark and unventilated, the windows sometimes being nailed and shut. To nail one’s bedroom window shut is to drive a nail into one’s coffin. WE SPEND MORE HOURS EACH DAY IN OUR BEDROOMS THAN IN ANY OTHER ROOM IN THE HOUSE; yet they are usually the smallest, least lighted, and least ventilated.    A WAR ON CONSUMPTION. 15     Sleeping out of doors is urged upon the consumptive, and it is probable that most of us would be in far better condition to resist tuberculosis if we slept out of doors a good portion of the year. .     A CHEAP TEMPORARY PORCH PROTECTED. BY AN AWNING AND SUPPORTED BY BRACES SET AT AN ANGLE.  Excessive hours of hard work, whether on the farm or in the factory, lower the vitality. INSUFFICIENT FOOD OR INDIGESTIBLE FOOD ALSO INJURES THE HEALTH. The steady drinking of alcoholic liquors, whether or not we become drunk, injures the body.  ‘WILL YOU DO YOUR PART?                The Light That Never Fails", null, null, null, "Metropolitan Life Insurance Company", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yrur~6wi7.irqu", "00000000-0000-0000-6F64-212DC3340995", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Disqualifications for Military Service in the United States", "9918573882206676X1", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. This section of the published report listed the general categories of physical deficits that would disquality a prospective soldier from service. Pages LI-LVI of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1.", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "6", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "INTRODUCTORY.  LI  DISQUALIFICATIONS FOR MILITARY SERVICE IN THE UNITED  STATES.  AND MORAL INFIRMITIES.  Manifest imbecility; dementia; dipsomania.  Conviction for felony.  Desertion, as evidenced by branding.  Obstinate malingering, (prevents re-enlistment.)  GENERAL PHYSICAL DISQUALIFICATIONS.  Feebleness of constitution.  Scrofulous diathesis.  Syphilis which has resisted treatment and has severely injured the health  Cancer, or the cancerous diathesis.  Habitual drunkenness.  SPECIAL DISQUALIFICATIONS.  FIRST CLASS  ORGANS OF SPECIAL SENSE AND ACCESSORY APPARATUS  A.-Affections of the auditory apparatus.  Deafness.  Inveterate chronic purulent otorrhoea.  Tumors, malignant disease or caries of mastoid cells, labyrinth, or tympanum.  Perforation of the membrana tympani.  Obliteration or imperforation of the auditory canal.  Malformation or loss of external ear.  JB.-Affections of the eye and its appendages.  Total loss of sight; loss of an eye, or loss of sight of right eye; cataract; loss of  crystalline lens of right eye.  Ophthalmia, when chronic, purulent, gonorrhoeal, or likely to be destructive in its  results.  Encanthis, if malignant; pterygium, if extensive.  Conicity of the cornea.  Opacity of the cornea; nebula, albugo, and leucoma, if upon the right eye and inter-  fering with vision.  Congenital defects of iris of right eye; rheumatic or syphilitic iritis; adhesions of  iris to the capsule of the lens; staphyloma scleroticae; glaucoma.  Myopia.  Strabismus of the right eye, if decided.  Ilydrophthalmia; exophthalmia. LII  INTRODUCTORY.  Fistula lachrymalis; epiphora; closure of duct or distention of sac.  Ptosis of right eye-lid; incessant spasmodic motion of lids; adhesions of eye-lids;  trichiasis of long standing; large encysted tumors.  Chronic abscess of the orbit.  C.-Affections of the olfactory apparatus.  Cancer of the integument; noli me tangere; erosive ulcers of the follicles.  Deformities of the nose greatly disfiguring the face, altering the voice, and impeding  respiration; loss of the whole or part of the nose.  Affections of the septum, permanent or chronic, sufficient to close the nasal fossae ;  polypus, if large enough to produce great deformity and embarrassment of  respiration.  Ozaena; purulent and fetid discharge from old intractable ulcerations.  D.-Aflections of the mouth and gustatory apparatus.  Hare-lip, simple, compound, or complicated.  Loss of the whole or part of either lip ; unsightly mutilations of the lips from wounds,  burns, or disease.  Loss of the whole or part of either maxilla; un-united fracture; ankylosis.  Deformities of either jaw, interfering with mastication, speech, or the tearing of the  cartridge.  Loss of the incisor and canine teeth of both jaws.  Cancerous or erectile tumors; cicatrices producing deformity.  Mutism.  Hypertrophy or atrophy of the tongue.  Stammering or stuttering, if inveterate.  Mutilation or partial or total loss of tongue.  Adhesion of tongue to parietes of mouth, or other adhesions preventing free motion.  Malignant disease of tongue; chronic and inveterate ulceration.  Congenital fissure of bones of the palate, or fissure produced by disease.  Salivary fistula; bucco-nasal fistula.  Chronic engorgement of the tonsils, sufficient to interfere with deglutition or phonation.  Great deformities of the face and loss of substance of the cheeks.  SECOND CLASS.  A.-Head and spinal column.  Imperfect ossification of the bones of the cranium, evidenced by the persistence of  the fontanelles, and sometimes separation and mobility of the sutures.  Monstrosity in size of the head; considerable deformity, the consequence of fracture.  Serious lesions of the skull, the consequence of complicated wounds, considerable  fractures, or the operation of trephining; caries and exfoliation involving the  whole thickness of the bone.  Injuries of cranial nerves affecting their functions. INTRODUCTORY.  L11I  Fungous tumors of the dura mater.  Caries of the spine; spina bifida; curvature in the cervical, dorsal, or lumbar region;  lumbar abscess; rickets; fracture and dislocation of the vertebrae.  Angular deformity, including gibbosity of the anterior and posterior part of the thorax.  B.-Affections of the cerebro-spinal nervous system.  Epilepsy; chorea.  Paralysis agitans; paraplegia; hemiplegia; paralysis of any part of the body.  Neuralgia, if intractable.  THIRD CLASS.  NECK AND CONTAINED ORGANS.  Chronic laryngitis; induration and scirrhus of epiglottis; polypus of the larynx;  aphonia due to any of these causes.  Dysphagia due to stricture of the oesophagus.  Goitre, if large enough to impede respiration.  Engorgement, scrofulous enlargement, and ulceration or abscess of the lymphatic glands.  Cicatrices producing deformity, retraction of jaw, and rigidity.  Fistula of larynx or trachea.  Wry-neck, if permanent in character.  Osseous degeneration of thyroid gland.  FOURTH CLASS  CHEST AND THORACIC ORGANS.  Malformation of chest or badly united fracture of ribs or sternum sufficient to interfere  with respiration; caries or necrosis of ribs; deficiency in extent of expansive  mobility; greatly diminished vital capacity; evident predisposition to phthisis.  Phthisis pulmonalis; chronic pneumonia; chronic pleurisy and emphysema; chronic  bronchitis ; asthma; haemoptysis.  Organic disease of the -heart and large arteries; hypertrophy; valvular insufficiency;  aneurism; serious and protracted functional derangement; dropsy dependent on  disease of heart  FIFTH CLASS.  ABDOMEN AND DIGESTIVE APPARATUS.  Chronic gastritis; chronic gastro-enteritis ; chronic disease of liver or spleen; engorge-  ment or tubercular infiltration of mesentery, (marasmus;) chronic diarrhoea;  chronic dysentery; taenia; chronic peritonitis, with or without effusion; ascites j  obesity.  Dyspepsia, if of long standing and accompanied by general emaciation, vomiting, &amp;c.  Haemorrhoids, if large, internal, bleeding, ulcerated, and painful.  Malformation or stricture of the rectum; prolapsus ani; fistula in ano; considerable  fissure of the anus; artificial anus.  Hernia in all situations.  Extensive cicatrices from incised wounds. L1V  INTRODUCTORY.  SIXTH CLASS.  GEN1T0-UR1NARY APPARATUS.  Loss of the penis ; permanent stricture of the urethra.  Loss of both testicles from any cause; permanent retraction of one or both testicles  within the external ring.  Malignant disease of testicle; scrofulous or syphilitic sarcocele; hydrocele, if large;  atrophy of testicle; varicocele and cirsocele, if large enough to impede walking,  or if it have produced atrophy of the corresponding testicle.  Epispadia and hypospadia, when not farther from the root of the penis than the middle.  Incontinence of urine; urinary fistula; discharge of urine by the umbilicus;  hsematuria, if evidence of organic disease.  Eversion of the bladder; loss of substance of the hypogastric region.  Chronic enlargement of the prostate; stone in the bladder; chronic cystitis of long  standing.  Abscess of kidney; fatty degeneration of kidney; closure of ureter by a calculus;  renal dropsy; diabetes.  Hermaphroditism.  Spermatorrhoea, if it have impaired the genei al health.  SEVENTH CLASS.  UPPER AND LOWER EXTREMITIES.  A.-Disqualifications common to both upper and lower extremities.  Chronic rheumatism, with swelling of the joints, enlargement of the surrounding  tissues, earthy deposits, contraction of the tendons, and wasting and loss of motion.  Chronic diseases of the joints.  Old or irreducible dislocations or false joints.  Severe sprains, resulting in impaired mobility.  Relaxation of the capsules or other ligaments of the joints; voluntary or involuntary  dislocation of the bones.  Complete or partial ankylosis of an important articulation.  Sinuses communicating with the osseous cavities, the articulations, and with the  thickness of spongy bones.  Dropsy of a joint.  Badly united fractures.  Defective or excessive curvature of long bones; rickets ; caries; necrosis; exostosis.  Atrophy of a limb; paralysis of a limb.  Extensive, deep, and adherent cicatrices.  Aneurism.  Contraction or permanent retraction of a limb or of a portion of a limb.  Loss of a limb or of an essential part thereof. INTRODUCTORY.  LV  B.-Disqualifications proper to upper extremities.  Extraordinary size of the hands, proceeding from a natural lymphatic engorgement, or  a general varicose state of the venous capillaries, or from habitually ulcerated  chilblains.  Fingers adherent or united, supernumerary, double, or branched; permanent flexion  or extension of one or more fingers, except the little finger, and irremediable loss  of motion of these parts.  Loss of the first phalanx of the thumb of the right hand.  Total loss of either thumb.  Total or partial loss of the index-finger of the right hand.  Loss of the first and second phalanges of the fingers of the right hand.  'Total loss of any two fingers of same hand.  Mutilation of the last phalanges of the fingers of either hand.  C.-Disqualifications proper to inferior extremities.  Varicose veins, voluminous and multiplied.  Chronic ulcers, if of long standing, not easily curable, and likely to be aggravated  by motion.  Extensive adherent cicatrices, if dark-colored and the result of former ulceration.  Lameness.  Badly united fracture, producing much shortening.  Knock-knees, if the deformity be excessive.  Club-feet; splay-feet, where no arch exists, the tuberosity of the scaphoid bone  touching the ground.  Ingrowth of the nail of the great toe, if deep and accompanied with inflammation or  ulceration.  The toes joined together, double, or branching.  That deformity in which the great toe crosses the other toes, and in which there is  great prominence of the articulation of the great toe and first metatarsal bone.  Over-riding and superposition of all the toes.  Loss of a great toe; loss of three toes of same foot.  Mutilation of the last phalanges of the toes of either foot.  The retraction or inflexion of all the toes of the same foot or of two toes.  The permanent retraction of the last phalanx of a toe in which the free border of the  nail bears upon the ground, or flexion at a right angle of the second phalanx of  the second toe upon the first, with ankylosis of the articulation.  Fetid sweat of the feet.  EIGHTH CLASS  SKIN AND APPENDAGES.  Chronic ekzema. 5  Herpes circinnatus; herpes capitis, when chronic.  Chronic pemphigus; scabies, when of long standing and herpetic in character.  Lepra; psoriasis ; pityriasis ; icthyosis.  Lupus serpiginosus; 1. devorans; cheloid tumors. LVI  INTRODUCTORY.  Porrigo; sycosis; the syphilides.  Alopaecia, if total.  Naevi; large, livid, hairy, and unsightly spots on the face  When the imperious need of obtaining men to fill up the dwindling regiments of  the national forces resulted in the passage of the enrollment-law, a concise code of  instructions to medical officers, for their guidance in examining recruits, formed a  part of the official regulations issued by the Provost-Marshal-General for the govern-  ance of the conscription. Although the more copious rules just given form the present  official standard, the instructions to enrolling surgeons are well worthy of preservation  for their own merit and for their pertinence to the history of the draft. In the  supplementary part of this volume will be found the expressed opinions of a large  number of the surgeons entrusted with the onerous duty of examining men drafted  or offered for service, as to the sufficiency and equitable adaptedness of this code for  the purpose intended. It met, for the most part, with their emphatic approval, the  alterations recommended being chiefly technical or comparatively unimportant.  INSTRUCTIONS FOR THE PHYSICAL EXAMINATION OF DRAFTED MEN AND SUBSTITUTES,  AND GENERAL REGULATIONS CONCERNING.1  The duty of inspecting men and of determining whether they are fit or unfit for  the military service of the country requires the utmost impartiality, skill, and circum-  spection on the part of the examining surgeon and board of enrollment; for upon the  manner in which this duty is performed will depend, in a very great degree, the  efficiency of the Army.  In the examination, the examining surgeons will bear in mind that the object of  the Government is to secure the services of men who are effective, able-bodied, sober,  and free from disqualifying diseases.  The .examining surgeons will also remember that the object of the drafted men,  in claiming exemption, may be to escape from service by pretended, simulated, or  factitious diseases, or by exaggerating or aggravating those that really exist, and that  the design of substitutes frequently is to conceal disqualifying infirmities. %  The examination by the examining surgeon is to be conducted in the day-time,  in the presence of the board of enrollment only, and in a room well lighted and  sufficiently large for the drafted man to walk about and exercise his limbs, which he  must be required to do briskly.  The man is to be examined stripped.  The surgeon will habitually conduct his examination of a man in the following  order, to ascertain:  1. Whether his limbs are well formed and sufficiently muscular; whether they  are ulcerated or extensively cicatrized; whether he has free motion of all his joints;  and whether there are any varicose veins, tumors, wounds, fractures, dislocations, or  sprains that would impede his marching, or prevent continuous muscular exertion.  2. Whether the thumbs and fingers are complete in number, are well formed,  and their motion unimpaired.  1 Revised regulations for the gorerninent of the Bureau of the Provost-Marshal-General. Washington, April 1, 18G4.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yc4g-syq8_r4x3", "00000000-0000-0000-C11D-A608D043EF2F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Instructions for the Physical Examination of Drafted Men and Substitutes, and General Regulations Concerning", "9918573882206676X2", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. This section of the published report described the medical examination procedure to be followed by the army surgeons. Pages LVI-LIX of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1.", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "4", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "LVI  INTRODUCTORY.  Porrigo; sycosis; the syphilides.  Alopaecia, if total.  Naevi; large, livid, hairy, and unsightly spots on the face  When the imperious need of obtaining men to fill up the dwindling regiments of  the national forces resulted in the passage of the enrollment-law, a concise code of  instructions to medical officers, for their guidance in examining recruits, formed a  part of the official regulations issued by the Provost-Marshal-General for the govern-  ance of the conscription. Although the more copious rules just given form the present  official standard, the instructions to enrolling surgeons are well worthy of preservation  for their own merit and for their pertinence to the history of the draft. In the  supplementary part of this volume will be found the expressed opinions of a large  number of the surgeons entrusted with the onerous duty of examining men drafted  or offered for service, as to the sufficiency and equitable adaptedness of this code for  the purpose intended. It met, for the most part, with their emphatic approval, the  alterations recommended being chiefly technical or comparatively unimportant.  INSTRUCTIONS FOR THE PHYSICAL EXAMINATION OF DRAFTED MEN AND SUBSTITUTES,  AND GENERAL REGULATIONS CONCERNING.1  The duty of inspecting men and of determining whether they are fit or unfit for  the military service of the country requires the utmost impartiality, skill, and circum-  spection on the part of the examining surgeon and board of enrollment; for upon the  manner in which this duty is performed will depend, in a very great degree, the  efficiency of the Army.  In the examination, the examining surgeons will bear in mind that the object of  the Government is to secure the services of men who are effective, able-bodied, sober,  and free from disqualifying diseases.  The .examining surgeons will also remember that the object of the drafted men,  in claiming exemption, may be to escape from service by pretended, simulated, or  factitious diseases, or by exaggerating or aggravating those that really exist, and that  the design of substitutes frequently is to conceal disqualifying infirmities. %  The examination by the examining surgeon is to be conducted in the day-time,  in the presence of the board of enrollment only, and in a room well lighted and  sufficiently large for the drafted man to walk about and exercise his limbs, which he  must be required to do briskly.  The man is to be examined stripped.  The surgeon will habitually conduct his examination of a man in the following  order, to ascertain:  1. Whether his limbs are well formed and sufficiently muscular; whether they  are ulcerated or extensively cicatrized; whether he has free motion of all his joints;  and whether there are any varicose veins, tumors, wounds, fractures, dislocations, or  sprains that would impede his marching, or prevent continuous muscular exertion.  2. Whether the thumbs and fingers are complete in number, are well formed,  and their motion unimpaired.  1 Revised regulations for the gorerninent of the Bureau of the Provost-Marshal-General. Washington, April 1, 18G4. INTRODUCTORY.  LVU  3. Whether the feet are sufficiently arched to prevent the tuberosity of the  scaphoid bone from touching the ground; whether the toes are complete in number,  do not overlap, are not joined together; and whether the great toes are free from  bunions.  4. Whether he has any inveterate and extensive disease of the skin.  5. Whether he is sufficiently intelligentis not subject to convulsions; and whether  he has received any contusion or wound of the head that may impair his faculties.  6. Whether his hearing, vision, and speech are good, and whether the eye and its  appendages are free from disqualifying diseases.  7. Whether he has a sufficient number of teeth in good condition to masticate his  food properly, and to tear his cartridge quickly and with ease. The cartridge is  torn with the incisor, canine, or bicuspid teeth.  8. Whether his chest is ample and well formed, in due proportion to his height,  and with power of full expansion.  9. Whethe? there is any structural or serious functional disease of the heart.  10. Whether the abdomen is well formed and not too protuberant; whether  either the liver or spleen is considerably enlarged; and whether the rectum and anus  are free from disqualifying diseases.  11. Whether the spermatic cords and testes are free from diseases which would  impair his efficiency; whether the testes are within the scrotum; and whether he has  any rupture.  12. Whether there is any organic disease of the kidney or bladder, or permanent  stricture of the urethra.  13. Whether his physical development is good, and constitution neither naturally  feeble nor impaired by disease, habitual intemperance, or solitary vice ; whether  he is free from phthisis, scrofula, and constitutional syphilis ; and whether he is epilep-  tic, imbecile, or insane.  Many of the physical defects above mentioned are insufficient to disqualify for  military service. In determining whether the man is fit or unfit for service, the board  must be governed by the list of diseases and infirmities enumerated in paragraph 85.  Paragraph 85.  The following diseases and infirmities are those which disqualify for military  service, and for which only drafted men are to be \"rejected as physically or mentally  unfit for the service,\" viz :  1. Manifest mental imbecility.  2. Insanity. This includes well-established recent insanity, with liability to a  recurrence.  3. Epilepsy. For this disability the statement of the drafted man is insufficient, and  the fact must be established by the duly-attested affidavit of a physician in  good standing, who has attended him in the disease within the six months  immediately preceding his examination by the board, and, in addition thereto,  by such other evidence as the board may require. LV111  INTRODUCTORY.  4. Paralysis, general or of one limb, or chorea; their existence to be adequately  determined. Decided atrophy of a limb.  5. Organic diseases of internal organs, which have so seriously impaired his general  health as to leave no doubt of his incapacity for military service, and which  prevents his pursuing any equally laborious occupation in civil life.  6. Developed tuberculosis.  7. Cancer; aneurism of the large arteries.  8. Inveterate and extensive disease of the skin, such as will necessarily impair his  efficiency as a soldier.  9. Permanent physical disability of such degree as to leave no doubt of the man's  unfitness for military service.  10. Scrofula, or secondary syphilis, which has so seriously impaired his general health  as to leave no doubt of the man's incapacity for military service.  11. Chronic rheumatism, unless manifested by positive change of structure, wasting of  the affected limb, or puffiness or distortion of the joints, does not exempt.  Impaired motion of joints and contraction of the limbs, alleged to arise from  rheumatism, and in which the nutrition of the limb is not manifestly im-  paired, are to be proved by examination while in a state of anaesthesia, induced  by aether only.  12. Total loss of sight of right eye; cataract of right eye; loss of crystalline lens of  right eye.  13. Partial loss of sight of both eyes, vision being so greatly impaired as to leave no  doubt of the man's inability to perform military duty. Serious permanent  diseases of the eye or eye-lids so manifestly affecting the use of the eyes as  to leave no doubt of the man's incapacity for military service. Nearsighted-  ness does not exempt.  14. Total loss of nose; deformity of nose so great as seriously to obstruct respiration;  ozaena, dependent on caries in progress.  15. Decided deafness. This disability must not be admitted on the mere statement of  the drafted man, but must be proved by the existence of positive disease or  by other satisfactory evidence, and it must be so decided as to leave no  doubt of the man's unfitness for military service. Chronic purulent otorrhoea.  16. Incurable diseases or deformities of either jaw, such as will necessarily greatly  impede mastication or speech. Ankylosis of the lower jaw ; caries of the  bones of the fa«e, if in progress; cleft palate, (bony;) extensive loss of sub-  stance of the cheeks, or salivary fistula.  17. Dumbness; permanent loss of voice; not to be admitted without clear and satis-  factory proof.  18. Total loss of tongue; hypertrophy, atrophy, mutilation, or obstinate chronic ulcera-  tion of the tongue, if sufficient in degree to interfere seriously with the use  of the organ.  19. Stammering, if excessive and confirmed; to be established by satisfactory evidence  under oath.  20. Total loss of all the front teeth, the eye-teeth, and first molars, even if only of  one jaw. INTRODUCTORY.  LIX  21. Tumors, or wounds of the neck, impeding respiration or deglutition ; fistula of  larynx or trachea ; torticollis, if of long standing and well marked.  22. Excessive deformity of the chest, or excessive curvature of the spine, sufficient to  prevent the carrying of arms and military equipments; caries of the spine,  ribs, or sternum, attended with ulceration.  23. Hernia.  24. Artificial anus; stricture of the rectum; prolapsus ani. Fistula in ano, if exten-  sive or complicated with visceral disease.  25. Old and ulcerated internal haemorrhoids, if in degree sufficient to leave no doubt  of the man's unfitness for military service. External haemorrhoids are no  cause for exemption.  26. Total loss or nearly total loss of penis; epispadia or hypospadia at the middle or  near the root of the penis.  27. Incurable permanent organic stricture of the urethra, in which the urine is passed  drop by drop, or which is complicated by disease of the bladder; urinary  fistula. Recent or spasmodic stricture of the urethra does not exempt.  28. Incontinence of urine is not, of itself, a cause for exemption. Stone in the bladder,  ascertained by the introduction of the metallic catheter, is a positive disquali-  fication.  29. Confirmed or malignant sarcocele; hydrocele, if complicated with organic disease  of the testicle. Varicocele is not, in itself, disqualifying.  30. Loss of a hand or foot.  31. Wounds which would manifestly incapacitate the man for military service; mus-  cular or cutaneous contractions from wounds, burns, or tumors, which  would prevent marching, or otherwise manifestly incapacitate the man for  military service.  32. Fractures, irreducible dislocations or ankylosis of the large joints, or chronic  diseases of the joints or bones, that would prevent marching, or otherwise  unfit the man for military service.  33. Total loss of right thumb ; loss of ungual phalanx of right thumb ; total loss of  any two fingers of same hand ; loss of the first and second phalanges of the  fingers of right hand. Permanent extension or permanent contraction of  two fingers of right hand; all the fingers adherent or united.  34. Club-feet; total loss of a great toe. Other permanent defects or deformities of  the feet, such as will necessarily prevent marching.  35. Varicose veins of inferior extremities, if large and numerous, and accompanied  with chronic swellings or ulcerations.  36. Chronic ulcers; extensive, deep, and adherent cicatrices of lower extremities.  No limits of stature are established for drafted men, beyond which they shall be  exempted from military service. The matter of stature should be considered by the  board only in the general examination as to the physical fitness of the man for military  service. •  The regulations issued by the principal governments of Europe, and those in force  in our own country, touching the physical qualifications of the recruit, are much alike", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-c79j-768b~zerz", "00000000-0000-0000-7AF9-0AD669C3B632", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Nomenclature of Diseases", "9918573882206676X3", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this discussion of the disease nomenclature used in the analysis. Pages 3-12 of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1.", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "10", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "REVIEW OF THE TABLES AND THEIR RESULTS.  \".If we are to devote our attention, before all things, to what can be measured and weighed,  the living man is the first object which demands our investigation. The 'average man' of Europe  having been determined by Quetelet, his system is now applied to races.\"-Carl Vogt.  Iii consulting the tables of Vol. II, it will be found of importance to bear in mind  the exact meaning to be attached to certain terms which appear in them. Some of  these terms are used in a more restricted sense than their ordinary employment would  seem to authorize, but the explanation is to be found in their original use by the Pro-  vost-Marshal-General's Bureau in the forms and reports required during the war.  When the law1 was enacted requiring an enrollment to be made of the entire male  population, within certain limits of age, with a view to compulsory military service, it  became quite common for men who had been enrolled, but who believed that they  were disqualified by physical infirmity, to present themselves voluntarily to the medi-  cal officer for examination. If their claim for exemption proved to be well founded,  their names were erased from the rolls; but if otherwise, they continued, like others,  to be liable to the draft. The effort to relieve the rolls of all unavailable material was  purposely encouraged by the authorities of each district, inasmuch as the quota of  men to be provided under any call bore relation to the total number enrolled. By  this prudent expurgation, the quota was diminished, and the supply of men from which  it was to be filled became correspondingly more available. The details of the exami-  nations, made from the motives described, form part of the tables, and the men, whether  found qualified for a soldier's life or not, are described as 11 Enrolled Men.\"  After the enrollment was completed, the quota of men due from each district under  each successive call was equitably supplied by lot, and the conscripts, whether retained  in the service or discharged, after examination by the surgeon, are spoken of as  \"Drafted Men.\"  The law provided that any citizen enrolled as liable to military duty might pre-  sent a substitute provisionally; and during the period of time for which the substitute  (if found qualified) was accepted, his principal was exempt from draft. In like manner,  a man who been drafted was able to obtain exemption from service if he succeeded  in furnishing a satisfactory substitute before reporting at the \" camp of rendezvous \"  In either case, if the substitute became liable to draft at any time thereafter, the liabil-  ity of the principal immediately recurred, and his name was again placed upon tho  rolls. Men offered in the manner described, whether accepted or rejected by the exam-  ining surgeon, form the \"Substitutes\" of the tables.  During the operation of the enrollment-law, volunteering by no means ceased.  1 Act for enrolling and calling out the national forces, approved March 3,1863.  3 4  DEFINITION OF TERMS NOMENCLATURE OF DISEASES.  Patriotic feelings continued to influence many to enlist, while others preferred the  credit of volunteering to the possibility of compulsory service under the draft. The  most effective inducements, however, were the large bounties offered by the State  and General Governments, which amounted to fully six hundred millions of dollars  during the war. In the tables, volunteers, whether rejected or accepted, are designated  as \"Recruits.\"  Drafted men, if found unfit for military service, were spoken of .in the reports and  tables as \"exempted\" and if otherwise as \"not exempted.\" The same terms are also  applied to enrolled men. Recruits and substitutes whose enlistment was voluntary  are described as \"accepted\" or \"rejected.\" The phrase \"found fit for service\" applies  to either or to all of the four classes.  The designation \" Colored Men,\" intended to describe exclusively the negro and  his hybrids, should not, perhaps, be admitted in scientific terminology on account of  its obvious lack of precision, and its equal applicability to the aboriginal inhabitants,  as well as to more than one foreign race found among us. Usage, however, in the  United States has so confined the term to the single meaning, and the reports upon  which these tables are based so constantly employ it, that it has been thought best to  retain it. Foreigners consulting these tables may need this explanation.  Nomenclature of Diseases.-The nomenclature of diseases adopted in this work  is, with some necessary modifications, that which was published by authority of the  Royal College of Physicians of London, in 18G9,1 and which was to a considerable  extent based upon the classification of Dr. Farr. The system of the latter had many  excellent characteristics, and, indeed, was adopted almost in its entirety as the form for  reports from hospitals during the war; but the more recent work of the English college  is in many respects its superior. It admits of the arrangement of disease in more  clearly-defined subdivisions, and in its classification of general diseases is more in  accordance with advanced pathology. It is quite possible to point out some faults in  this nosology, but taken as a whole it is the best and most practical yet devised. When,  too, it is considered that the English terms are employed, by direction of the registrar-  general of England, in the very thorough system of registration which has been in  force in that kingdom for over thirty-five years, and that it is also the official standard  for use in the British army and navy, it becomes obvious that the opportunity of con-  venient comparison renders its employment highly desirable in our army-reports and  the medical statistics of civil life.2  In explanation of some peculiarities in the classification of disease in the patho-  logical tables of this work, it is to be said that the early returns from examining surgeons  were not characterized by the uniformity and precision which afterward prevailed. In  many instances, the causes of exemption were described in such indefinite terms as to  render the classification a matter of no slight difficulty, and yet the omission of such  returns from the tables would necessarily have vitiated the result. The inaccuracies  referred to are most observable in the description of diseases of the viscera. Such  1 The nomenclature of diseases, drawn up by a joi.it committee appointed by the Royal College of Physicians of  London, (subject to decennial revision,') 8vo, London, 1869.  2 A recent order has directed that this nomenclature shall be exclusively employed in the reports of the United  States marine-hospitals. r 5  NOMENCLATURE OF DISEASES.  expressions as \"disease of the heart,\" \"disease of the lungs,\" \"disease of the liver,\"  are specimens of the mode of reporting causes of exemption in many instances. A  careful examination of this class of returns, and some necessary correspondence relat-  ing to them, made it evident that they were capable of being assigned to the two  groups of acute and chronic diseases of the organ. There still remained, however, a  small number of cases in which, although organic disease was in some manner indi-  cated, the particular organ affected had not been specified. Under the heading  \" Unclassified \" these form a group described as \" Organic disease of internal organs.\"  Their number is only 183; but a French critic, unaware of the explanation just given,  has especially commented upon this portion of our nomenclature in an article marked  by much ability but containing some errors and misapprehensions.1  1 Le recrutement dans Varmee federate des ftats-Unis, pendant la guerre de secession, par M. Ely, mededn-major de Ira  classc. RecueildeM4in.de mddecine, de chirurgie et de pharmacie militaires, 3me s4rie, tome xxii, p. 1, Paris, 1869.  This article consists of a review of the \"Final Report of the Provost-Marshal-General,\" printed in 1866, and which, it  will be remembered, contained the first part of the statistical tables of which the present work forms the completion.  M. Ely comments with emphasis upon the harshness and severity that must have resulted from the enforcement of  the rules as to exemption for physical disability, \" under which,\" he says, \" the American surgeons were compelled to  leave none outside the ranks of the Army bat men afflicted with incurable disease. Those whose maladies were suscep-  tible of cure might seek that cure in the field. Men blind in the left eje, or in the first stage of phthisis; those suffer-  ing from osseous caries which happened to be stationary ; with uncomplicated anal fistula, or external haemorrhoids,  and the like, were considered to be fit for service, as well as all those who were suffering from diabetes, albuminuria,  &amp;c., whose diseases had not as yet made them absolute invalids. The injunct ions are formal, and the selected phrases are  underlined and reiterated, such as manifest, grave, evident, or established incapacity.\" The writer also expresses his sur-  prise that myopia should not exempt, and that liability to hernia from relaxation of the inguinal ring should not be  regarded as disqualifying. He proceeds to point out the different spirit of the French and English regulations, under  which it is the object of the surgeon to exclude from the army men even threatened with disease. Divested of their  rhetorical exaggeration, there is still some justice in these criticisms. The chief medical officer of the Bureau strongly  advised the making of certain alterations in the list of disqualifications, and among them the very obvious ones alluded  to by M. Ely. The pressure of public business, and other reasons not now needful to be detailed, retarded the design  until the collapse of the rebellion made it no longer necessary. Some of M. Ely's objections proceed from incorrect  translation, as where he supposes '•well-established recent insanity, with a liability to recurrence,\" to mean a case  where the service has recourse upon the man in the future. The emphatic terms, also, whose frequent use he criticises,  by no means apply always to the disease but more often to the proof of its existence : thus, in the case just alluded to, the  expression \" well-established\" refers to the evidence that insanity did recently exist, and not, as M. Ely supposes, to the  degree of development of the disorder.  M. Ely more than once expresses his astonishment that the use of anaesthetics should be allowed in cases of sup-  posed simulation of disease. Yet the French code expressly permits their employment upon the soldier in hospital  who may be suspected of feigning disability for the purpose of obtaining a discharge^1] The drafted man was, with us,  held to be actually in the service from the moment the lot fell to his name; if a disqualification were found to exist, it,  entitled him to his discharge, and there seems to be no reason why a soldier, owing his whole period of service to the  Government, should not be subjected to the same scrutiny as he from whom a remainder only is due. M. Ely errs in  stating that anaesthesia was commonly resorted to in the examinations; its aid was allowed only in cases of professed  rheumatic contraction of joints when unattended w ith perceptible alteration of form or structure. As a matter of fact,  however, the length of time required in the process, especially for the application of aether, rendered the permission  nugatory when from forty to sixty men had to be examined each day.  M. Ely asserts that the exigence with which men even partially diseased were compelled to take their places in  the ranks deprives the statistics of all value as regards the number of those exempted in relation to the population.  The inference which he wishes to make is, doubtless, that the figures representing the military aptitude of the nation,  if drawn therefrom, would be unreliable. To this it may be replied, generally, ihat the facts do not warrant his con-  clusions. It is true that the rulings of the medical instructions wrere curtly worded, but the examining surgeons. wTere  men, selected for their experience and ability, who knew well that the object of the Government was to obtain men  able to endure the hardships of a prolonged campaign, and that the degree of incapacity attendant upon any disorder  was left to their judgment. They also knew that each recruit would be rigorously re-examined at the camp of rendez-  vous by the surgeon in charge, and that if considered unfit for service he would.be returned to his district with a repri-  mand, implied or expressed, to the medical officer who had approved him. That certain defects were not to be looked  upon as causes tor exemption was, as already stated, a matter of regret; but it is also true that these were mostly  defects of function, congenital or acquired, and not disabilities arising from disease.  [* J Instruction pour servir de guide aux offiders de sante, &amp;c., p. 24, folio, Paris, 1862. 6  NOMENCLATURE OF DISEASES.  The following is the classification finally adopted as most suitable for the enumer-  ation of the disqualifications for military service:  GENERAL DISEASES.  t  A. -Erysipelas.  Fever.  B. -Cancer.  Chronic rheumatism.  General dropsy.  Scurvy.  Syphilis.  Non-malignant tumors.  Scrofula.  Phthisis pulmonalis.  DISEASES OF THE NERVOUS SYSTEM.  DISEASES OF THE BRAIN AND ITS MEMBRANES  Acute disease of brain.  Chronic disease of brain.  Sun-stroke.  DISEASES OF THE NERVES.  Paralysis.  FUNCTIONAL DISEASES OF THE NERVOUS SYSTEM.  Chorea.  Epilepsy.  Neuralgia.  Stammering.  DISORDERS OF THE INTELLECT  Chronic alcoholism.  Imbecility.  Insanity.  Solitary vice.  DISEASES AND INJURIES OF THE EYE AND EYELIDS.  DISEASES AND INJURIES OF THE EYE.  Cataract of right eye.  Loss of crystalline lens of right eye. NOMENCLATURE OF DISEASES  7  Loss of sight of right eye.  Loss of sight of left eye.  Partial loss of sight of both eyes.  Diseases of the eyes.  DISEASES OF THE EYELIDS.  Diseases of the eyelids.  DISEASES OF THE EAR.  Chronic purulent otorrhoea.  Deaf-dumbness.  Deafness.  DISEASES AND INJURIES OF THE NOSE.  Deformity of nose.  Loss of nose.  Ozaena.  DISEASES OF THE CIRCULATORY SYSTEM.  DISEASES OF THE HEART AND ITS MEMBRANES.  Acute disease of heart.  Chronic disease of heart.  DISEASES OF THE BLOOD-VESSELS.  Diseases of the Arteries.  Aneurism.  Diseases of the Veins.  Varicose veins.  DISEASES OF DUCTLESS GLANDS.  DISEASES OF THE THYROID GLAND.  Goitre.  DISEASES OF THE RESPIRATORY SYSTEM.  DISEASES OF THE LARYNX.  Fistula of larynx.  FUNCTIONAL AFFECTIONS OF THE LARYNX.  Loss of voice. NOMENCLATURE OF DISEASES.  DISEASES OF THE TRACHEA AND BRONCHI.  Bronchitis.  Fistula of trachea.  DISEASES OF THE LUNG.  Acute disease of lung.  Chronic disease of lung.  ' DISEASES OF THE PLEURA.  Chronic pleurisy.  DISEASES OF THE DIGESTIVE SYSTEM.  DISEASES AND INJURIES OF THE JAW.  Ankylosis of jaw.  Disease or deformity of jaw.  DISEASES, MALFORMATIONS, AND INJURIES OF THE TEETH, GUMS, AND ALVEOLI.  Loss of teeth.  DISEASES AND INJURIES OF THE TONGUE.  Loss of tongue.  DISEASES OF THE FAUCES AND PALATE.  Cleft palate.  DISEASES OF THE SALIVARY GLANDS.  Salivary fistula.  DISEASES OF THE STOMACH.  Acute disease of stomach.  Chronic disease of stomach.  DISEASES OF THE INTESTINES.  Chronic diarrhoea.  Hernia.  Hernia, umbilical.  Hernia, ventral.  Hernia, right inguinal.  Hernia, left inguinal. 9  NOMENCLATURE OF DISEASES.  Hernia, double inguinal.  Hernia, right femoral.  Hernia, left femoral.  Hernia, double femoral.  DISEASES OF THE RECTUM AND ANUS  Fistula in ano.  Haemorrhoids.  Prolapsus ani.  Stricture of rectum.  DISEASES OF THE LIVER.  Acute disease of liver.  Chronic disease of liver.  DISEASES OF THE SPLEEN.  Acute disease of spleen.  Chronic disease of spleen.  DISEASES OF THE URINARY SYSTEM.  DISEASES OF THE KIDNEY.  Acute disease of kidney.  Chronic disease of kidney.  DISEASES OF THE BLADDER.  Acute disease of bladder.  Chronic disease of bladder.  Calculus.  Incontinence of urine.  DISEASES OF THE URETHRA.  Stricture of urethra.  Urinary fistula.  DISEASES OF THE GENERATIVE SYSTEM  DISEASES AND INJURIES OF ORGANS OF GENERATION.  Diseases of Penis.  Epispadia.  Hypospadia  Gonorrhoea.  Loss of penis. 10  NOMENCLATURE OF DISEASES.  Diseases of Tunica Vaginalis.  Hydrocele.  Sarcocele.  Varicocele.  Diseases of Testicle.  Acute disease of testicle.  Chronic disease of testicle.  Retention of testicle.  DISEASES OE ORGANS OE LOCOMOTION.  DISEASES OF BONES.  Chronic disease of bones.  DISEASES AND INJURIES OF THE JOINTS.  Ankylosis of joints.  Chronic diseases of joints.  Dislocation of joints.  DISEASES OF THE SPINE.  Curvature of spine.  DISEASES OF THE MUSCULAR SYSTEM.  Diseases of Muscle.  Atrophy of limb.  Diseases of Tendon.  Muscular contractions.  Club-foot.  Wry-neck.  DISEASES OF THE CELLULAR TISSUE.  Abscess.  Obesity.  DISEASES OF THE CUTANEOUS SYSTEM.  Cutaneous contractions.  Disease of skin.  Ulcers. NOMENCLATURE OF DISEASES.  11  CONDITIONS NOT NECESSARILY ASSOCIATED WITH GENERAL OR  LOCAL DISEASE.  Deficient size of chest.  Deformity of chest.  Permanent physical debility.  Relaxed inguinal rings.  Over age.  Under age.  Under size.  LOCAL INJURIES.  LOCALITY OF INJURY NOT SPECIFIED.  Fractures.  Loss of limb.  W ounds.  INJURIES AND MALFORMATIONS OF UPPER EXTREMITIES.  Defects or deformities of hand.  Loss of thumb.  INJURIES AND MALFORMATIONS OF LOWER EXTREMITIES.  Defects or deformities of foot.  Loss of great toe.  UNCLASSIFIED.  Organic disease of internal organs.  There are some minor peculiarities of this classification, a recollection of which  will be found of assistance in obtaining a clear understanding of the tables.  Thus, men who claimed to be suffering from rheumatism were not exempted unless  the affected limb exhibited evidence of change of structure, such as wasting of the  limb, or puffiness of the joint.  By syphilis is to be understood the secondary form of that disease, with impaired  constitution ; primary syphilitic ulcers did not exempt.  The term chronic alcoholism applies to gross habitual intemperance; delirium  tremens did not exempt. In fact, drunkenness is not mentioned as a disqualification in  the official instructions; but an impaired constitution, the result of the constant abuse  of stimulants, or of indulgence in the habit of masturbation, was an authorized ground  for exemption.  By loss of teeth is to be understood the total loss of the incisors, canines, and first  molars at least of one jaw. 12  NOMENCLATURE OF DISEASES NATIVITIES.  The heading chronic diarrhoea includes some cases of chronic dysentery ; the two  diseases having been generally combined in the returns  It will be observed that a certain number of cases of hernia are recorded without  specification of the variety. These form a part of the early returns before alluded to,  and arc necessarily included, although in an unsatisfactory form of description, in  order that the ratio of the total number of cases of hernia to the whole number of  men examined might remain unaffected.  External haemorrhoids were not admitted to be a cause for exemption, but internal  piles, if ulcerated and of long standing, disqualified. By the term lioemorrhoids, there-  fore, the latter are to be understood.  Under the first draft, the medical instructions admitted varicocele and incontinence  of urine as disqualifications for service; under succeeding drafts, these disorders were  not allowed to exempt. Their relation, therefore, to the whole number of men exam-  ined must be considered as below the correct ratio.  By stricture of the urethra must be understood severe or inveterate cases. Recent  or spasmodic stricture did not exempt.  The cases of epispadia or hypospadia are altogether those in which the opening of  the urethra was at the middle of the penis, or still nearer its root.  No definite measurements were established under the instructions of the Bureau  as the minima of girth of chest or of stature, but these points were left to the judgment  of the medical officer. It may be stated that as a general rule men of less circumfer-  ence of chest than thirty inches, or of less height than sixty-two inches, were rejected.  The cases tabulated as under size comprise those of deficient stature.  The age of drafted men, which defined their liability to service, was always  inquired into at the time of their enrollment. The columns headed over age and under  age are made up entirely from the cases of recruits {volunteers) and substitutes. These  men, desirous of procuring admission into the service, in order to obtain bounty or  substitute-money, constantly endeavored to conceal the fact of their being either below  or beyond the limits of competent age.  By loss of thumb, in the division of \" Injuries and malformations of the upper  extremities,\" is to be understood the loss of one phalanx, or of the entire thumb of the  right hand. The loss of any two fingers of either hand, or of the first and second  phalanges of the fingers of the right hand, the permanent extension or permanent con-  traction of two fingers of the right hand, or adhesion of all the fingers of same, are  those only which are included in the group of defects or deformities of hand.  In the next division,'comprising \" Injuries and malformations of lower extremi-  ties,\" the loss of great toe applies to that of either foot. By defects or deformities of foot  is meant club-foot or such other permanent defects or deformities of the feet as would  necessarily prevent marching.  Some of the headings in the tables of disease may seem needlessly comprehensive  when compared with the cases following them, but it was thought best to adhere to  the wording of the classification selected as a standard.  Nativities.-The nativities represented in this work are twenty-four in number;  three of these, however, are the distinct races existing in the United States, namely,", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8sti-5i8v-rzci", "00000000-0000-0000-06B6-8B381F1D3893", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart IV: Phthisis Pulmonalis", "9918573882206676X4", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for phthisis pulmonalis (tuberculosis of the lungs) in relation to marital status, color, age, height, and national origin. Chart IV of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "(lass I.  ( \"h a rt I]  Phthisis Piihiioiialis  Zzz its tv/fi/iott fo Sori til 4'o/ith tioti .Hortiftlr.rion, At/e, Heiiflit titi el .Vtt h\\\\'t/V;s/itnx tttt/  f/lr tut tuber t'Att/tti tier/ ,d tn/ f/tr rttlio rr/er/rt/ /trr/DOO r.rti/tt i tutl.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cdd6.yn29_yih5", "00000000-0000-0000-C704-7F32C1BD20A8", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart II: Syphilis", "9918573882206676X5", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for syphilis in relation to marital status, color, age, height, and national origin. Chart II of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1.  Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "t'f<!././ I  Chart //  Syphilis  /n its re/tttion to Soritft Com/itio/i.Compt'e.riott, .4(/e. //ett/ht oh//Xoti\\\\i/y;s/ioni/ft/  f/te ntnntjer e/AYtt/tine/f,hml fhe rtrfio re/ecletl//er7000 rAvuHutr/l.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-htw7_htme-xjrt", "00000000-0000-0000-C4FF-D2286AD3F09D", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart XIV: Hernia", "9918573882206676X6", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for hernia in relation to marital status, color, age, height, and national origin. Chart XIV of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "/ 7,/.«v /.  /Z////.I7C  Hernia  /// i/sivl/f/ion /o Sori/f/ ton//i//o/t .//>////>/f.i fo/i. .h/e. //rif/ht a/(</Xhftutf/  f/lr /ttftuber r.iv///////rJytft// f/tr rsttio rr/re/eti//rr/t/tjt) r.rtttuttirt/.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7k3i_bajv.d3uz", "00000000-0000-0000-5AF0-3F063E5E5503", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart XXXIV: Disease", "9918573882206676X7", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for all diseases in relation to occupation. Chart XXXIV of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1.  Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Class //.  Disease  thaif ELF/f.'  /// /Zs* re/ct I ton to //ctit/tn/t oit; s/toyvitic/ f/te nttrti/tcr edO/fHnrd. out/  t/ie rci/io re/rrtrct /tor ////Pl pAv/miner/.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6gd7_kjbf~i763", "00000000-0000-0000-29D8-4C0E5733AEED", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart XXVI: Syphilis", "9918573882206676X8", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for syphilis in relation to occupation. Chart XXVI of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "rioss 11.  I'loirt .[.I I /  In z/.s- retntiem to Oreupdtihn , sfawinp the number eanmened, nnrt  the ret tin rejected per Ittttt) ea vmi in ert.  Svphilis", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fm2x~yqe5_if77", "00000000-0000-0000-5327-30213049B8DC", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart XXVII: Phthisis Pulmonalis", "9918573882206676X9", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for phthisis pulmonalis (tuberculosis of the lungs) in relation to occupation. Chart XXVII of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "(Piss JI.  fhart.miL  Phthisis Puhnonalis  In t/s to Oecupntiofi; sJtowin# the nu/filter ejcntntnrcl. and  the ratio rejected per 1000. ejcarn iaed.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bq7v.bmek_xidw", "00000000-0000-0000-077D-FB2950B9949F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart XXXVII: Phthisis Pulmonalis", "9918573882206676X10", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for phthisis pulmonalis (tuberculosis of the lungs) in relation to locality. Chart XXXVII of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "t/assj//  Chart .XAA ] 71.  /// ttx rei/tf/oH to Lorfi/iiv.shoH ins/ the t/t/t/iherotDr/tttrtf ttte/t evt/t/ti/ted.  a/tfi i/te //tt t/e.sit/tt/i /ntio rejrrtes/.  Phthisis Puliiioii.ilis  /n ifsrrfrtfiow foffirsanirSfafrstn theirf/urr font nt on 7/i \\\\f sion s.  7n ifs trhifion to f/tr suinr 7(771 ionol (inoi iiiffi fftr Affrs/fiaiiy  i 77a/t(//' /bf • ifs frnfm/ Di i i si on.  7f/ its rr/fifion to Physiro tror/ni/th DvitPh f'ston s.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-vxm8~rux3-r4cc", "00000000-0000-0000-1F77-B13589B5060B", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart XL: Hernia", "9918573882206676X11", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for hernia in relation to locality. Chart XL of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1.  Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "e/n.w ///.  r/itirt AL.  IltH-iiia  hi its relation to L oeahty tshoivintf the nit in her e.vanitiie/1 , ansi the  ratio re/eetert perJOOt) e.va muted.  mtss/n.  S\\\\ pli ilis  iJt.trf XL I.  hi its rctttfiori to Locality;sitoirin<7 the tnHtiber cayi/ii////t7,anet the  ratio rejected per1000eAYtttnttetf.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qynm.8sp6-5aky", "00000000-0000-0000-3C4A-25FEBC342857", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart XLI: Syphilis", "9918573882206676X12", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for syphilis in relation to locality. Chart XLI of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "e/n.w ///.  r/itirt AL.  IltH-iiia  hi its relation to L oeahty tshoivintf the nit in her e.vanitiie/1 , ansi the  ratio re/eetert perJOOt) e.va muted.  mtss/n.  S\\\\ pli ilis  iJt.trf XL I.  hi its rctttfiori to Locality;sitoirin<7 the tnHtiber cayi/ii////t7,anet the  ratio rejected per1000eAYtttnttetf.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jc3k~anxx-yn6g", "00000000-0000-0000-4DCE-DBAD291A92DF", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chart LVI: Disease", "9918573882206676X13", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this chart showing the number of recruits rejected for all disease in relation to locality. Chart LVI of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "rtnss nr.  ZZz/// Z V/  Disease1  hi its re/tUton to L ortthty; s/imtiii </ the mt tn tier r.vttttii neU ,u/i/i the  tttfio rrjretert //er J000e.wit/iineU'.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5bka~jx9w~7xvn", "00000000-0000-0000-4E30-8E4A3E833BEC", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Maps-Plate VI: Phthisis Pulmonalis", "9918573882206676X14", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this map showing the prevalence of phthisis pulmonalis (tuberculosis of the lungs) in the various U. S. regions, based on exams of drafted men. Maps-Plate VI of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents), Cartographic materials", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Plate VI.  Il JATSTKATING BY GRADATION OF COLOR  THE PREVALENT OF  PHTHISIS PULMONALIS.  (CONGRESSIONAL DISTRICTS.)  (DRAFTED MEN.)  Lith. by Julius Bieu.K.Y  Statistics, Medical atal Anthropological.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-f7eh_dqdu~b9wd", "00000000-0000-0000-875E-B2C07B79BDFB", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Maps-Plate XI: Hernia", "9918573882206676X15", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this map showing the prevalence of hernia in the various U. S. regions, based on exams of drafted men. Maps-Plate XI of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents), Cartographic materials", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Plate XL.  ILLUSTRATING m' GRADATION OF COLOR  THE PREVALENCE OF  II E R N I A.  (CONGRESSIONAL DISTRICTS.)  (DRAFTED MEN.)  Matisticn. Medical and Anthropological.  Lilli, bv Julius Bien,Ji Y", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6jns~su69-8ahs", "00000000-0000-0000-D179-396B089C07C9", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Maps-Plate IV: Syphilis", "9918573882206676X16", null, "1875", "1875", "The Provost-Marshal-General's Office managed recruiting for the Union forces during the last two years of the Civil War (1863-1865), standardizing the medical exams and making other improvements to ensure better fitness in new troops. This process generated over a million detailed medical records, which after the war were compiled and analyzed to provide a valuable statistical profile of the physical and medical condition of American men. The published report included this map showing the prevalence of syphilis in the various U. S. regions, based on exams of drafted men. Maps-Plate IV of J. H. Baxter, Comp., Statistics, medical and anthropological, of the Provost-Marshal-General's Bureau, derived from records of the examination for military service in the armies of the United States during the late war of the rebellion. Vol 1. Part 2, Charts and Maps.", "Monographs, Excerpts, Charts (graphic documents), Cartographic materials", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Plate. IV.  ILLUSTRATING BY GRADATION OF COLOR  THE prevalence: OF  S Y P II I L I S.  (CONGRESSIONAL DISTRICTS.!  (DRAFTED MEN.)  G i.  Statistics. Medical and Anthropological.  I.ith. Iry  Julius Bien,N.Y.", "United States. Provost Marshal General's Bureau", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-s7ac_fbzd_w6ni", "00000000-0000-0000-400D-E762362E8998", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Form E - USSC examination questions (early series)", "9918573882206676X17", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" They used this list of examination questions in the first year of the project (1863-64) and later revised and expanded it for the last part of the war. Pages 218-220 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "3", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "CHAPTER VIII.  MEAN DIMENSIONS OF BODY.  1. History of the Investigation.  In the early part of the year 1863, an extensive series of in-  quiries as to the physical and social condition of our soldiers was  prepared by Mr. Olmsted, the General Secretary of the Commis-  sion, and Mr. Elliott, the Actuary. These were intended to include  the most important physical dimensions and personal characteris-  tics, and the necessary apparatus was procured without delay.  Similar investigations had already been undertaken, to some ex-  tent, by Professor Henry, in behalf of the Smithsonian Institution,  who had caused apparatus to be constructed for the purpose; and  new instruments for measuring were made at the Coast Survey  office, under the supervision of the Vice President of the Commis-  sion and Superintendent of the Coast Survey, the late Professor  Bache. Two inspectors were appointed, and charged with the  duty of obtaining the desired measurements and information for as  many men as possible. One of these, Dr. S. B. Buckley, was  assigned to the army of the Potomac, while the other, Mr. Risler,  measured soldiers in Washington City. The latter was, after a  month's service, relieved by Mr. E. B. Fairchild, who was sta-  tioned first at a camp on one of the islands in New York harbor,  and subsequently at that for rebel prisoners at Point Lookout in  Maryland.  The schedule to be filled out by the examiner was in two parts,  one pertaining solely to physical characteristics, and such other  questions as might be supposed to be of importance in connection  with these, and the other having only a bearing on the purely  moral and social condition of the same men. The former series  only is here discussed, the blank schedule containing them, and  known as Form E, having been as follows : -  1. Number of soldier in order of examination?  2. Name of soldier ?  Rank? MEAN DIMENSIONS OF BODY.  219  3. Regiment?  4. Entire height (in stockings - inches and tenths) ?  5. Height from ground to lower part of neck (7 th cervical vertebra) ?  6. Height to perinaeum ?  7. Breadth of neck ?  8. Breadth of shoulders ?  9. Breadth of pelvis ?  10. Circumference of chest over the nipple (under the coat and vest -  inches and tenths) ?  11. Circumference of waist?  12. Length of arm - from arm-pit to tip of middle finger ?  13. Capacity of chest (cubic inches) ?  14. Weight (lbs. and half lbs.) without coat, hat, arms, or accoutre-  ments ?  15. Dynamometer?  16. In the opinion of Inspector, from appearance and statements of  subject, is he of American stock of three or more generations ? (In  cases where this question cannot be answered with confidence, affir-  matively or negatively, it will be best not to pursue the examination.)  17. If so, period of immigration of ancestry ? (Detail of both sides  desirable.)  18. Where born - country or State ?  \" county ?  \" parish or town ?  19. If foreign born, year of arrival in this country ?  Supposed about ?  20. Country of birth - of father ?  \" \" of mother ?  \" \" of grandparents ?  21. Enlisted - when ?  where ?  for what period ?  22. Conjugal relation (as single, married, or widower) ?  23. Age (last birthday) ?  24. Former occupation ?  25. Hair - color?  Bald?  \" slightly ?  If so, at what age did baldness become distinct ?  26. Eyes - color ?  \" distance between pupils ?  \" prominent ?  27. Complexion?  28. Pulse (regular), beats per minute ?  29. Respiration (number of inspirations per minute) ? 220  MEAN DIMENSIONS OF BODY.  30. Muscular development ?  31. State if in usual vigor?  if reduced by disease ?  \" wounds ?  \" recent exertion ?  \" hardship ?  \" poor fare ?  32. Is he, when ordinarily well, a tougher and more vigorous man than  before he entered the army ?  Less so ?  33. Condition of teeth ?  Number lost ?  Number decayed ?  Number filled ?  34. Head - circumference about frontal eminence and greatest projec-  tion of occiput ?  Distance between the condyloid process of lower jaw over os  frontis - longest measurement ?  Distance between condyloid processes over parietal bones ?  Distance from frontal eminence to protuberance of occiput ?  35. Facial angle?  The questions of which the numbers are omitted here belonged  to the social series.  Of examinations and measurements made in conformity with  his schedule, there are existing very nearly 8000, which will be  specified in detail hereafter.  In June 1864 the author of this treatise was appointed Actuary  of the Commission, and the following passage is quoted from his  first report, made after an examination into the statistical materials  of the Commission, and their condition, and dated 1864, July 12.  \" Of the reports of physical and social condition of soldiers not quite  7200 have been received, namely, about 5200 for national, and 1970 for  rebel soldiers. . . . The results of the physical inspections are  tabulated for all the 1970 rebel prisoners, and for 3277 of the United  States soldiers ; also for about 760 returns from the convalescent camp.  \" A cursory examination of these returns has impressed me forcibly  not only with the great value of the work, but also with the importance  of some more distinct understanding and interchange of ideas between  different inspectors, if their results are to be combined or compared  with one another. Those questions which are necessarily general in  their nature have been answered by the inspectors according to their  individual interpretation of somewhat vague words, and it appears to me  essential that some arbitrary directions be prescribed for their guidance,", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-erwg-jnmm-2mpb", "00000000-0000-0000-F41A-4AA7D44CFD46", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Form EE - USSC examination questions (later series)", "9918573882206676X18", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" This is the revised and expanded list of exam questions used from 1864 through mid-1865. Pages 223-225 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "3", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "MEAN DIMENSIONS OF BODY.  223  [Form EE.]  SANITARY COMMISSION.  INDIVIDUAL INSPECTION.  1. Number of soldier in order of examination ?  2. Name of soldier ?  Rank ?  3. Regiment?  4. Entire height (in stockings-inches and tenths)?  Distance from tip of middle finger to level of upper margin of  patella (in \" attitude of the soldier \") ?  5. Height to lower part of neck (spine of the prominent, i. e., 7th  cervical vertebra) ?  5|. Height to knee (middle of patella) ?  6. Height to perinaeum ?  6|. Perinaeum to most prominent part of pubes ?  7. Breadth of neck ?  7|. Girth of neck ?  8. Breadth of shoulders between acromion processes ?  9. Breadth of pelvis between crests of ilia ?  10. Circumference of chest across the nipples -  a. Full inspiration?  b. After expiration?  10|. Distance between nipples ?  11. Circumference of waist above hips?  11|. Circumference around hips on level with trochanters?  12 a. Length of arm - from tip of acromion to tip of middle finger?  b. Distance from middle of top of sternum to tip of middle fin*  ger, arm extended ?  c. Distance from tip of acromion to extremity of elbow?  13. Capacity of chest in cubic inches (i. e., amount exhaled after full  inhalation) ?  14. Weight (lbs. and half lbs.) without coat, hat, arms, or accoutre-  ments ?  14|. Weight (from memory) at enlistment?  15. Dynamometer ?  16. In the opinion of the Inspector, from appearance and statements of  subject, is he of American stock of three or more generations ?  17. If so, period of immigration of ancestry? (Detail of both sides  desirable.)  18. Where born - country or State ?  \" county ?  \" parish or town ? 224  MEAN DIMENSIONS OF BODY.  19. If foreign born, year of arrival in this country?  Supposed about ?  20. Country of birth - of father ?  \" of mother ?  \" of grandparents?  21. Enlisted - when ?  where ?  for what period ?  22. Conjugal relation (as single, married, or widower) ?  23. Age (last birthday) ?  24. Former occupation or occupations ?  25. Hair - color?  amount ?  texture ?  If bald, at what age did baldness become distinct ?  26. Eyes - color ?  distance between outer angles ?  \" \" inner angles ?  prominent ?  27. Complexion ?  28. Pulse (regular) beats per minute ?  29. Respiration (number of inspirations per minute, when quiet) ?  30. Muscular development?  31. Is he in usual vigor ?  reduced by disease ?  \" wounds ?  \" recent exertion ?  \" hardship ?  \" poor fare ?  32. Is he, when ordinarily well, a tougher and more vigorous man than  before he entered the army ?  33. Condition of teeth ?  Number lost?  34. Head - a. Circumference about frontal eminence, and greatest pro-  jection of occiput?  b. Distance between the condyloid processes of lower jaw  over os frontis, longest measurement ?  c. Distance between condyloid processes over parietal  bones ?  d. Distance between condyloid processes over occipital pro-  tuberance ?  e. Distance from frontal eminence to protuberance of oc  ci put ?  f. Width between angles of jaws ?  g. Width between condyloid processes ? MEAN DIMENSIONS OF BODY.  225  35. Facial angle ?  36. Foot- a. Length from tip of great toe to extremity of heel?  b. Length from tip of great toe to hollow above heel ?  c. Thickness at instep ?  d. Circumference around heel and anterior ligament?  51. Was he, before the war, given to athletic recreations, and if so,  what kind ?  55. Education - Limited common school ?  Good common school ?  High school ?  Professional ?  57. Distance of distinct vision for small pica double-leaded type ?  58. Does he distinguish colors correctly ?  If not, describe the irregularity ?  To secure uniformity in the mode of measurement by different  examiners, Dr. Buckley, whose experience and scientific attain-  ments had already proved serviceable in the examinations under  Form E, was appointed Chief Examiner, and all the other gen-  tlemen engaged upon the work went through some days' practice  in measuring with him. The following printed instructions were  also furnished to each examiner.  INSTRUCTIONS FOR EXAMINATION OF INDIVIDUALS. - [Form EE.]  The persons examined should not be selected, but should be taken  indiscriminately, - by companies and regiments, when possible.  The object of Question 4|, is to determine the point on the outer  side of the thigh corresponding with the tip of the middle finger, in the  \" attitude of the soldier.\" It is best measured with the calipers.  Question cannot be answered by means of the andrometer, but  may be omitted, as also may Question 10|, when no opportunity is found  for examination of the individual without clothing. Such opportunities  are never to be lost; although the ordinary examination requires merely  the removal of hat, coat, waistcoat, and boots, and loosening the shirt  at the breast.  The Girth of neck (Question 7|) is to be taken around the pomuni  Adami.  The Circumference of chest (10) is to be measured under all the  clothing; the Distance between nipples (10|), taken with calipers.  For ascertaining the Capacity of the chest (13), the lungs are to be  fully inflated, and then as completely emptied as may be, by breathing  through the tube of the spirometer. The results of three consecutive  trials are to be recorded.  The Questions 28 and 29, as to the number of pulsations and inspi-", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-sag9_db59-zani", "00000000-0000-0000-332A-C02654B5E3C7", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Classification of disease cases in the volunteer army", "9918573882206676X19", null, "1861", "1861", "The United States Sanitary Commission, a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. In December 1861, the USSC sent a report to the Secretary of War, including this tally of illness from disease and violence, comparing the American figures with those from the British casualties in the Crimean War (1852-54). The authors noted that disease disabled far more troops than did violence, in both conflicts. Page 49 of A Report to the Secretary of War of the Operations of the Sanitary Commission, and upon the Sanitary Condition of the Volunteer Army, its Medical Staff, Hospitals, and Hospital Supplies. Washington, DC: McGill and Witherow, 1861.", "Excerpts, Monographs", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "49    Mortality from disease, in the Royal Navy of Great Britain, is  140 per cent, greater in time of war than of peace ; rising from  an annual rate of 15 or 16 to one of 37 or 38 per 1,000 strength.  The principal increase of  the deaths in the navy, in time of war,  is from disease; the amount of increase from casualties being com-  monly quite inconsiderable.      The following  statement exhibits a classification of the cases  of disease in the  volunteer  army during a  portion of the cam-  paign, showing, also,  the  per centage of casualties  of all  kinds  (wounds,  accidents, &c.)  for the  same  period,  compared with  like returns from the  army of the Crimea, from April 10, 1854,  to June 30,1856 :                    Army of the     Army of Army of the Crii                      Potomac.        the West        Ap. 10, '54, to J 80,1866.  Zymotic disease,      (P      er cent.)       - 61.1  76.4    69.8  Constitutional,                a       - 1.2   .6      .5  Local,           a       - 30.7  17.3    15.6  Developmental,         a       - 3.4   3.5     .1  Violence,                a       - 3.6   2.2     14.0  All cases               _       100.0   100.0   100.0    Two  most important facts appear  on the  face  of this table :  first, the immense disproportion between  cases of disease and of  violence, fully justifying all that has been asserted as  to the loss  an army in  the  field  must expect to sustain  from these causes  respectively.; and, secondly, the great excess of zymotic diseases,  nearly all of which are, in a greater or less degree, preventible by  proper precautions.   For instance, typhus can be almost certainly  averted by systematic attention to cleanliness and ventilation, small-  pox by vaccination,  and  malarious diseases (intermittent fever,  &c.) by quinine.  It  seems apparent, therefore, that it is within  the power of Government, either by the action of the War De-              4", "United States Sanitary Commission", null, null, "McGill & Witherow", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-apuk.rrsc-mtwm", "00000000-0000-0000-3B7F-5FC6478BA258", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Diseases and Casualties of the Army Statistically Classified", "9918573882206676X20", null, "1861", "1861", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. In December 1861, the USSC sent a report to the Secretary of War, including this detailed breakdown of disease and other medical cases occurring in the Union Army during the first seven months of the war. Pages 54-59 of A Report to the Secretary of War of the Operations of the Sanitary Commission, and upon the Sanitary Condition of the Volunteer Army, its Medical Staff, Hospitals, and Hospital Supplies. Washington, DC: McGill and Witherow, 1861.", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "6", "pages", "Text", "English", "The National Library believes this item to be in the public domain.", "Public domain", null, null, "t.»                             54  Diseases and  Casualties of the Army statistically classified.  1-3    4      l    2    3    4  Diseases, etc.  ALL CASES...........    SPECIFIED CASES.               (Classes.)  Zymotic Diseases............  Constitutional Diseases..  Local Diseases..............  Developmental Diseases.  Violence.......................  Miasmatic  Enthetic...  Dietic......  Parasitic..  (Orders.)  Diathetic...  Tubercular.  Nervous System.........  Organs of Circulation.  Respiratory Organs...,  Digestive Organs.......  Urinary Organs........  Generative Organs......  Organs of Locomotion.  Integumentary System.  Not occurring in the army.  Diseases of Nutrition.........  Accident and  >  Battle        f  Homicide.........  Suicide............  ( Punishment and 1  1  Execution       f    Causes not specified.  NUMBER OF CASES TREATED.   >>a   a s  5 °  15,439    15,439  9,437    193  4,737    520    552  8,821    551     53     12  aff  s- in  12,215    12,087   9,228      77  ,2,086    427    269  9,065    132     30      1     86    107    1,122     51    817  1,757    107     97    149    637  520  551  427  268      1  12855  DISEASES AND CASUALTIES—Continued.  Diseases, etc.               (Diseases.)           CLASS  I.—Zymotic.           Order 1.—Miasmatic.    Small-Pox.........................  Varioloid...........................  Mcas'es.............................  Scarlet fever......................  Quinsy..............................  Mumps..............................  Influenza...........................  Catarrh.............................  Ophthalmia.......................  Typhoid fever....................  Typhus..............................  Congestive fever.................  Continued fever..................  Erysipelas.........................  Carbuncle..........................  Dysentery.........................  Diarrhoea....... ...................  Cholera morbus...................  Cholera Asiatica..................  Intermittent fever................  Remittent fever...................  Yellow fever......................  Rheumatism.......................  All other fevers...................            Order 2.—Enthetic.    Gonorrhoea.........................  Syphilis.............................  Bubo ................................  Stricture of urethra.............  Cachexia...........................             Order 3.—Dietic.    Scurvy..............................  Alcholism..........................            Order 4.—Parasitic.    Worms..............................  NUMBER OF CASES TREATED.  n  308  159   54   29    1  224        482  -        1  183        20  127       54  17        -  626        171  97       140  156      131  1        14  - 18  - 39  37        16  81        4  618        527  3,C67   3,362  259   23  1 _  1,178     2,868  639   839  720      163  190      193   4  49  12  63  57   6   3   3  21   9  1356  DISEASES AND  CASUALTIES—Continued.      CLASS  II.—Constitutional.            Order 1.—Diathetic.    Gout.......................................  Lumbago.................................  Anasarca.................................  Cancer....................................  All other diseases of this order.. ..          Order 2.—Tubercular.    Scrofula...................................  Phthisis, (consumption of lungs)..  Hsemoptysis.............................  Anaemia.................................          CLASS III.—Local.         Order 1.—Nervous System.    Apoplexy.................................  Headache................................  Inflammation of Brain ...............  Chorea, (St. Vitus' dance)..........  Epilepsy..................................  Sun-stroke..............................  Spinal irritation.........................  Mania.....................................  Melancholy..............................  Neuralgia.................................  Paralysis.................................  Nyctalopia...............................  Hemeralopia......*.....................  Retinitis, (inflammation of retina)  Iritis....................... ..............  Amaurosis................................  Cataract..................................  Earache...................................  Otitis, (inflammation of ear)........  Otorrhcea, (discharge from ear)...  Deafness..................................  Delirium tremens.......................  Nostalgia, (home sickness)........,.  Toothache................................  Tetanus....................................  All other diseases of this order....,,  1     -       1  73 19      92  10        6       16  2 -       2  14 7       21  51        19      70  21        11      32  21        15      36     3  281     7     3   29   31     9     3     7  120     4     1     97     5       4  105   67   75   10   12    185     64  51  73   2  29DISEASES AND  CASUALTIES—Continued.  Diseases, etc.    CLASS  III.—Local—Continued.      Order 2.—Organs of Circulation.  Aneurism......................................  Angina pectoris..............................  Carditis........................................  Endocarditis..................................  Pericarditis...................................  Inflammation of Vein......................  Varix ..........................................  Hematocele..................................  All other  diseases of the organs of    circulation.................................     Order 3.—Respiratory Organs.    Asthma........................................  Bronchitis acute.............................     \"      chronic..........................  Laryngitis.....................................  Pleurisy.......................................  Pneumonia, (inflammation of lungs)..  Hydrothorax.................................  Epistaxis, (bleeding at the nose).......  All other diseases of respiratory organs      Order 4.—Digestive Organs.    Constipation................................  Colic..........................................  Dyspepsia...................................  Enteritis, (inflammation of bowels)..  Gastritis, (inflammation of stomach)  Hsematemesis..............................  Inflammation of liver, acute...........       \"'        \"     chronic.........  Fistula.......................................  Jaundice.....................................  Peritonitis..............................-•••  Splenitis, (inflammation of spleen)...  Hernia.......................................  Haemorrhoids...............................  Prolapsus ani...............................  Ascites ......................................  Other diseases of digestive organs...  NUMBER OF CASES TREATED.  a£  20    3  350        140  65       14  22        2  112        29  45        41  27        _  176        47  629       505  334      82  158       19  19        2  39 18  8 2  38 218  32       42  35        -  33 50  15        -  2  30  97        9  141        33  '12       -  1  _  164        227  58  DISEASES AND CASUALTIES—Continued.  Diseases, etc.    CLASS III.—Local—Continued.         Order 5.— Urinary Organs.    Calculus......................................  Inflammation of bladder.................  Diabetes.....................................  Enuresis.....................................  Ischuria et  Dysuria........................  Inflammation of kidney........  ..........  Ulcus penis non syphiliticum...........  Other diseases of the urinary  organs.        Order 6.—Generative Organs.    Varicocele....................................  Orchitis......................................  Sarcocele.....................................,  Hydrocele....................................      Order 7.—Organs of Locomotion.    Hydrarthrus.................................  Anchylosis..................................,  Exostosis......................................  Necrosis......................................  Other diseases of this order.............      Order 8.—Integumentary System.    Abscess........................................  Whitlow, or felon...........................  Phelgmon.....................................  Ulcer...........................................  Tumor..........................................  Other diseases of  the integumentary    system.......................................         CLASS IV.—Developmental.       Order  1-3.—(Not  applicable to  the                 Army.)         Order 4.—Diseases of Nutrition.  4 | Atrophy and debility........................  NUMBER OF CASES TREATED.  a s  10   6   6   7  23  25  19  11  27  63   2   5   17   13    4    3  112  133   79   63  116    7    239  520  a^   5  10  19     2   2     1    24  51   6  20  50  78  42759  DISEASES AND CASUALTIES—Continued.        Q a o   Diseases, etc. NUMBER OF CASES TREATED.                  to CO <                     Cm O O IT1 <! ° cm . o -jjj     \"3 fcjO *<  V        1 4 5   CLASS V.—Violence.      20 5 1 135 15 1 21 51 84 23 50 6 60 79  5 2 102 6 1 14 33 24 8 18 5 36 14       25                                    5             Compression \" ............................                     3                                     237           Fracture.......................................                 21                                    2                                     35                                    84                                    108 31                                        68                                    11 96                                                                               93                                      V                   -       1       1                                       V                   1       -       1                                                           -       128     128                                       On the next page is a table by which the distribution, accord-  ing to  Statistical Classes, of  the  diseases  and casualties of the  same  portion of the forces of the United States (1861) may be  compared with those of the British army when in the Crimea.", "United States Sanitary Commission", null, null, "McGill & Witherow", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qetr_ezhr_fnen", "00000000-0000-0000-2E03-C287C0446904", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Table of Mean Heights at Each Age, by States of Enlistment", "9918573882206676X21", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" This table collated data on stature as related to age and home states. Pages 94-95 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "94  STATURES.  TABLE I.  JTean Heights at each Age,  by States of Enlistment.  Total  35 &amp; over  31-34  co  o  to  c©  to  CD  to  to  Ci  to  Ox  to  to  CO  to  to  to  h-*  0  c©  00  HJ  Under 17  Age last  birthday  ■  52314  68.122  7081  68.587  3482  68.555  1264  68.461  1143  68.561  1668  68.559  1696  68.618  1904  68.447  2167  68.489  2420  68.432  2947  68.547  3539  68.339  5411  68.279  34(55  68.226  4390  67.856  9524  66.966  134  66.917  79  63.701  Maine  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Cl  Ci  Ci  ©  ©  ©  ©  ©  •'I  o  r* c©  co  •4  • to  7* B  M co  • 90  • co  Ci co  ©  '..GO  IO  V rr>  New Hamp-  o §  ox ci  QD Ci  © 4*-  SS  •4 co  Cl c'  S W  ? &amp;  o S  co  Ox to  to 00  CO 00  CO °  tO 00  CO  to co  CD G5  0 -'I  © to  CO  4*. ©  shire  to  Ci  CD  )-*  o  to  \"4  OX  Ox  •4  to  co  to  o M  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Cl  Ci  Ci  ©  ©  ©  ©  ©  CD >-u  •4  QD  CD  t  M to  1 1  ©  © t ,  Ci £  M S  • cn  O co  • CH  © co  c©  >- ft  O §  • GO  • co  CD  CD S  M 8  ■4 01  © 0  W OT  M 3  Vermont  4- co  *4 CP  © co  Ci  © GJi  o °  © \"  O CTI  4x GO  •4  © -4  4*  to CO  Ox  to  to  co  to  CD  CO  Ci  »-•  Ox  GO  CD  *•)  •'I  to  H-'  ©  Ci  Ci  Ci  Ci  Ci  Ci  Ci  ci  Ci  Ci  Cl  Ci  ©  ©  ©  ©  ©  M cp  7* £  M H-'  M i-»  \"4  7* co  • m  Ci  • S  M tO  • CO  M go  M cp  M tO  OO  Cl co  © CP  • co  Ox . i  to  Massachu-  O co  CO lu  I--1  4*. <3i  to s  IO cp  to  co co  tO co  to -1  to CO  co  O 65  co 5S  © 0  ©  4x GO  OT. co  O  C© °  Hj Cn  >-i CP  00 «O  Ox GO  0  © Q0  © 4-  CO W  © 05  co  © CT  4k  c©  Ox  Ci  Ci  ©)  ©  t-1  c©  \"4  1-1  ©  Ox  co  ©  Ci  M O1  Ci  *4 co  Ci  Ci  Ci  Ci  Cl  Cl  Cl  <t to  Ci  M tO  Ci  •M co  Ci  Ci 4»  ©  Ci to  ©  © co  ©  Ci  ©  Ox  ©  co  Rhode Island  b co  8  • IO  CO CH  to go  w £  •  CO  GO  !|X , '  • co  CO CO  CO CO  W O  O  © 0  © y  © to  05 &amp;  0 £  and Connec-  CD g  ►u cp  CO G71  CO co  CD 00  Ci co  W> &amp;  to  HU CO  © CO  OX GO  © Ci  IO  l-1 GP  ©  O> M  4x C'  00  ticut  co °  Ci  ©  ©  Ox  Ci  IO  ©  c©  CD  \"4  to  ©  c©  ©  © LJ  Ci  © 1-i  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  05 I-*  Si _  05 KI  ©  ©  .8800  7.08  7' S  w w  te 25  $  co £j  ©  CP >-t  k 8  65 00  k 8?  65 o  ci  O< GO  M oo  CO tS  to GO  r1 8  CO Cl  00  £S OT  r4 go  £ §  O  oo 2  © K  M §  Ox »_>  to 8  CD GP  co  to  CD  New York  OX GO  00  Ox  CO  \"4  Ox  to °  Cl  H- °  0  Ox  co  -4  O  ©  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Ci  Cl  Cl  Ci  Ci  ©  ©  ©  ©  ©  © QO  Ci to  ©  ©  Ci  © 7  Ci j  Ci  Ci  Cl 1-u  Cl  Ci  Cl  ©  © »-*  CP  to  OX co  CD -4  • CP  CD -1  © s  OT> 65  Ci 22  <1 Ci  OX SP  ■4 to  *4 CP  *4 o  © OT  • ©  © co  CO CP  O  © Qo  to fe  ■cd S  New Jersey  J-i to  co  Ox 00  co  CD M  65 \"  *©  GO  oc *■  GO  0  © GO  © CP  O  to  GO  Qi 01  o  GO  CD  •4  CD  co  *-1  Ox  Ci  ro  c©  M  to  0  CD  © ,  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  ci  Cl  Ci  ©  ©  ©  ©  ©  r4 to  Ox 0:1  *4 CH  Ci to  • °2  M to  Ox co  M to  Ox to  to  u< o  g?  4*.  4*. co  W OT  r» 2  CO to  M O1  4-1 CP  © -1  © 8  w g  co ®  Ox b-4  . CO  Ox -1  41  5.0  Pennsylva-  w 2  7;  ©  CO GO  •4 00  co  tO 4**  © CO  CO  OX  h-1 co  OX GO  © 4-  © 65  © -4  ©  CO  Ox  c©  Ox  o  C©  Cl  to  OX  ©  44  <©  ©  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Ci  Cl  Cl  Ci  ©  ©  ©  ©  ©  ©  7333  7.31  1001  7.74  14  u t°  Ci Gli  w \"  r* to  M 00  £  s s  to  •4 ci  Ot  r* to  00 S  co 05  c© g  c© °  CP  Ci  CP  *G>  Z4 00  Cl  cp  CO g>  qo 01  Cl  © a  Ox  c© 8  © ©  00  © oo  co  6tZ  09  Maryland  to  v|  to  •'I  CD  Ci  •'I  to  >-*  to  QD  ©  ©  Ox  ©  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Cl  ©  ©  ©  ©  ©  ©  9°  4x 2  GO K>  • , CO  -4 cp  CO hU  © 3  ©  k 8  CD  c© 2S  00  Ci  GO  • CP  00  00 on  © o  c©  • s°  ° hS  CD t-»  Ci M  00 1-1  •  Ox -1  CD  © §  CD  w S  £  CO  94  5.3  West Vir-  to g  CO CO  CO CO  U-U \"''t  00 \"  Cl  Cl ~ '  >5. \"  Ci  © CO  © 00  CD  co  Ox  gima  Ox 05  QD  H4  QD  _w_  _qx  to  _Ci  Q  CD  to  ©  OX  Ox  © STATURES.  95  TABLE I. - (Continued.')  Mean Heights at each Age,  by States of Enlistment.  Total  31-34  35 &amp; over  30  29  to  00  to  to  Ci  to  OX  bO  to  co  to  to  to  HU  to  0  ©  00  H-*  Under 17  Age last  birthday  23 993  68.160  3031  68.817  1342  68.784  642  68.927  490  68.763  742  68.894  716  68.802  861  168.899  1026  68.530  1212  68.907  1383  68.707  1619  68.608  1794  68.426  2069  68.274  2099  67.685  4563  66.662  66.119  s  144  65.625  Kentucky  ©  Ci ,  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Cl  Ci  ©  © u-l  ©  ©  gg ss  OO St  Ci hu  9° to  03 co  O So  9° to  O  Ci Ci  9° co  9° co  ©  9° £2  tO q.  Ci  00  to £  OX 03  00 rfx  © tc  ® O,  S  HU  9° 05  M 8  9° -i  © §3  CD 05  oo  oo 2  03 CO  co  O' Sc  to H-  © <x>  QX  do  §  ba §  0  Ohio  00 0°  ©  (-*  CD  Ox  Ci  to  co  Ox  00  Cl  OD  00  £*•  © 1-4  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Ci  Ci  © HJ  X IO  ©  ©  .18 25  8.06  $» £  9° 00  » \"  9° w  9° to  CD o  9° co  » S  tci g  CD  M 8?  r, to  © co  OD  bx o  oo o  O to  9° ©  &amp; S  CD  oo  OO CT  CO  w o  ox O'  2 8?  Ox  ©  ©  I  270  4.59  Indiana  tO HU  tC  to  Ci  oo  Ci  Ox  Ci  00  co  HU  03  0  188507  67.970  1  23 421  68.306  13453  68.454  5257  68.344  4833  68.452  6646  68.398  | 6647  68.421  7753  68.426  9097  68.387  1 10118  68.441  OD £  p  Ci  13024  68.396  14 919  68.247  13130  68.121  16 547  67.620  28 635  66.638  66.004  2527  1168  64.326  Illinois  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Cl  Cl  ©  ©  ©  ©  to  T4 co  2g  B 3  e« 00  £ 00  00  Ci  co o  OX  do co  oo  oo  oo  ®s  OO j_4  O CH  Ox co  00  S 05  oo co  CD  b3  o co  O oo  do 3  bO o  2 so  © to  03 S  HU to  co  OX £  03 Ox  ©  05  8  298  5.72  Michigan  Ox  HU  Ox  03  Ox  h  Ox  Ox  Ox  to  O  03  00  O'  to  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  ©  ©  ©  ©  M 01  Ci  ™ OO  W g  *.  So 25  Li oo  CD ?  °°  CO 03  OO  ± g  Ci 05  © 8  to  to  o  ►U OX  9° to  O co  CD CO  9° 03  2-&amp;  ® g  03 oo  03  o< w  © 00  00 S  ©  w  2  243  5.24  Wisconsin  &amp; \"  to  CD  Ci  to  00  to  4u.  03  to  O  to  ©  Qx  •m  Ci  O'.  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Cl  Cl  ci  ©  ©  ©  ©  Ci £  bO -I  1475  17.77  00  . to  O  t-*  I4 to  oo g  Ss  OX b-  § §  to  HU Ox  Ox  to  9° co  M S  r* cn  00 S  to 00  00  r* co  0  ©  52 S  Ox  bx  OD  05  HU  O'  03 2  HU  Minnesota  Ox  »-*  Ci  CD  ©  CD  to  Ci  00  Ci  HU  ©  ©  ©  Ox  4u  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  ci  ©  ©  ©  ©  ©  3 g  00  co to  cd ,  &amp; _  <x x  co  00 to  OO  00  00  OD >_x  90 to  00 !_!  ® 2  Ox  CO  © g  do £  M CO  o s  (X>  ox 2  M cn  © s  ©  © 3  co 2  © s  DO  00 qo  ©  UJ  Iowa  03  Ci' co  M do  00 °  co *4  co 03  CD 03  O C5  HU CO  Ci co  HU QO  O O'  M 05  © 05  Ox  OD 10  Ci  Ci  HU  tO  Ox  I-*  to  Ci  -■1  to  ©  ©  IO  Hu  4--  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  Ci  ©  ©  ©  ©  ©  So 3  © *•  w S  CIO co  k g  00  03 ll  bO Ci  00 _  • . co  b3  M ci  CD  tO &amp;  Ci 03  9° to  Ci 03  00 g  H- to  9° to  S »»  Ci 05  9° w  P3 JO  £ 8  b» co  9° co  w 2  00 Ip-  00  to  tO 05  90 05  tg g  do 0  © to  © -1  © QC  QX O  ©  HU  to  s  05  to  do co  ox  Missouri  w>  00  Ci  co  *-  CD  Ox  Ox  CD  *•1  to  Ci  ©  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Ci  Cl  ©  ©  ©  ©  O'  f» JO  691  16.98  03  O £  kl  to O  o \"  o *■*  Ci  CD HU  S 00  Ci  £8?  k o  co 00  QQ <35  Ci  bi w  Ci 1  ci  to  b3  ©  OD '  ©  03  \"00  O< HU  w B  to  0  to  24  9.25  Louisiana  ! ■  Ci  ci  00  HU  co  Hu  CD  HU  CO  OD  Cl  ©  ©  03  00", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ypt5-var8-vnnz", "00000000-0000-0000-99F8-1E3C1CE78EFD", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Classification of nativities in USSC statistical tables", "9918573882206676X22", null, "1869", "1869", "The USSC used 18 classes of nativity in many of the statistical tables generated by their research. This page of Gould's \"Investigations\" explained the designations. Page 92 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "92  STATURES.  organizations) was decidedly greater than that of the recruits who  subsequently enlisted, and it is therefore a source of much regret  that so large a proportion of our material is afforded by the latter  only. The successive \" counts \" for different States indicate a  decided tendency to diminution of the average stature as the war  went on ; and it is doubtless owing to this fact that the mean stat-  ures afforded by the present investigation, which comprises all  those volunteers whose statures were recorded, range much higher  than those given in the excellent report1 of Dr. Baxter, Chief Med-  ical Officer of the Provost Marshal General's Bureau, inasmuch  as these latter are deduced from the statistics of less than 350 000  soldiers, all of whom were recruits, substitutes, or drafted men.  The regulations prescribe that the heights be taken with a meas-  uring rod, while the men are without clothing. There is doubtless  some difference in the average results obtained by different exam-  ining surgeons, but these must vanish from the mean of measure-  ments by so many different officers. The most appropriate distri-  bution of the soldiers according to nativities was a question of  some difficulty, since it was necessary to decide upon the mode  of distribution, before the relative numbers were known. The  following eighteen classes were adopted, and although this division  might with advantage be somewhat modified in the light of our  present knowledge, it has, to preserve uniformity, been retained  for all the statistics of the Commission.  A. The six New England States.  B. New York, New Jersey, and Pennsylvania.  C. Ohio and Indiana.  D. Michigan, Wisconsin, and Illinois.  E. Slave States, not including F and G2.  F. Kentucky and Tennessee.  Gr Free States west of the Mississippi.  G2. Slave States west of the Mississippi.  H. British America, exclusive of Canada.  I. Canada.  J. England.  K. Scotland.  L. Ireland.  M. France, Belgium, and Switzerland.  N. Germany.  O. • Scandinavia.  P. Spain, Portugal, and Spanish America.  Q. Miscellaneous.  1 Pages 698, 699.", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-u8hb_ie58_se2i", "00000000-0000-0000-0EC5-69C182408A0D", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Tables of hair color of soldiers examined", "9918573882206676X23", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" These tables recorded hair colors found in the soldiers examined and related them to other factors. Pages 186-193 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "8", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "186  COMPLEXIONS : COLOR OF HAIR AND EYES.  volunteers are not included in our statistics ; while of the recruits,  the omissions are generally of the later ones, inasmuch as the  clerks in transcribing usually followed the order in which the de-  scriptions were recorded, and ceased collecting when the number  transcribed seemed adequate for the purposes in view.  The results of these researches are presented in two modes :  first, according to the States by which the troops were furnished,  and secondly, according to the nativity of the men, without refer-  ence to the State of enlistment. The assortment by nativities is  identical with that employed for the investigation of statures, there  being eighteen classes for wdiite soldiers.  o o  2. Color of Hair.  TABLE I.  Color of Hair. Original Volunteers,  by States.  State of Enlistment  Black  Dark  Brown  Light  Sandy  Red  Gray  Totals  Maine . . .  6 178  13 352  11 681  6 189  1 186  203  502  39 291  New Hampshire  2 178  3 371  7 297  4 224  754  163  124  18 111  Vermont . .  1 995  2 234  5 351  2 420  489  105  112  12 706  Massachusetts.  2 114  4 556  6 621  4 644  516  133  103  18 687  Connecticut  2 306  3 727  5 716  3 592  713  133  278  16 465  Pennsylvania .  3 263  8 968  5 964  5 431  1 316  762  272  25 976  West Virginia  2 412  4 234  1 981  4 447  567  494  147  14 282  Kentucky . .  2 202  4 384  1 076  5 185  458  504  100  13 909  Ohio ....  8 835  15 392  12 780  15 190  2 170  2 579  323  57 269  Indiana . . .  8 197  18 166  8 429  17 347  2 077  3 425  518  58 159  Illinois . . .  10 170  15 722  15 864  19 548  2 120  3 706  688  67 818  Michigan . .  1 073  1 829  3 347  2 085  274  291  54  8 953  Wisconsin . .  3 918  4 812  12 461  7 622  1 103  240  267  30 423  Iowa ....  2 491  3 212  4 051  2 954  591  185  452  13 936  Missouri . .  4 341  6 390  6 992  8 529  2 256  383  442  29 333  Total . . .  61 673  110 349  109 611  109 407  16 590  13 306  4 382  425 318 187  COMPLEXIONS: COLOR OF HAIR AND EYES.  TABLE II.  Color of Hair.  Recruits, try States  State of Enlistment  Black  Dark  Brown  Light  Sandy  Red  Gray  Totals  Maine . . .  2 591  4 742  8 130  3 085  492  162  131  19 333  New Hampshire  1 430  570  5 487  866  267  66  75  8 761  Vermont . .  1 304  1 566  3 945  1 535  340  63  41  8 794  Massachusetts.  2 797  5 730  10 374  5 047  741  382  284  25 355  Connecticut .  1 943  2 937  7 672  2 096  543  206  191  15 588  New York . .  5 985  11 655  22 264  9 269  1 718  650  612  52 153  Pennsylvania .  5 376  14 406  13 900  9 352  1 866  1 241  376  46 517  West Virginia  484  1 053  219  870  71  67  21  2 785  Kentucky . .  701  1 937  365  2 294  188  205  23  5 713  Ohio ....  875  1 872  1 975  1 900  232  266  39  7 159  Indiana . . .  671  2 052  1 173  1 498  271  309  51  6 025  Illinois . . .  244  444  748  441  43  67  21  2 008  Michigan . .  1 832  3 745  8 396  3 757  556  491  84  18 861  Wisconsin . .  2 560  2 610  8 766  4 169  647  114  156  19 022  Iowa ....  644  1 127  1 327  993  244  51  40  4 426  Missouri . .  68  131  149  154  34  3  3  542  Total . . .  29 505  56 577  94 890  47 326  8 253  4 343  2 148  243 042 188  complexions: color of hair and eyes.  TABLE III.  Color of Hair.  U. S. Soldiers, by States.  State of Enlistment  Black  Dark  Brown  Light  Sandy  Bed  Gray  Totals.  Maine . . .  8 769  18 094  19 811  9 274  1 678  365  633  58 624  New Hampshire  ' 3 608  3 941  12 784  5 090  1 021  229  199  26 872  Vermont . .  3 299  3 800  9 296  3 955  829  168  153  21 500  Massachusetts.  4 911  10 286  16 995  9 691  1 257  515  387  44 042  Connecticut  4 249  6 664  13 388  5 688  1 256  339  469  32 053  New York . .  5 985  11 655  22 264  9 269  1 718  650  612  52 153  Pennsylvania .  8 639  23 374  19 864  14 783  3 182  2 003  648  72 493  West Virginia  2 896  5 287  2 200  5 317  638  561  168  17 067  Kentucky . .  2 903  6 321  1 441  7 479  646  709  123  19 622  Ohio ....  9 710  17 264  14 755  17 090  2 402  2 845  362  64 428  Indiana . . .  8 868  20 218  9 602  18 845  2 348  3 734  569  64 184  Illinois . . .  10 414  16 166  16 612  19 989  2 163  3 773  709  69 826  Michigan . .  2 905  5 574  11 743  5 842  830  782  138  27 814  Wisconsin . .  6 478  7 422  21 227  11 791  1 750  354  423  49 445  Iowa ....  3 135  4 339  5 378  3 947  835  236  492  18 362  Missouri . .  4 409  6 521  7 141  8 683  2 290  386  445  29 875  Total . . .  91 178  166 926  204 501  156 733  24 843  17 649  6 530  668 360 189  COMPLEXIONS: COLOR OF HAIR AND EYES.  TABLE IV.  Color of Hair.  Original Volunteers, by Nativities.  Nativity  Black  Dark  Brown  Light  Sandy  Red  Gray  Totals  A  13 364  24 852  33 042  19 551  3 195  819  1 060  95 883  B  9 821  18 266  19 607  15 766  2 824  2 172  984  69 440  C  16 616  30 631  21 117  31 315  4 212  5 383  496  109 770  D  3 935  6 157  6 417  8 216  797  1 304  52  26 878  E  3 315  5 516  2 841  5 274  689  708  289  18 632  F  3 925  7 147  2 387  7 704  918  986  283  23 350  Gi  170  269  347  329  34  27  2  1 178  g2  947  1 558  1244  1 967  357  135  19  6 227  H  258  513  698  410  73  19  20  1 991  I  1 162  1419  2 109  1075  186  114  44  6 109  J  1 043  1 954  3 015  2 230  429  249  165  9 085  K  260  450  599  501  106  59  55  2 030  L  2 637  4 875  5 795  3 743  966  513  455  18 984  M  298  375  420  312  50  38  33  1 526  N  3 325  5 387  8 490  9 144  1449  673  359  28 827  0  131  314  740  1 178  152  59  13  2 587  P.  17  15  4  3  -  -  -  39  Q  449  651  739  689  153  48  53  2 782  Total  61 673  110 349  109 611  109 407  16 590  13 306  4 382  425 318 190  COMPLEXIONS : COLOR OF HAIR AND EYES.  TABLE V.  Color of Hair.  Recruits, by Nativities.  Nativity  Black  Dark  Brown  Light  Sandy  Red  Gray  Totals  A  5 273  9 016  19 060  8 020  1 277  472  378  43 496  B  10 449  21 767  32 510  16 898  3 009  1 669  698  87 000  C  1 961  4 367  4 666  4 067  668  543  65  16 337  D  1 129  1 600  3 653  2 012  219  174  13  8 800  E  885  1 877  1 281  1 310  178  143  54  5 728  F  765  1 865  599  2 055  195  189  29  5 697  Gi  67  149  211  174  20  5  7  633  G'j  97  177  188  150  23  7  -  642  H  494  879  1 645  472  92  52  22  3 656  I  1 736  2 248  4 806  1 278  277  106  59  10 510  J  1 022  1 937  4 335  1 568  333  152  105  9 452  K  259  523  1 192  400  134  66  39  2 613  L  2 888  5 696  11 105  3 481  1 039  522  449  25 180  M  362  443  631  163  34  12  18  1 663  N  1 469  3 251  7 417  4 242  573  192  176  17 320  0  61  178  703  712  129  20  8  1 811  P  113  101  59  11  2  1  4  291  Q  475  503  829  313  51  18  24  2 213  Total  29 505  56 577  94 890  47 326  8 253  4 343  2 148  243 042 191  COMPLEXIONS : COLOR OF HAIR AND EYES.  TABLE VI.  Color of Hair.  U. 8. Soldiers, by Nativities.  Nativity  Black  Dark  Brown  Light  Sandy  Red  Gray  Totals  A  18 637  33 868  52 102  27 571  4 472  1 291  1 438  139 379  B  20 270  40 033  52 117  32 664  5 833  3 841  1 682  156 440  C  18 577  34 998  25 783  35 382  4 880  5 926  561  126 107  D  5 064  7 757  10 070  10 228  1 016  1 478  65  35 678  E  4 200  7 393  4 122  6 584  867  851  343  24 360  F  4 690  9 012  2 986  9 759  1 113  1 175  312  29 047  Gi  237  418  558  503  54  32  9  1 811  Gg  1 044  1 735  1 432  2 117  380  142  19  6 869  H  752  1 392  2 343  882  165  71  42  5 647  I  2 898  3 667  6 915  2 353  463  220  103  16 619  J  2 065  3 891  7 350  3 798  762  401  270  18 537  K  519  973  1 791  901  240  125  94  4 643  L  5 525  10 571  16 900  7 224  2 005  1 035  904  44 164  M  660  818  1051  475  84  50  51  3 189  N  4 794  8 638  15 907  13 386  2 022  865  535  46 147  0  192  492  1 443  1 890  281  79  21  4 398  P  130  116  63  14  2  1  4  330  Q  924  1 154  1 568  1 002  204  66  77  4 995  Total  91 178  166 926  204 501  156 733  24 843  17 649  6 530  668 360  The corresponding relative proportions for each State and each  nativity may be more readily seen from the following tables, in  which the several numbers are reduced to the scale of 1000. The  degree of reliance to be placed upon these results may be readily  estimated by reference to the tables of absolute numbers, from  which they are deduced. 192  COMPLEXIONS : COLOR OF HAIR AND EYES.  TABLE VII.  Color of Hair.  Proportionate Numbers for different States.  State of Enlistment  Black  Dark  Brown  Light  Sandy  Red  Gray  Totals  Maine . . .  149  309  338  158  29  6  11  1 000  N. Hampshire.  134  147  476  189  38  9  7  1 000  Vermont . .  153  177  432  184  39  8  7  1000  Massachusetts.  111  234  386  220  28  12  9  1000  Connecticut .  132  208  418  177  39  11  15  1 000  New York . .  115  223  427  178  33  12  12  1 000  Pennsylvania .  119  322  274  204  44  28  9  1 000  West Virginia  170  310  129  311  37  33  10  1000  Kentucky . .  148  322  74  381  33  36  6  1 000  Ohio ....  151  268  229  265  37  44  6  1 000  Indiana . . .  138  315  150  294  36  58  9  1000  Illinois . . .  149  232  238  286  31  54  10  1 000  Michigan . .  105  200  422  210  30  28  5  1 000  Wisconsin . .  131  150  429  239  35  7  9  1 000  Iowa ....  171  236  293  215  45  13  27  1 000  Missouri . .  147  218  239  291  77  13  15  1000  Total . . .  136  250  306  235  37  26  10  1000 COMPLEXIONS : COLOR OF HAIR AND EYES.  193  TABLE VIII.  Color of Hair.  Proportionate Numbers for different Nativities.  Nativity  Black  Dark  Brown  Light  Sandy  Red  Gray  Totals  A  134  243  374  198  32  9  10  1 000  B  130  256  333  209  37  24  11  1 000  C  147  278  204  281  39  47  4  1 000  D  142  217  282  287  29  41  2  1 000  E  172  304  169  270  36  35  14  1000  F  162  310  103  336  38  40  11  1 000  G1  131  231  308  278  30  17  5  1 000  G2  152  253  208  308  55  21  3  1 000  H  133  247  415  156  29  13  7  1000  I  174  221  416  142  28  13  6  1 000  J  111  210  396  205  41  22  15  1 000  K  112  209  386  194  52  27  20  1 000  L  125  239  383  164  45  23  21  1 000  M  207  256  330  149  26  16  16  1 000  N  104  187  345  290  44  19  11  1 000  0  43  112  328  430  64  18  5  1 000  P  394  352  191  42  6  3  12  1 000  Q  185  231  314  201  41  13  15  1000  Total  136  250  306  235  37  26  10  1000", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-32tx-5p8u-rx6s", "00000000-0000-0000-21DF-BCD09132B2F8", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Tables of eye color of soldiers examined", "9918573882206676X24", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" These tables recorded eye colors found in the soldiers examined and related them to other factors. Pages 194-201 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "8", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "194  COMPLEXIONS: COLOR OF HAIR AND EYES.  3. Color of Eyes.  TABLE IX.  Color of Eyes.  Volunteers by States.  State of Enlistment  Blue  Gray  Hazel  Dark  Black  Totals  Maine . . .  17 847  6 820  6 783  2 828  5 013  39 291  New Hampshire  9 692  2 957  2 327  1 599  1 536  18 111  Vermont . .  7 222  1 833  860  1 288  1 503  12 706  Massachusetts  9 477  3 279  3 101  1 515  1 316  18 688  Connecticut .  8 274  3 418  1 227  2 083  1 462  16 464  Pennsylvania  8 330  9 176  3 261  4 098  1 111  25 976  West Virginia  6176  3 644  1 118  1 819  1 526  14 283  Kentucky . .  6 388  3 085  1 291  1 321  1 823  13 908  Ohio....  22 698  16 601  6 680  6 523  4 766  57 268  Indiana. . .  24 714  14 928  7 690  5 557  5 258  58 147  Illinois . .  30 275  16 608  8 137  7 213  5 571  67 804  Michigan . .  4 534  1 980  915  611  905  8 945  Wisconsin  16 256  6 343  2 995  2 834  1 995  30 423  Iowa ... .  6 620  3 192  1 669  1 210  1 241  13 932  Missouri . .  13 505  7 175  3 372  3132  2 129  29 313  Total . . .  192 008  101 039  51 426  43 631  37155  425 259 complexions: color of hair and eyes.  195  TABLE X.  Color of Eyes.  Recruits by States.  State of Enlistment  Blue  Gray  Hazel  Dark  Black  Totals  Maine . . .  8 971  3 220  4 525  1 275  1 342  19 333  New Hampshire  3 575  2 225  2 183  420  358  8 761  Vermont . .  4 723  1 355  894  820  1 004  8 796  Massachusetts  12 783  4 839  4 532  1 834  1 367  25 355  Connecticut .  6 984  3 874  2 746  1 219  763  15 586  New York . .  24 342  13 314  3 910  7 326  3 261  52 153  Pennsylvania .  14 829  16 626  7 047  6 743  1 273  46 518  West Virginia  1 158  754  325  325  223  2 785  Kentucky . .  2 754  1 230  494  591  644  5 713  Ohio....  2 632  2 261  1055  691  519  7 158  Indiana . .  2 374  1 653  1 237  474  286  6 024  Illinois . . .  897  518  334  169  89  2 007  Michigan . .  9 977  4 261  1 673  1 749  1 200  18 860  Wisconsin . .  10 101  3 658  2 240  1 758  1 265  19 022  Iowa . . .  1 857  1 196  706  362  305  4 426  Missouri . .  236  133  66  63  45  543  Total . .  108 193  61 117  33 967  25 819  13 944  243 040 196  complexions: colob of hair and eyes.  TABLE XL  Color of Eyes.  U. 8. 8oldiers by States.  State of Enlistment  Blue  Gray  Hazel  Dark  Black  Totals  Maine . . .  26 818  10 040  11 308  4103  6 355  58 624  New Hampshire  13 267  5 182  4 510  2 019  1 894  26 872  Vermont . .  11 945  3 188  1 754  2 108  2 507  21 502  Massachusetts  22 260  8 118  7 633  3 349  2 683  44 043  Connecticut .  15 258  7 292  3 973  3 302  2 225  32 050  New York . .  24 342  13 314  3 910  7 326  3 261  52 153  Pennsylvania .  23 159  25 802  10 308  10 841  2 384  72 494  West Virginia  7 334  4 398  1443  2 144  1749  17 068  Kentucky . .  9142  4 315  1 785  1 912  2 467  19 621  Ohio ....  25 330  18 862  7 735  7 214  5 285  64 426  Indiana. . .  27 088  16 581  8 927  6 031  5 544  64 171  Illinois . . .  31 172  17126  8 471  7 382  5 660  69 811  Michigan . .  14 511  6 241  2 588  2 360  2 105  27 805  Wisconsin . .  26 357  10 001  5 235  4 592  3 260  49 445  Iowa . . .  8 477  4 388  2 375  1 572  1 546  18 358  Missouri . .  13 741  7 308  3 438  3195  2 174  29 856  Total. . .  300 201  162 156  85 393  69 450  51 099  668 299 complexions: color of hair and eyes.  197  TABLE XII.  Color of Eyes.  Volunteers by Nativities.  Nativity  Blue  Gray  Hazel  Dark  Black  Totals  A  47 633  16 632  13 295  8 321  10 001  95 882  B  29 193  18 837  8 280  8 126  4 994  69 430  C  45 834  29 033  13 565  11 339  9 989  109 760  D  11 901  6 415  3 428  2 512  2 606  26 862  E  8 206  4 545  1 904  2 028  1 944  18 627  F  10 777  5 209  2 506  2 113  2 740  23 345  Gi  479  324  176  98  97  1174  G2  2 746  1 504  785  585  605  6 225  H  1015  379  309  158  129  1 990  I  2 760  1 290  698  677  684  6 109  J  4 514  2 078  1 091  862  538  9 083  K  1 012  535  225  159  98  2 029  L  9 820  5 004  1 873  1 452  836  18 985  M  522  364  228  257  155  1 526  N  12 819  7 674  2 610  4 267  1 458  28 828  0  1 764  444  137  182  60  2 587  P  6  5  8  10  10  39  Q  1007  767  308  485  211  2 778  Total  192 008  101 039  51 426  43 631  37155  425 259 198  complexions: color or hair and eyes.  TABLE XIII.  Color of Eyes.  Recruits by Nativities.  Nativity  Blue  Gray  Hazel  Dark  Black  Totals  A  21 890  7 777  7 558  3 318  2 956  43 499  B  35 737  24 954  10 248  11 632  4 432  87 003  C  6 747  4 464  2 470  1 547  1 110  16 338  D  4 112  2 021  877  923  867  8 800  E  2 321  1 518  816  653  421  5 729  F  2 689  1223  530  617  638  5 697  Gi  236  189  112  53  43  633  G2  243  168  93  72  65  641  H  1 602  769  786  285  214  3 656  I  4 415  - 2 331  1 856  1 109  799  10 510  J  4 2351  2 325  1 548  877  463  9 445  K  1 206  645  376  224  161  2 612  L  12 486  7 073  3 367  1 614  639  25179  M  525  353  385  225  175  1 663  N  7 700  4 463  2 321  2 229  609  17 322  0  1246  310  141  80  34  1811  P  73  56  46  55  61  291  Q  733  478  437  306  257  2 211  Total  108 193  61117  33 967  25 819  13 944  243 040 COMPLEXIONS: COLOR OR HAIR AND EYES.  199  TABLE XIV.  Color of Eyes.  IT. S. Soldiers by Nativities.  Nativity  Blue  Gray  Hazel  Dark  Black  Totals  A  69 523  24 409  20 853  11 639  12 957  139 381  B  64 930  43 791  18 528  19 758  9 426  156 433  C  52 581  33 497  16 035  12 886  11 099  126 098  D  16 013  8 436  4 305  3 435  3 473  35 662  E  10 527  6 063  2 720  2 681  2 365  24 356  F  13 466  6 432  3 036  2 730  3 378  29 042  Gi  715  513  288  151  140  1 807  Ga  2 989  1 672  878  657  670  6 866  H  2 617  1 148  1 095  443  343  5 646  I'  7 175  3 621  2 554  1 786  1 483  16 619  J  8 746  4 403  2 639  1 739  1 001  18 528  K  2 218  1 180  601  383  259  4 641  L  22 306  12 077  5 240  3 066  1 475  44 164  M  1047  717  613  482  330  3 189  N  20 519  12 137  4 931  6 496  2 067  46 150  0  3 010  754  278  262  94  4 398  P  79  61  54  65  71  330  Q  1 740  1245  745  791  468  4 989  Total  300 201  162 156  85 393  69 450  51 099  668 299 200  COMPLEXIONS: COLOR OF HAIR AND EYES.  TABLE XV.  Color of Eyes.  Proportionate Numbers for different States.  State of Enlistment  Blue  Gray  Hazel  Bark  Black  Totals  Maine . . .  458  171  193  70  108  1000  New Hampshire  494  193  168  75  70  1 000  Vermont . .  555  148  82  98  117  1000  Massachusetts.  506  184  173  76  61  1 000  Connecticut .  476  228  124  103  69  1000  New York . .  467  255  75  140  63  1000  Pennsylvania .  319  356  142  150  33  1 000  West Virginia  430  258  84  126  102  1000  Kentucky . .  466  220  91  97  126  1 000  Ohio ....  393  293  120  112  82  1 000  Indiana . . .  422  258  139  94  87  1 000  Illinois . . .  447  245  121  106  81  1000  Michigan . .  522  224  93  85  76  1 000  Wisconsin . .  533  202  106  93  66  1000  Iowa ....  462  239  129  86  84  1000  Missouri . .  460  245  115  107  73  1000  Total . . .  449  243  128  104  76  1000 COMPLEXIONS : COLOR OF HAIR AND EYES.  201  TABLE XVI.  Color of Eyes.  Proportionate Numbers for different Nativities.  Nativity  Blue  Gray  Hazel  Dark  Black  Totals  A  499  175  150  83  93  1 000  B  415  280  119  126  60  1000  C  417  266  127  102  88  1000  D  449  237  121  96  97  1000  E  432  249  112  110  97  1 000  F  464  221  105  94  116  1 000  Gi  396  284  159  84  77  1000  Ga  435  243  128  96  98  1 000  H  464  203  194  78  61  1 000  I  432  218  154  107  89  1000  J  472  238  142  94  54  1 000  K  478  254  129  83  56  1 000  L  505  274  119  69  33  1000  M  328  225  192  151  104  1 000  N  445  262  107  141  45  1 000  0  684  172  63  60  21  1000  P  239  185  164  197  215  1000  Q  349  250  149  158  94  1000  Total  449  243  128  104  76  1000", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rymb~7ask.8tc6", "00000000-0000-0000-53E1-845F4C004CB2", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Tables of skin color of soldiers examined", "9918573882206676X25", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" These tables recorded the skin colors (\"complexions\") of the soldiers and related them to other variables. Pages 202-205 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "4", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "202  COMPLEXIONS: COLOR OF HAIR AND EYES.  4. Complexions.  TABLE XVII.  Complexions. By States.  State of  Volunteers  Recruits  Enlistment  Dark  Light  Me-  dium  Totals  Dark  Light  Me-  dium  Totals  Maine . . .  17 002  21 175  1 106  39 283  5142  13 013  1 173  19 328  New Hampshire  5 900  11 310  898  18 108  3 352  3 744  1 659  8 755  Vermont . .  4 307  7 340  1052  12 699  2 746  5 368  647  8 761  Massachusetts.  6 171  11 899  608  18 678  8 060  15 882  1 395  25 337  Connecticut .  5 124  10 782  549  16 455  4 793  8 849  1 939  15 581  New York . .  -  -  -  -  13 523  23 879  14 712  52 114  Pennsylvania .  9 061  14 789  2 125  25 975  15 748  24 478  6 292  46 518  West Virginia  4 783  9 498  2  14 283  878  1 907  -  2 785  Kentucky . .  4 584  9 325  -  13 909  1729  3 984  -  5 713  Ohio ....  18 310  38 916  44  57 270  1942  5 195  22  7 159  Indiana . . .  21 165  34 426  2 489  58 080  2 099  3 733  190  6 022  Illinois . . .  22 451  42 105  3 241  67 797  581  1 344  78  2 003  Michigan . .  2 357  6 582  16  8 955  4 557  14 287  16  18 860  Wisconsin . .  8 906  21 515  2  30 423  5 927  13 095  -  19 022  Iowa ....  4 584  5 388  3 964  13 936  1376  1 799  1 251  4 426  Missouri . .  8 879  20 138  314  29 331  160  380  3  543  Total . . .  143 584  265 188  16 410  425 182  72 613  140 937  29 377  242 927 complexions: color of hair and eyes.  203  TABLE XVIII.  Complexions. By Nativities.  Nativity  Volunteers  Recruits  Dark  Light  Medium  Totals  Dark  Light  Medium  Totals  A  34 815  57 375  3 673  95 863  12 217  28 190  3 063  43 470  B  22 945  43 017  3 470  69 432  25 689  47 776  13 492  86 957  C  36 766  68 875  4 098  109 739  4 818  10 634  881  16 333  D  8 434  17 523  911  26 868  2 447  6 058  292  8 797  E  6 753  11 444  428  18 625  2 051  3 243  426  5 720  F  8 247  14 613  488  23 348  1 753  3 795  149  5 697  Gi  369  602  206  1 177  175  291  167  633  Ga  1 935  4 205  80  6 220  219  371  48  638  H  699  1 186  104  1 989  1 238  2 054  363  3 655  I  2 425  3 431  249  6 105  3 929  5 573  1 005  10 507  J  2 732  5 998  352  9 082  2 773  5 325  1 351  9 449  K  645  1 297  88  2 030  663  1 514  435  2 612  L  6 291  11 752  927  18 970  7 423  13 482  4 272  25 177  M  703  769  52  1 524  863  578  222  1 663  N  8 381  19 273  1 147  28 801  4 807  9 804  2 701  17 312  0  447  2 079  61  2 587  338  1 332  140  1 810  P  25  14  -  39  193  48  48  289  Q  972  1 735  76  2 783  1 017  869  322  2 208  Total  143 584  265 188  16 410  425 182  72 613  140 937  29 377  242 927 204  complexions: color of hair and eyes.  TABLE XIX.  Complexions.  U. S. Soldiers by States.  State of  Enlistment  Absolute  Relative  Dark  Light  Medium  Total  Dark  Light  Medium  Total  Maine . . .  22 144  34 188  2 279  58 611  378  583  39  1 000  New Hampshire  9 252  15 054  2 557  26 863  345  560  95  1 000  Vermont . .  7 053  12 708  1 699  21 460  329  592  79  1 000  Massachusetts  14 231  27 781  2 003  44 015  323  631  46  1000  Connecticut .  9 917  19 631  2 488  32 036  309  613  78  1 000  New York . .  13 523  23 879  14 712  52 114  260  458  282  1 000  Pennsylvania .  24 809  39 267  8 417  72 493  342  542  116  1 000  West Virginia  5 661  11 405  2  17 068  332  668  0  1 000  Kentucky . .  6 313  13 309  0  19 622  322  678  0  1 000  Ohio....  20 252  44111  66  64 429  314  685  1  1 000  Indiana. . .  23 264  38 159  2 679  64 102  363  595  42  1 000  Illinois . . .  23 032  43 449  3 319  69 800  330  622  48  1 000  Michigan . .  6 914  20 869  32  27 815  249  750  1  1 000  Wisconsin . .  14 833  34 610  2  49 445  300  700  0  1 000  Iowa....  5 960  7187  5 215  18 362  325  391  284  1 000  Missouri . .  9 039  20 518  317  29 874  303  686  11  1000  Total . . .  216 197  406 125  45 787  668 109  324  608  68  1000 COMPLEXIONS: COLOR OF HAIR AND EYES.  205  TABLE XX.  Complexions.  U. S. Soldiers by Nativities.  Nativity  Absolute  Relative  Dark  Light  Medium  Total  Dark  Light  Medium  Total  A  47 032  85 565  6 736  139 333  338  614  48  1000  B  48 634  90 793  16 962  156 389  311  581  108  1 000  C  41 584  79 509  4 979  126 072  330  631  39  1000  D  10 881  23 581  1 203  35 665  305  661  34  1 000  E  8 804  14 687  854  24 345  362  603  35  1 000  F  10 000  18 408'  637  29 045  344  634  22  1 000  Gi  544  893  373  1 810  301  493  206  1 000  G2  2 154  4 576  128  6 858  314  667  19  1 000  H  1937  3 240  467  5 644  343  574  83  1 000  I  6 354  9 004  1254  16 612  383  542  75  1 000  J  5 505  11 323  1703  18 531  297  611  92  1 000  K  1 308  2 811  523  4 642  282  605  113  1 000  L  13 714  25 234  5 199  44 147  311  571  118  1 000  M  1 566  1 347  274  3 187  491  423  86  1 000  N  13 188  29 077  3 848  46 113  286  631  83  1 000  0  785  3 411  201  4 397  178  776  46  1 000  P  218  62  48  328  665  189  146  1 000  Q  1 989  2 604  398  4 991  398  522  80  1 000  Total  216 197  406 125  45 787  668 109  324  608  68  1 000", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dfca_h2k5.yz68", "00000000-0000-0000-9199-8060757178F7", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "General Principles of Insurance and Tables of Mortality", "9918573882206676X26", null, "1858", "1858", "The Mutual Benefit Life Insurance Company published a small handbook in 1858 to explain life insurance to policyholders. This section covered general principles and provided some of the tables of mortality then in use. Pages 10-17 of Mutual Benefit Life Insurance Company, Life Insurance, its nature, origin, and progress: a plain explanation of the principles. . .. New York: John A. Gray, 1858.", "Monographs, Excerpts", null, "Background Information", "10", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "10     general principles explained.  New York,\" and the \" New England Life Insur-  ance Company of Boston.\" The next were the  \" State Mutual, of Worcester, Mass.,\" the \" Mu-  tual Benefit, of  Newark,\"  and the \" New York  Life Insurance  Company,\" of New York City,  established in 1845, since that date several other  companies have been  established  in  different  sections of the country.        GENERAL PRINCIPLES OF INSURANCE.    Notwithstanding the  natural weakness of the  human frame, and  the obvious insecurity and  brevity of life, it is  clear  that, in  the almost  boundless resources of the  mind of man, Provi-  dence has  furnished him with a variety of intel-  lectual devices, which, in a very great degree,  compensate  for  those  imperfections.   By the  improvement of  the mechanical arts, the feeble  strength of individuals is increased to an almost  unlimited extent; and  the  simplification of the  sciences has produced a correspondent facility  and power in their  acquirement.   Thus, much  more is done and learned, with  greater effect, in  less time,  and  with far less  labor, than couldgeneral principles explained.     11  formerly have  been done or learned by  any  then-existing means; which  economy of time  and strength, as it were, lengthens life, by leav-  ing space  in  it  for other,  or more  profound  pursuits.  In like manner, the proverbial uncer-  tainty  of life, which  appears to be altogether  beyond the reach of  human  control, is not  without  a  consolatory  remedy, at least, inso-  much as relates to the prevention of that distress  which the  destitution of those we leave behind  us adds to death,  or to the occurrence of  a sud-  den, heavy, or unexpected, calamity.   It  seems,  therefore, highly probable that  mankind were  subjected to  infirmity,  shortness of  life, and  continual mutability, partly for the purpose of  inducing the unceasing exertion of  the counter-  acting  powers of ingenuity  and prudence;  in  the diligent improvement of which  consists one  of the greatest moral benefits of the most high  ly-cultivated conditions of society.     It is not intended here to enter into any fur  ther explanation of the advantages to be derived  from this continual improvement, or to enforce  any of the  ordinary rules of prudence ; but only  to exhibit the qualities and importance of that12     general principles explained.  branch of it, which relates to the securing of a  safe termination to an uncertain event, as far as  that object  is to be effected  by the means of  Life-Insurances.   The  nature and  design  of  these contracts  not being either generally  01  accurately understood, prevents their very great  value  being duly appreciated; but it will  be  found, when the principles upon which they are  founded are  rightly comprehended, that it is im-  possible to entertain too high an  estimation  of  their utility, or to conceive too wide an extent  for their application.   Their  vital principle is  future security;  and their chief employment, at  present, is to secure a provision  for those  who  depend upon some particular individual for sup-  port, whenever their protector shall be removed  by  death.   For  though,  in a multitude of in-  stances, the  first burst of sorrow is far too deep  and  violent  to admit of any other feeling than  that of anguish,  yet it cannot be  doubted  that  even such distress is soon rendered more acute,  when the survivors reflect that they are left  in  the world destitute as well as friendless; and  calamity is always most afflicting, when the dis-       general principles explained.     13  traction of grief is  harassed by the  anxieties  and privations of want.     As tne present treatise is intended to afford  the fullest information upon  the subject in  the  plainest terms,  perhaps the  first  point to be  made clear, is the way in which the issue of an  event  depending upon  the  inscrutable will of  Providence, can be ascertained by a human be-  ing, with any probability of being correct in the  result.  The chance of the duration of life, in  single instances, is of very great uncertainty;  but as  all  human knowledge is extremely lim-  ited, when it attempts to institute a minuteness  of inquiry,  the calculation  can only be made  upon a very wide and general view of the  oc-  currence of such destruction, within  a certain  time and space.   When this view is sufficiently  extensive, even those events  which  are consid-  ered and called purely accidental and fortuitous,  are discovered to have certain relations and to  be governed by certain laws.  Thus,  the num-  ber of births, marriages, and deaths; the rela-  tive proportions of the sexes, are, under similar  circumstances, found to be nearly equal during  equal periods of time : whence it is easy, from14         tables  of mortality.  observations made upon them, to determine the  amount which an individual should pay for the  securing of a certain sum to be paid to his sur-  viving relatives at his death.             TABLES  OF MORTALITY.      An estimate of the average duration of hu-  man life, at all ages, is formed from observations  among large masses of mankind during an ex-  tended period of time; such observations con-  stituting the  materials,  or data,  necessary for  constructing  what  are  technically termed Ta-  bles  of Mortality.  A right understanding of  the construction and use of  these tables consti-  tutes a most important part of an accurate no-  tion of tho nature and benefits of life-insurance ;  a particular explanation of them shall, therefore,  now be given, as naturally following the pre-  ceding notices of the principles of chances, and  as the first elementary point of information con-  nected with the actual subject of these  pages.     The  first table of  mortality, which had any  pretensions to  accuracy  and utility,  was de-  duced  from a  series  of five  annual tables ofTABLE  OF MORTALITY.          15  births and deaths, with the ages and sexes dis-  tinguished, kept for the years 1687, 1688, 1689,  1690, and 1691, by Dr.  Neumann,  in the  city  of Breslau, in Silesia.   These, however, omitted  to state the entire number of the  population;  but the place  itself was  considered at the time  to afford a fair standard of ordinary mortality,  on account of the uniformity in numbers and  seclusion  of the inhabitants, who, being chiefly  engaged in the linen manufacture,  were visited  by few strangers, and remained resident on the  same  spot for one generation after  another.  From these registers, Dr. Edmund Halley con-  structed the  first  tolerably correct table for as-  certaining the true values of life-annuities, which  had been previously  calculated from hypotheti-  cal and uncertain data.   He assumed, from his  examination,  that  the  advances  of birth and  death are equal and uniform, which, however,  was soon discovered to be erroneous; but his  tables and rules will nevertheless  be sufficient  to make the reader acquainted with a few very  simple calculations, illustrating the relative du-  ration of life and its consequent value.     Considerable improvement was made by sev-16          TABLE OF  MORTALITY.  eral parties, subsequent to this period, up to the  time of the construction of  the Northampton  Tables, by Dr. Price, in 1769.  About the year  1800, a set of Swedish Tables was published,  founded on returns, both of the number  living  and of the deaths in Sweden and Finland for  20 years, ending with 1795.    The Carlisle Table, which is the one now  most in use, in England and  this  country, for  the calculation of life-insurance premiums, was  constructed by Mr. Joshua Milne, from obser-  vations  made by  Dr.   Heysham,   at Carlisle,  England, during a period of nine years, ending  in 1787.    The best actuaries in  England and this coun-  try, concur in opinion as to the decided superi-  ority of the Carlisle over the Northampton  tables of Mortality, and it is generally conceded  that these tables more closely exhibit the  state  of longevity as  it exists in the Northern and  Eastern States than any of the others.OOQ0MOl6iihUKll-'OO01M0)S*.MK)l-OtD(Xl^OlnK«li'-O100l)')O0lit'Ut0l-Ot000'401Cn*MK)HO    Age.   Breslau,  Dr. Halley,  frMt«o>^^aooe-i-«teuifcin«o^»i(roooooMBau*ik«uo>oi^^05&ogoiOfew3ggg  wt(i!Ottt»ioin>5io«isa)Sh-ajiM-»J*'00iwoffli«ioWh-S4.r-caio-ii-iio^ooa»ito*0'-oiutawwo    goo oo>— *s  4^ .£. ^ ^ ^ »  lSS2JSi»SH^U10CJlOWF-Cl^«OWO*.CD»SO)0*.^t30)i-OOMfc*5i>-WOOO  Northamp-     ton.   Dr. Price.  Carlisle,  Dr. Hey-   sham.  Sweden,  Dr. War-   lientin.  © © © © co o    »-2'£00^Gbl>^Ut<9HO(2Q0^OU1^UtS  OOOCOOOOCOOOOi--K!*.0!0  HHMiot5uu*wae>«)i>ioooi-Ki«*kiiia^-jmooo-Mu*ti(ji  COOOOOOOOH*Wt5W**01-^<WW                                    pHHMHMMMMWI->WHH|9IOI3K!l9)9  MMtoiouuiU*.tn(noM^aj(Doci---BaikJ-wos^oojogp K \"\"i!? Sf ™  WClOUOtJtOClWOOWHCCOlWtDOWBaiWO^if'i-ffiOHiOCJWO^rf.HCO  MUMMMHUMI  fc-wl-tSUUIiUW(»-l(JlIBO(SWtllO100b- tittlC-lfcO- Ki^O'O-tgOtOOOi—JOlOCjS  IOOOOOOOOOOM»UWa03^*o!hSS3fflSS!!icilO)S«0(OU-)OiS*W^IDr-IOUWKll5f-OtOaO-Oa   Breslau,  Dr. Halley.  Northanip     ton.   Dr. Price.  Carlisle.  Dr. Hey-   sham.  Sweden,  Dr. War-   gentin.  » 2  3,3 O    Ml    !s5  M — O    re °        g CO g    \"S3  EL a o-  ~» S    ||S,        sg i  ft n  >  a  f  H  O  *S    o    H  >  era     ,10     general principles explained.  New York,\" and the \" New England Life Insur-  ance Company of Boston.\" The next were the  \" State Mutual, of Worcester, Mass.,\" the \" Mu-  tual Benefit, of  Newark,\"  and the \" New York  Life Insurance  Company,\" of New York City,  established in 1845, since that date several other  companies have been  established  in  different  sections of the country.        GENERAL PRINCIPLES OF INSURANCE.    Notwithstanding the  natural weakness of the  human frame, and  the obvious insecurity and  brevity of life, it is  clear  that, in  the almost  boundless resources of the  mind of man, Provi-  dence has  furnished him with a variety of intel-  lectual devices, which, in a very great degree,  compensate  for  those  imperfections.   By the  improvement of  the mechanical arts, the feeble  strength of individuals is increased to an almost  unlimited extent; and  the  simplification of the  sciences has produced a correspondent facility  and power in their  acquirement.   Thus, much  more is done and learned, with  greater effect, in  less time,  and  with far less  labor, than couldgeneral principles explained.     11  formerly have  been done or learned by  any  then-existing means; which  economy of time  and strength, as it were, lengthens life, by leav-  ing space  in  it  for other,  or more  profound  pursuits.  In like manner, the proverbial uncer-  tainty  of life, which  appears to be altogether  beyond the reach of  human  control, is not  without  a  consolatory  remedy, at least, inso-  much as relates to the prevention of that distress  which the  destitution of those we leave behind  us adds to death,  or to the occurrence of  a sud-  den, heavy, or unexpected, calamity.   It  seems,  therefore, highly probable that  mankind were  subjected to  infirmity,  shortness of  life, and  continual mutability, partly for the purpose of  inducing the unceasing exertion of  the counter-  acting  powers of ingenuity  and prudence;  in  the diligent improvement of which  consists one  of the greatest moral benefits of the most high  ly-cultivated conditions of society.     It is not intended here to enter into any fur  ther explanation of the advantages to be derived  from this continual improvement, or to enforce  any of the  ordinary rules of prudence ; but only  to exhibit the qualities and importance of that12     general principles explained.  branch of it, which relates to the securing of a  safe termination to an uncertain event, as far as  that object  is to be effected  by the means of  Life-Insurances.   The  nature and  design  of  these contracts  not being either generally  01  accurately understood, prevents their very great  value  being duly appreciated; but it will  be  found, when the principles upon which they are  founded are  rightly comprehended, that it is im-  possible to entertain too high an  estimation  of  their utility, or to conceive too wide an extent  for their application.   Their  vital principle is  future security;  and their chief employment, at  present, is to secure a provision  for those  who  depend upon some particular individual for sup-  port, whenever their protector shall be removed  by  death.   For  though,  in a multitude of in-  stances, the  first burst of sorrow is far too deep  and  violent  to admit of any other feeling than  that of anguish,  yet it cannot be  doubted  that  even such distress is soon rendered more acute,  when the survivors reflect that they are left  in  the world destitute as well as friendless; and  calamity is always most afflicting, when the dis-       general principles explained.     13  traction of grief is  harassed by the  anxieties  and privations of want.     As tne present treatise is intended to afford  the fullest information upon  the subject in  the  plainest terms,  perhaps the  first  point to be  made clear, is the way in which the issue of an  event  depending upon  the  inscrutable will of  Providence, can be ascertained by a human be-  ing, with any probability of being correct in the  result.  The chance of the duration of life, in  single instances, is of very great uncertainty;  but as  all  human knowledge is extremely lim-  ited, when it attempts to institute a minuteness  of inquiry,  the calculation  can only be made  upon a very wide and general view of the  oc-  currence of such destruction, within  a certain  time and space.   When this view is sufficiently  extensive, even those events  which  are consid-  ered and called purely accidental and fortuitous,  are discovered to have certain relations and to  be governed by certain laws.  Thus,  the num-  ber of births, marriages, and deaths; the rela-  tive proportions of the sexes, are, under similar  circumstances, found to be nearly equal during  equal periods of time : whence it is easy, from14         tables  of mortality.  observations made upon them, to determine the  amount which an individual should pay for the  securing of a certain sum to be paid to his sur-  viving relatives at his death.             TABLES  OF MORTALITY.      An estimate of the average duration of hu-  man life, at all ages, is formed from observations  among large masses of mankind during an ex-  tended period of time; such observations con-  stituting the  materials,  or data,  necessary for  constructing  what  are  technically termed Ta-  bles  of Mortality.  A right understanding of  the construction and use of  these tables consti-  tutes a most important part of an accurate no-  tion of tho nature and benefits of life-insurance ;  a particular explanation of them shall, therefore,  now be given, as naturally following the pre-  ceding notices of the principles of chances, and  as the first elementary point of information con-  nected with the actual subject of these  pages.     The  first table of  mortality, which had any  pretensions to  accuracy  and utility,  was de-  duced  from a  series  of five  annual tables ofTABLE  OF MORTALITY.          15  births and deaths, with the ages and sexes dis-  tinguished, kept for the years 1687, 1688, 1689,  1690, and 1691, by Dr.  Neumann,  in the  city  of Breslau, in Silesia.   These, however, omitted  to state the entire number of the  population;  but the place  itself was  considered at the time  to afford a fair standard of ordinary mortality,  on account of the uniformity in numbers and  seclusion  of the inhabitants, who, being chiefly  engaged in the linen manufacture,  were visited  by few strangers, and remained resident on the  same  spot for one generation after  another.  From these registers, Dr. Edmund Halley con-  structed the  first  tolerably correct table for as-  certaining the true values of life-annuities, which  had been previously  calculated from hypotheti-  cal and uncertain data.   He assumed, from his  examination,  that  the  advances  of birth and  death are equal and uniform, which, however,  was soon discovered to be erroneous; but his  tables and rules will nevertheless  be sufficient  to make the reader acquainted with a few very  simple calculations, illustrating the relative du-  ration of life and its consequent value.     Considerable improvement was made by sev-16          TABLE OF  MORTALITY.  eral parties, subsequent to this period, up to the  time of the construction of  the Northampton  Tables, by Dr. Price, in 1769.  About the year  1800, a set of Swedish Tables was published,  founded on returns, both of the number  living  and of the deaths in Sweden and Finland for  20 years, ending with 1795.    The Carlisle Table, which is the one now  most in use, in England and  this  country, for  the calculation of life-insurance premiums, was  constructed by Mr. Joshua Milne, from obser-  vations  made by  Dr.   Heysham,   at Carlisle,  England, during a period of nine years, ending  in 1787.    The best actuaries in  England and this coun-  try, concur in opinion as to the decided superi-  ority of the Carlisle over the Northampton  tables of Mortality, and it is generally conceded  that these tables more closely exhibit the  state  of longevity as  it exists in the Northern and  Eastern States than any of the others.OOQ0MOl6iihUKll-'OO01M0)S*.MK)l-OtD(Xl^OlnK«li'-O100l)')O0lit'Ut0l-Ot000'401Cn*MK)HO    Age.   Breslau,  Dr. Halley,  frMt«o>^^aooe-i-«teuifcin«o^»i(roooooMBau*ik«uo>oi^^05&ogoiOfew3ggg  wt(i!Ottt»ioin>5io«isa)Sh-ajiM-»J*'00iwoffli«ioWh-S4.r-caio-ii-iio^ooa»ito*0'-oiutawwo    goo oo>— *s  4^ .£. ^ ^ ^ »  lSS2JSi»SH^U10CJlOWF-Cl^«OWO*.CD»SO)0*.^t30)i-OOMfc*5i>-WOOO  Northamp-     ton.   Dr. Price.  Carlisle,  Dr. Hey-   sham.  Sweden,  Dr. War-   lientin.  © © © © co o    »-2'£00^Gbl>^Ut<9HO(2Q0^OU1^UtS  OOOCOOOOCOOOOi--K!*.0!0  HHMiot5uu*wae>«)i>ioooi-Ki«*kiiia^-jmooo-Mu*ti(ji  COOOOOOOOH*Wt5W**01-^<WW                                    pHHMHMMMMWI->WHH|9IOI3K!l9)9  MMtoiouuiU*.tn(noM^aj(Doci---BaikJ-wos^oojogp K \"\"i!? Sf ™  WClOUOtJtOClWOOWHCCOlWtDOWBaiWO^if'i-ffiOHiOCJWO^rf.HCO  MUMMMHUMI  fc-wl-tSUUIiUW(»-l(JlIBO(SWtllO100b- tittlC-lfcO- Ki^O'O-tgOtOOOi—JOlOCjS  IOOOOOOOOOOM»UWa03^*o!hSS3fflSS!!icilO)S«0(OU-)OiS*W^IDr-IOUWKll5f-OtOaO-Oa   Breslau,  Dr. Halley.  Northanip     ton.   Dr. Price.  Carlisle.  Dr. Hey-   sham.  Sweden,  Dr. War-   gentin.  » 2  3,3 O    Ml    !s5  M — O    re °        g CO g    \"S3  EL a o-  ~» S    ||S,        sg i  ft n  >  a  f  H  O  *S    o    H  >  era     ,18             TABLE  OF  MORTALITY.           COMPARATIVE  EXPECTATIONS  OF LIFE.      Showing  the  Expectation or Average duration  of  life, deduced from    Original Tables, prepared under the superintendence of a committee    of eminent Actuaries  in England, and  compared with the Carlisle,    Equitable, and Northampton Tables.     Male    Female                                        Lives—  JAves—                                  North-  ■o    Town,  Town,  Gene-   Ad-     Car-    Equi-   amp-  © o5    Coun-   Coun-   ral     justed  lisle   table   ton.  i       try and try and Expe-   Expe-   Expe-   Expe-   Expe          Irish   Irish   rience. rience. rience. rience. rience.  o    Expe-   Expe-                                         rience. rience.                                   20  39.84   35.86   40.97   41.49   41.46   41.06   33.43  21     39.29   36.01   40.45   40.79   40.75   40.33   32.90  22     38.70   36.20   39.92   40.09   40.04   39.60   32.39  23     37.98   35.41   39.18   39.39   39.31   38.88   31.88  24     37.41   34.81   38.54   38.68   38.59   38.16   31.36  23     36.63   34.41   37.84   37.98   37.86   37.44   30 85  26     35.88   33.79   37.13   37.27   37.14   36.73   30.33  27     35.23   33.14   36.42   36.56   36.41   36.02   29.82  28     34.63   33:07   35.76   35.86   35.69   35.33   29.30  29     33.96   32.61   35.06   35.15   35.00   34.65   28.79  30     33.17   31.73   34.25   34.43   34.34   33.98   28.27  31     32.44   31.04   33.50   33.72   33.68   33.30   27.76  32     31.73   30.51   32.75   33.01   33.03   32.64   27.24  33     30.92   29.86   31.98   32.30   32.36   31.98   26.72  34     30.21   29.60   31.27   31.58   31.68   31.32   26.20  35     29.52   29.07   30.55   30.87   31.00   30.66   25.68  36     28.87   28.88   29.90   30.15   30.32   30.01   25.16  37     28.15   28.30   29.20   29.44   29.64   29.35   24 64  38     27.49   27.62   28.51   28.72   28.96   28.70   24.12  39     26.81   27.00   27.79   28.00   28.28 . 28.05   23.60  40     26.06   26.36   27.07   27.28   27.61   27.40   23.08  41     25.42   25.84   26.41   26.56   26.97   26.74   22.56  42     2470    25.34   25.68   25.84   26.34   26.07   22.04  43     24.00   24.57   24.98   25.12   25.71   25.40   21.54  41     23.34   23.94   24.26   24.40   25.09   24.75   21.03  45     22.63   23.21   23.55   23.69   24.46   24.10   20.52  46     21.98   22.60   22.85   22.97   23.82   23.44   20 02  47     21.24   21.97   22.12   22.27   23.17   22.78   19.51  48     20.62   21.16   21.41   21.56   22.50   2212    19.00  49     20.08   20.69   20.79   20.87   21.81   21.47   18.49  1 Completed OO ~5 <I ~j <I -4 -3 ~J -4 <I <l ©fliaaoi 05CJCJOSOS Cncncn&ncn en Cn en en en Ana O to 00-10s en >fcC0*0*-o tOCCMOU »Ut9MO tOOO-JOSCn ifcUtSHOl Age.        ;&. .fa. cn Cn Cn OS OS OS *q CS CO OOtOtOO© H-«JOWU CO rf* Cn Cn CS CS -1 GO 00 tO Lj to — ji.b;© cn to k. bo co bo co to \"co bo to to »&. to ife. to ►&. © as to ^iu©^j>&. Cn tOOSCOCOCO •J-lUtB*. h-COCOOO-4 -JOCStO-4 (CVtSOlH ^IOUUh  Male Lives— Town, Coun-try and Irish Expe-rience.  J*. [^ J* >&. Cn Cn Cn OS OS <l -^ popoto©© ►-» ►- JO CO CO rf^ >*^ cn as as <1 GO CO to © -1 bo cn ^-4 a* co <i ~ as co to co bo cn © as o ao \"*. Ui ^i kj<jwb-J cn co ao ►&. © cn © as ao cn —i to to co r-co ao cn as © © to-i*—©oo cototo-jao con-©asm       Female Lives— Town, Coun-try and Irish Expe-rience.  •*»> Cn Cn Cn OS OS OS ^ <? 00 GO GO tO © © *-- ►— *£> K> UU *. rffc. Cn OS OS <! CO 00 to © -j ow^j©£ oPiSr^rlS£! to •*»• © en © en © os k> be co to as to bo en — <i'&■•— Cn -J ^1 -*J GO CS t*^ OS tC> CO © tO OS CO r—> CO ©tOOD^h- GO GO til CO W © OS tO OS »-•   Gene-ral Expe-rience.  J*, CnCnCnOSOS OS-3-3 po 00 to to to © © WKSfcOCOCO rfufkCnCSOS -^JCOOOtO© m S^S^Jfe 2£ i2°C5 j- cn © *• to *■ to cn b cs ~ l~i co to cn k> bo cn i- bo in ►-< oo toNsasi—oo oscs-io*. ©-loscs-i h-ui-ooM -i-itotscs ocsta©oo Ad-justed Expe-rience.  cn en as as as <t <i -q po co to to © © *-»-. *o te> co co *. AOioa^) ooootoS — S3 2S tS !fc 52 ° K r^ jr P1 b: ;} to -J to 4 io bo co bo co to in Vo bo en io to b co :-■ »— ©fcO©to*-* cotoascnoo ©cocn-3to ©h-h-torf* *ocn*-ooo Go-^aoto*- Car-lisle Expe-rience.  20.83 20.20 19.59 19.00 18.43 17.85 17.28 16.71 16.15 15.60 15.06 14.51 13.96 13.42 12.88 12.35 11.83 11.32 10 82 10.32 9.84 9.36 8 88 8.42 7.97 7.52 7.08 6.64 6.20 5.78 5.38        Equi-table Expe-rience.  *&> encnenpsas ps <j <i oo oo to to © © © w~ toioco eo4*->fe-cncn asas-j-i-3 r1 HSi?1't;V to Jo ki U b b cn to '.&. bo co co k> ^-J Vo 'as :-■ b l-> en b cn © cn to en *- oo co ao 4* tsu»-to cn © as to co en h- go en >— co en co o co cs >«». N> © to    North-amp-ton Expe-rience.  /", "Mutual Benefit Life Insurance Company", null, null, "Gray, John A. (John Abercrombie), 1818-1903", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-db3m~zsg3~y3hi", "00000000-0000-0000-1AF5-8761C5296CA6", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Medical Examination by an Experienced Examiner", "9918573882206676X27", null, "1858", "1858", "The Mutual Benefit Life Insurance Company published a small handbook in 1858 to explain life insurance to policyholders.  This section described the importance of the medical exam and the conditions that might disqualify an applicant for life insurance. Page 44-58 of Mutual Benefit Life Insurance Company, Life Insurance, its nature, origin, and progress: a plain explanation of the principles.... New York: John A. Gray, 1858.", "Monographs, Excerpts", null, "Background Information", "15", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "44         MEDICAL  EXAMINATIONS.           MEDICAL EXAMINATIONS.           BY  AN  EXPERIENCED  EXAMINER.      Until the establishment of the present prac-  tice of appointing a medical officer to look after  the interests of a  life-insurance company, great  uncertainty existed in relation  to  the insura-  bility of many lives that  were  taken as  good  risks.  This uncertainty became often the cause  of protracted litigation ;  and  the  opinions  of  learned jurists on the bench, and the decisions  of juries were not unfrequently founded  upon  imperfectly ascertained  facts, and   loose and  careless opinions  supplied by an agent  or bro-  ker : such as, \" he believed it to be a good life,\"  or that,  \" the party was in a reasonably good  state of health,\" and a host of similarly express-  ed opinions—as vague as they were unsatisfac  tory.*  Often these would arise from positive      * Stackpole v. Simon. Sitt. at Guildhall Hall. Vac. 1779  Ross v. Bradshaw, 1. Blac. Rep. 312.MEDICAL  EXAMINATIONS.         45  ignorance,  at other  times,  from  collusion  or  carelessness on the part of one who was repre-  sented as being the usual medical  attendant of  the  party  whose life was proposed to be  in  sured.*     A remarkable instance of the effect of an  ab-  sence  of a personal examination by an officer  of a company, occurred in England in the year   1824.   The Duke of Saxe Wiemar insured his   life for £5,000 sterling, and died  in a year or   two after  effecting  the insurance.   After  the   amount of the policy had been paid,  it was as-   certained  that he  had been  wounded in  the   spine, and  that he  was both  idiotic and para-   lytic at the time of effecting the insurance, but   had furnished the office with  two certificates,     tie from a physician  and  another from a  sur-   geon, stating that he was at that time in sound   health: on which certificates the policy was is-   sued.   In a suit to recover the money paid, the   company   was non-suited,  as the  transaction   was in accordance with their method of busi-   ness.   Had  there  been a medical examiner at-  * Aveson v. Lord Kincaird and others, 6 East,4b         MEDICAL  EXAMINATIONS.   tached to the company, a policy on such a  life   never would have been issued.      The certificate of the usual medical attend-   ant is often of little importance beyond furnish-   ing information  as  to  the past state  of health   of the party, or as to his family predisposition,   for the simple reason  that he may never have   had an opportunity of investigating the condi-   tion of his patient in  the  mode needed for life-   insurance.   The writer has known instances of   physicians giving certificates of good  health,   and upon having their attention directed to  the   results of a  physical  examination, frankly  ac   knowledge that they were ignorant until  that   time of any predisposition to disease in their pa-   tients.  An instance worthy of note occurred to   the writer where the physician had been an inti-   mate friend of the applicant for twenty years,   during which time he had not  experienced any   indisposition,  but  had always  had the appear-   ance of ruddy health.   An examination  of the   chest detected unequivocal symptoms of the ex-   istence of incipient phthisis.   The attention ol   the medical friend, who had accompanied him  to  the office, was directed to them; he at onceMEDICAL  EXAMINATIONS.         47  admitted the accuracy of the diagnosis, and ac-  knowledged that the case was not an  insurable  one.  He subsequently-reported  that  this  indi-  vidual had, about six months after his examina-  tion, suffered from severe  pulmonary haemor-  rhage with every  prospect of phthisis.   Other  instances could be given where the parties died  of the same disease—the predisposition being  unsuspected by the physician  solely from his  not having had occasion to investigate the con-  dition of his patient with reference to pulmona-  ry diseases.     The experience of  the London Equitable In-  surance Company furnishes some very  striking  facts in illustration of the importance of a per-  sonal examination by an officer of the company.  At an early period of their  business,  when the  deaths  amounted  to  331, those  caused  by con-  sumption were 122, at the rate of 7. 7 per thou-  sand.   In the  year 1832,  the total number of  deaths was 4,095; while the number  from  con-  sumption was 339. In the first period the appli-  cations were very carelessly investigated, while  during a part of the last  mentioned period a   careful personal examination was  made  by a48         MEDICAL EXAMINATIONS.   suitably  qualified  person  appointed by the of-   fice.      It has happened also that persons have pre«   sented themselves for examination who never   nave had any medical attendant, and who never   suspected the existence of any disease in them-   selves, and  yet have given evidence of such a   state of system, that there existed no reasona-   ble presumption that they would ever reach the   period of their \"expectation.\"  An early death   has proved the accuracy of this opinion in many   an instance.   It was formerly required of the   party, in case he had no physician, to procure   a certificate from  « some other \" medical man;   but it is  evident that but little information of   value  could  be obtained from  such a source.   The medical officer appointed by the company   supplies now any deficiency in the medical tes-   timony, and offers the best security both for the   insured and the insurers—to the former  as fur-   nishing to a certain extent a warranty, and to   the latter a security that  every means has been   used to obtain none but good risks.    When  a person applies for examination he  should bring with him,  1st, a declaration signed           MEDICAL EXAMINATIONS.         49  by himself; 2d, another signed by a friend; and  3d, one from  his medical attendant; all giving  an  accurate  statement of his health.   These  documents are furnished in blank by the com  pany.  An error has arisen on the part of some  medical gentlemen in supposing that when they  fill up the blank that is intended for them, that  they are  furnishing the information they impart,  at the request of the company.  This mistake  has  doubtless arisen from  a common practice  of the officers and agents,  who, in wishing to  save the applicant  a  little  trouble,  send the  blank  certificate  to the  physician;  the latter  supposing it to come from  the company are in  some instances unwilling to furnish the required  information  without a fee.  All these docu-  ments, however, being the  proofs which the ap-   plicant  is required to  supply and to bring with   him to be filed in the office, must be obtained   by him  at his own expense.  In addition  to   these  evidences, the agent is supposed  to be-   come  to some extent responsible, and in one   company at least, he is required to fill up an of-   ficial  certificate  embracing his knowledge of  350  MEDICAL  EXAMINATIONS.   the party, and his opinion as to the safety of the   risk in question.     In no case should a person be examined un-   less all these papers are  submitted, complete, to   the  medical examiner; for  they might contain   some important fact necessary to a correct de-   cision.   With these  documents before him  he   will proceed to a personal examination.     The external form and general  aspect often   convey a vast amount of information to one ac-   customed  to  associate these  with  health and   sickness.   The habits of life, which may be as-   certained by a few questions, will naturally lead   to others ; the trade, occupation, profession, etc.,   of the applicant come under this head of exam-   ination, and will enable the  examiner  to  draw   his inference as to their effects  on the general   health.   The condition of the brain and spinal   marrow will also be thus learned;  for this can   be ascertained to a sufficient extent  by the gait,   manner of speech, and want of control  over the   muscular system, exhibited in tremor, and other   irregular movements,  together with questions  on these points which may suggest themselves.MEDICAL  EXAMINATIONS.         51  [   In the examination of the chest, although it is  \\\\ of great importance, yet it is not necessary to  1 ascertain anything more than that there is a de-   parture from health ; it is not required that any   detail of unfavorable symptoms be recorded, if   any appear.   The first step in the investigation,   is to percuss the chest in all its parts, and then  I to apply the  Stethescope, or the ear, to ascer-   tain whether or not there exists a healthy res-   piration.  Consider  the  capacity of the  chest,   whether narrow or expanded—whether there is   any unusual  depression in the subclavian re-   gions—whether the chest expands equally  on   inspiration—any Bronchophony, particularly in   the subclavian and mammary regions.   The   action  of the heart is also ascertained by aus-   cultation : ascertain whether  the  rythm of the   heart is natural, and confined within the natural   limits—whether the impulse of  the heart is co-   incident with the first sound, and  of natural in-   tensity.  When  making a report, the state of   the heart may be recorded \"Normal,\" or \"Ab-   normal,\" as the case may be.      The pulse, besides being regular, should never   beat over 90 in a minute : if it cannot be  re-  52         MEDICAL EXAMINATIONS.    duced to this by changing to a recumbent posi-    tion, rest, and time being given  for  the  com-    posure of the nervous system, and if necessary,    two or three examinations,  on different days—    the applicant is to be rejected.  A very careful    investigation into the condition of the heart and   lungs should be made,  whenever the pulse  is   found to beat uniformly  over 80.     An habitually rapid pulse is generally indica-   tive  of some  organic   affection.   The  pulse   should always be regular in its beats, and never   intermitting.     Spitting of blood, if it arise in a person of  a   consumptive family, must be  a reason  for re-   jection.   If, however, it arises from a  mere   congestion  of  the  mueous membrame of the   bronchial tubes, it need not be a cause for declin-   ing the risk; a  sufficient time, however, must   elapse to ascertain,  together with  an  examina-   tion of the chest, that it is of this nature.  Bleed-   ing from the lungs from violent straining, need   not be a reason for rejecting the applicant__al-  lowing, as in the former instance, a long time  to pass to  prove that it is but a local and tempo-  rary affection, and has not produced any diseaseMEDICAL  EXAMINATIONS.         53  If either of these should be followed by cough or  occasional difficulty of respiration, the party of  course, must be rejected.  It is needless to men-  tion that more than usual care must be taken  in the physical examination  of such persons.    Occasional difficulty of breathing, or asthma,  must always  be rejected.  The causes of these  affections are often obscure, and  as they arise  from some obstruction in one  or  other  of the  vital organs, as  the  lungs  or  heart, safety re-  quires that such  cases should not be received.     There is an affection of the  bowels which is  often suddenly fatal; a person affected with it  is by no means a safe risk:  it is an obstruction,  sometimes known as colic, bilious colic, or spas-  modic  colic;  any person  affected with  this,  ought to be rejected.     In making up  a report to be transmitted to  the parent office, there need be no  remark made  beyond  the conclusions at which  the examiner  has arrived, a brief statement and expression of  opinion, which  ought to be unqualified, is  all  that is required.   Whenever there is any state-  ment of positive disease, it would be well for54  MEDICAL  EXAMINATIONS.   the  examiner to refer  to  it in a note to show   that it has received his  special attention.     The existence of tumors, hernia, hemorrhoids,   fistula,  &c, should be ascertained,  and  their   condition ascertained by inspection.  Fistula is   a good reason for rejection.  A hernia need not   render the party incapable of  availing  himself   of the benefits of life-insurance provided  it be   properly secured by a truss, and the occupation   of the individual be such as not to  endanger its   protrusion by excessive straining.     When inquiring into  the usual occupation of   the applicant, the questions relating to exposure   to insalubrious climates will naturally arise.  If   the party has been within the tropics, or in any   part of the  world  known to  be  at times un-   healthy, it will be necessary to inquire whether   or not he has had any endemic diseases which   have prevailed—and if so, if  they have  left any   enlargement of the liver or spleen, or any other   permanent effection of an important viscus.    Hereditary tendency  to disease  is an impor-  tant part of an  examination.   There are  how-  ever,  but  four  that appear  to have a  bear-  ing upon  the duration  of  life;  these are  conMEDICAL  EXAMINATIONS.         55  sumption, disease of the  heart, insanity and  epilepsy.  Although diseases are termed heredi-  tary, yet they are, more properly speaking, pre-  dispositions.   Hence, the actual development  of disease requires  some co-operating circum-  stances, and it is not in every instance of the  parent  having either of  the  above named dis-  eases that the applicant should be pronounced  ineligible to life-insurance.       Consumption.—Consumption does not always  descend from the parent to the child.   But if  either  parent has been effected with it, and the   offspring exhibits a slender form, or has a nar-   row  or badly formed  chest,  or an habitually   rapid  pulse,  the  predisposition  is  evidently so   strong, that some slight exposure or irregularity   of living, or a constrained occupation in a close   atmosphere,  will almost surely  develop the dis-   ease.      Many persons exhibit no such tendency, and,   therefore, ought not to be rejected, because one   of the  family may have  had the disease.  It is   different, however, if two parents have died of   it or one parent and one child.  Under these56  MEDICAL  EXAMINATIONS.   circumstances the applicant ought not to be in-   sured until he has passed  the period at which   he is likely to be  affected with the  disease.   Now it appears from records carefully  made,   that  more persons die of consumption between   the ages of 18  and 35 years than at any other   age, the mortality from this cause sinking very   rapidly to comparatively a  small  number after   the last mentioned age.  Hence, it is advisable   that  the party should have reached the age of   comparative exemption.  Forty   years,  there-   fore,  is a good age that he should be required to   attain before he  is examined ; some have lim-   ited  the period to thirty-five years.  It is  of   course supposed that all symptoms, both rational   and   physical, which indicate a  predisposition   to phthisis, be absent.       Disease  of the Heart—There are  many ex-   amples on record of this predisposition inherited   from  parents.   One of a family of high rank in   Italy,  which, for four successive  generations,   were  effected  with  aneurism,  or  morbid en'   largement  of the heart.   When  a parent has  died of this affection, it would be safe  to examMEDICAL EXAMINATIONS.         57  ine an applicant with more than ordinary care ;  advancing age in this disease will be more likely  to aid in the development, than tend to lessen  the predisposition, as is the case in phthisis.       Insanity.—The  hereditary predisposition in  this affection is so  strong that it has been dis-  tinctly traced in six sevenths  of the cases in a  lunatic asylum.   One instance of insanity in  a family must be a cause of rejection.       Epilepsy.—Epilepsy in a parent need not be  in every instance a cause of rejection.   Its ex-  istence should lead to a very  scrutinizing  in-  quiry into  the present condition of the party and  his mode of living and occupation.   No positive  rule can  be laid down ; it must be left to the  sound judgment of the examiner.       General Indications of Longevity.   A well  proportioned stature without  being  too tall,  but rather  of  the middle height,  and  tolera-  bly stout.   The  complexion  not  too  florid;  too much  ruddiness, at least in  youth, is seldom  a  sign of longevity.  The hair should not be58         MEDICAL  EXAMINATIONS.  black, but rather approaching  to fair ;  the skin  strong, but not coarse.  The head not too large.  He should have  prominent veins on the limbs  and the shoulders rather round than flat.  The  neck is neither very long nor short.  The stom-  ach of such a person does not project—the hands  are large, but not too deeply cleft.   The foot is  rather thick than  long, and the inferior limbs  are firm  and round.   The chest is broad and  arched—the voice strong,  and  the faculty of re-  taining the breath for a considerable time with-  out inconvenience or difficulty,  is  one of the  most marked signs.   In general there is a com-  plete harmony of proportion among all the parts  of the body.44         MEDICAL  EXAMINATIONS.           MEDICAL EXAMINATIONS.           BY  AN  EXPERIENCED  EXAMINER.      Until the establishment of the present prac-  tice of appointing a medical officer to look after  the interests of a  life-insurance company, great  uncertainty existed in relation  to  the insura-  bility of many lives that  were  taken as  good  risks.  This uncertainty became often the cause  of protracted litigation ;  and  the  opinions  of  learned jurists on the bench, and the decisions  of juries were not unfrequently founded  upon  imperfectly ascertained  facts, and   loose and  careless opinions  supplied by an agent  or bro-  ker : such as, \" he believed it to be a good life,\"  or that,  \" the party was in a reasonably good  state of health,\" and a host of similarly express-  ed opinions—as vague as they were unsatisfac  tory.*  Often these would arise from positive      * Stackpole v. Simon. Sitt. at Guildhall Hall. Vac. 1779  Ross v. Bradshaw, 1. Blac. Rep. 312.MEDICAL  EXAMINATIONS.         45  ignorance,  at other  times,  from  collusion  or  carelessness on the part of one who was repre-  sented as being the usual medical  attendant of  the  party  whose life was proposed to be  in  sured.*     A remarkable instance of the effect of an  ab-  sence  of a personal examination by an officer  of a company, occurred in England in the year   1824.   The Duke of Saxe Wiemar insured his   life for £5,000 sterling, and died  in a year or   two after  effecting  the insurance.   After  the   amount of the policy had been paid,  it was as-   certained  that he  had been  wounded in  the   spine, and  that he  was both  idiotic and para-   lytic at the time of effecting the insurance, but   had furnished the office with  two certificates,     tie from a physician  and  another from a  sur-   geon, stating that he was at that time in sound   health: on which certificates the policy was is-   sued.   In a suit to recover the money paid, the   company   was non-suited,  as the  transaction   was in accordance with their method of busi-   ness.   Had  there  been a medical examiner at-  * Aveson v. Lord Kincaird and others, 6 East,4b         MEDICAL  EXAMINATIONS.   tached to the company, a policy on such a  life   never would have been issued.      The certificate of the usual medical attend-   ant is often of little importance beyond furnish-   ing information  as  to  the past state  of health   of the party, or as to his family predisposition,   for the simple reason  that he may never have   had an opportunity of investigating the condi-   tion of his patient in  the  mode needed for life-   insurance.   The writer has known instances of   physicians giving certificates of good  health,   and upon having their attention directed to  the   results of a  physical  examination, frankly  ac   knowledge that they were ignorant until  that   time of any predisposition to disease in their pa-   tients.  An instance worthy of note occurred to   the writer where the physician had been an inti-   mate friend of the applicant for twenty years,   during which time he had not  experienced any   indisposition,  but  had always  had the appear-   ance of ruddy health.   An examination  of the   chest detected unequivocal symptoms of the ex-   istence of incipient phthisis.   The attention ol   the medical friend, who had accompanied him  to  the office, was directed to them; he at onceMEDICAL  EXAMINATIONS.         47  admitted the accuracy of the diagnosis, and ac-  knowledged that the case was not an  insurable  one.  He subsequently-reported  that  this  indi-  vidual had, about six months after his examina-  tion, suffered from severe  pulmonary haemor-  rhage with every  prospect of phthisis.   Other  instances could be given where the parties died  of the same disease—the predisposition being  unsuspected by the physician  solely from his  not having had occasion to investigate the con-  dition of his patient with reference to pulmona-  ry diseases.     The experience of  the London Equitable In-  surance Company furnishes some very  striking  facts in illustration of the importance of a per-  sonal examination by an officer of the company.  At an early period of their  business,  when the  deaths  amounted  to  331, those  caused  by con-  sumption were 122, at the rate of 7. 7 per thou-  sand.   In the  year 1832,  the total number of  deaths was 4,095; while the number  from  con-  sumption was 339. In the first period the appli-  cations were very carelessly investigated, while  during a part of the last  mentioned period a   careful personal examination was  made  by a48         MEDICAL EXAMINATIONS.   suitably  qualified  person  appointed by the of-   fice.      It has happened also that persons have pre«   sented themselves for examination who never   nave had any medical attendant, and who never   suspected the existence of any disease in them-   selves, and  yet have given evidence of such a   state of system, that there existed no reasona-   ble presumption that they would ever reach the   period of their \"expectation.\"  An early death   has proved the accuracy of this opinion in many   an instance.   It was formerly required of the   party, in case he had no physician, to procure   a certificate from  « some other \" medical man;   but it is  evident that but little information of   value  could  be obtained from  such a source.   The medical officer appointed by the company   supplies now any deficiency in the medical tes-   timony, and offers the best security both for the   insured and the insurers—to the former  as fur-   nishing to a certain extent a warranty, and to   the latter a security that  every means has been   used to obtain none but good risks.    When  a person applies for examination he  should bring with him,  1st, a declaration signed           MEDICAL EXAMINATIONS.         49  by himself; 2d, another signed by a friend; and  3d, one from  his medical attendant; all giving  an  accurate  statement of his health.   These  documents are furnished in blank by the com  pany.  An error has arisen on the part of some  medical gentlemen in supposing that when they  fill up the blank that is intended for them, that  they are  furnishing the information they impart,  at the request of the company.  This mistake  has  doubtless arisen from  a common practice  of the officers and agents,  who, in wishing to  save the applicant  a  little  trouble,  send the  blank  certificate  to the  physician;  the latter  supposing it to come from  the company are in  some instances unwilling to furnish the required  information  without a fee.  All these docu-  ments, however, being the  proofs which the ap-   plicant  is required to  supply and to bring with   him to be filed in the office, must be obtained   by him  at his own expense.  In addition  to   these  evidences, the agent is supposed  to be-   come  to some extent responsible, and in one   company at least, he is required to fill up an of-   ficial  certificate  embracing his knowledge of  350  MEDICAL  EXAMINATIONS.   the party, and his opinion as to the safety of the   risk in question.     In no case should a person be examined un-   less all these papers are  submitted, complete, to   the  medical examiner; for  they might contain   some important fact necessary to a correct de-   cision.   With these  documents before him  he   will proceed to a personal examination.     The external form and general  aspect often   convey a vast amount of information to one ac-   customed  to  associate these  with  health and   sickness.   The habits of life, which may be as-   certained by a few questions, will naturally lead   to others ; the trade, occupation, profession, etc.,   of the applicant come under this head of exam-   ination, and will enable the  examiner  to  draw   his inference as to their effects  on the general   health.   The condition of the brain and spinal   marrow will also be thus learned;  for this can   be ascertained to a sufficient extent  by the gait,   manner of speech, and want of control  over the   muscular system, exhibited in tremor, and other   irregular movements,  together with questions  on these points which may suggest themselves.MEDICAL  EXAMINATIONS.         51  [   In the examination of the chest, although it is  \\\\ of great importance, yet it is not necessary to  1 ascertain anything more than that there is a de-   parture from health ; it is not required that any   detail of unfavorable symptoms be recorded, if   any appear.   The first step in the investigation,   is to percuss the chest in all its parts, and then  I to apply the  Stethescope, or the ear, to ascer-   tain whether or not there exists a healthy res-   piration.  Consider  the  capacity of the  chest,   whether narrow or expanded—whether there is   any unusual  depression in the subclavian re-   gions—whether the chest expands equally  on   inspiration—any Bronchophony, particularly in   the subclavian and mammary regions.   The   action  of the heart is also ascertained by aus-   cultation : ascertain whether  the  rythm of the   heart is natural, and confined within the natural   limits—whether the impulse of  the heart is co-   incident with the first sound, and  of natural in-   tensity.  When  making a report, the state of   the heart may be recorded \"Normal,\" or \"Ab-   normal,\" as the case may be.      The pulse, besides being regular, should never   beat over 90 in a minute : if it cannot be  re-  52         MEDICAL EXAMINATIONS.    duced to this by changing to a recumbent posi-    tion, rest, and time being given  for  the  com-    posure of the nervous system, and if necessary,    two or three examinations,  on different days—    the applicant is to be rejected.  A very careful    investigation into the condition of the heart and   lungs should be made,  whenever the pulse  is   found to beat uniformly  over 80.     An habitually rapid pulse is generally indica-   tive  of some  organic   affection.   The  pulse   should always be regular in its beats, and never   intermitting.     Spitting of blood, if it arise in a person of  a   consumptive family, must be  a reason  for re-   jection.   If, however, it arises from a  mere   congestion  of  the  mueous membrame of the   bronchial tubes, it need not be a cause for declin-   ing the risk; a  sufficient time, however, must   elapse to ascertain,  together with  an  examina-   tion of the chest, that it is of this nature.  Bleed-   ing from the lungs from violent straining, need   not be a reason for rejecting the applicant__al-  lowing, as in the former instance, a long time  to pass to  prove that it is but a local and tempo-  rary affection, and has not produced any diseaseMEDICAL  EXAMINATIONS.         53  If either of these should be followed by cough or  occasional difficulty of respiration, the party of  course, must be rejected.  It is needless to men-  tion that more than usual care must be taken  in the physical examination  of such persons.    Occasional difficulty of breathing, or asthma,  must always  be rejected.  The causes of these  affections are often obscure, and  as they arise  from some obstruction in one  or  other  of the  vital organs, as  the  lungs  or  heart, safety re-  quires that such  cases should not be received.     There is an affection of the  bowels which is  often suddenly fatal; a person affected with it  is by no means a safe risk:  it is an obstruction,  sometimes known as colic, bilious colic, or spas-  modic  colic;  any person  affected with  this,  ought to be rejected.     In making up  a report to be transmitted to  the parent office, there need be no  remark made  beyond  the conclusions at which  the examiner  has arrived, a brief statement and expression of  opinion, which  ought to be unqualified, is  all  that is required.   Whenever there is any state-  ment of positive disease, it would be well for54  MEDICAL  EXAMINATIONS.   the  examiner to refer  to  it in a note to show   that it has received his  special attention.     The existence of tumors, hernia, hemorrhoids,   fistula,  &c, should be ascertained,  and  their   condition ascertained by inspection.  Fistula is   a good reason for rejection.  A hernia need not   render the party incapable of  availing  himself   of the benefits of life-insurance provided  it be   properly secured by a truss, and the occupation   of the individual be such as not to  endanger its   protrusion by excessive straining.     When inquiring into  the usual occupation of   the applicant, the questions relating to exposure   to insalubrious climates will naturally arise.  If   the party has been within the tropics, or in any   part of the  world  known to  be  at times un-   healthy, it will be necessary to inquire whether   or not he has had any e", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7f29.kjm4-w9cr", "00000000-0000-0000-8038-ED341DE8D42F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Instructions for USSC examiners", "9918573882206676X28", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" The USSC provided this guidance to its examiners who worked with Form EE. Pages 225-227 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "3", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "MEAN DIMENSIONS OF BODY.  225  35. Facial angle ?  36. Foot- a. Length from tip of great toe to extremity of heel?  b. Length from tip of great toe to hollow above heel ?  c. Thickness at instep ?  d. Circumference around heel and anterior ligament?  51. Was he, before the war, given to athletic recreations, and if so,  what kind ?  55. Education - Limited common school ?  Good common school ?  High school ?  Professional ?  57. Distance of distinct vision for small pica double-leaded type ?  58. Does he distinguish colors correctly ?  If not, describe the irregularity ?  To secure uniformity in the mode of measurement by different  examiners, Dr. Buckley, whose experience and scientific attain-  ments had already proved serviceable in the examinations under  Form E, was appointed Chief Examiner, and all the other gen-  tlemen engaged upon the work went through some days' practice  in measuring with him. The following printed instructions were  also furnished to each examiner.  INSTRUCTIONS FOR EXAMINATION OF INDIVIDUALS. - [Form EE.]  The persons examined should not be selected, but should be taken  indiscriminately, - by companies and regiments, when possible.  The object of Question 4|, is to determine the point on the outer  side of the thigh corresponding with the tip of the middle finger, in the  \" attitude of the soldier.\" It is best measured with the calipers.  Question cannot be answered by means of the andrometer, but  may be omitted, as also may Question 10|, when no opportunity is found  for examination of the individual without clothing. Such opportunities  are never to be lost; although the ordinary examination requires merely  the removal of hat, coat, waistcoat, and boots, and loosening the shirt  at the breast.  The Girth of neck (Question 7|) is to be taken around the pomuni  Adami.  The Circumference of chest (10) is to be measured under all the  clothing; the Distance between nipples (10|), taken with calipers.  For ascertaining the Capacity of the chest (13), the lungs are to be  fully inflated, and then as completely emptied as may be, by breathing  through the tube of the spirometer. The results of three consecutive  trials are to be recorded.  The Questions 28 and 29, as to the number of pulsations and inspi- 226  MEAN DIMENSIONS OF BODY.  rations in a minute, must both be answered before the trial of the Dy-  namometer (15), which would derange the normal condition. The res-  pirations are of course to be counted without the knowledge of the  individual. It is recommended that they be noted immediately after  the arm-measurements (13), when the person examined is not suspect-  ing a change in the order of questions as printed; and before the  trials with the Spirometer. The precautions for insuring accurate an-  swers are self-evident.  In answer to Question 16, state the stock, if possible (as English,  Irish, French, etc.) ; if not, state the race, unless Caucasian, (as Afri-  can, Malay, etc.) ; or if of mixed races, and what.  In Trades (Question 24), the journeyman is to be distinguished  from the master in all cases - as, Baker (journeyman); Carpenter  (master). Laborers are to be described according to the nature of  their employment - as Agricultural Laborer, Railway Laborer. The  term Farmer should be applied only to those who have themselves  owned or rented land. The sons of farmers, living on the farm and  working on it, may be returned \" Farmers' sons.\" Descriptions of oc-  cupation should be precise - they are too often incomplete : for exam-  ple, engine feeder, engine driver, not engineer; brass founder, iron  founder, not founder simply; commercial clerk, lawyer's clerk, not  clerk, simply. If a Mechanic, state the Branch of manufacture ; if a  Shopkeeper or Salesman, state the kind of business.  The Color of the Hair (25), may be described as Black, Dark-Brown,  Brown, Light-Brown, Sandy, Red, Gray (if gray, the original color  should also be ascertained and recorded) ; its Amount, as Thick,  Medium, Scanty, or the degree of baldness indicated ; its Texture, as  Straight, Wavy, or Curly, and as Coarse, Medium, or Fine.  The Color of the Eyes (26), - as Blue, Gray, Hazel, Light-Brown,  Dark-Brown, Black.  The Complexion (27), -as Fair, Ruddy, Medium, or Dark.  The Muscular Development (30), - as Large, Moderate, or Defi-  cient.  In the Measurements of Head (34), - the lengths under the hair are  desired. The measures a and b refer to the \" frontal eminence,\" or most  prominent part of the forehead above the superciliary ridge. But the  distance e should be measured from the angle of the skull between the  eyebrows to that at the base behind. The widths f and g are to be  taken with calipers; the other measures with the tape.  The Length and Thickness of Foot (36), are to be measured with cal-  ipers.  In answering Question 55, record the apparent degree of actual cul-  ture or intelligence, rather than the mode in which it was obtained.  The Facial Angle (35), has its center at the alveolar process, and the  angle desired is included between lines drawn to the orifice of the ear,  and to the \"frontal eminence\" as above defined. MEAN DIMENSIONS OF BODY.  227  The lines entitled \"Objects of the Examination \" are printed on the  back of the Forms EE in small-pica double-leaded, and may be used  for Question 57.  The object of Question 58 is to determine the comparative frequency  of what is called color-blindness, by ascertaining whether green can be  distinguished easily from red, yellow from blue, etc.  AH measurements are to be noted in inches and tenths, so far as pos-  sible ; and if for any reason it should not be found practicable to obtain  satisfactory and accurate answers, it is better to make a dash against the  question, omitting the answer entirely, than to record an uncertain re-  sult.  In examining negro troops, give, as answer to Question 30, an esti-  mate of the proportion of black blood, such as Ftill Black, Mulatto,  Quadroon, Octroon ; as well as of the negro race, if this can be dis-  criminated. In answer to Question 55, a statement of the apparent in-  telligence may be given, such as Very low, Low, Average, Quick, etc.;  - the ordinary white private soldier being taken as the standard of  comparison. Also state whether he can read or write, or both, well or  imperfectly; and when this was learned.  The blanks, when filled, are to be sent to the Statistical Department  of the Commission, at Washington, - weekly, if possible. Not more  than one hundred sets of measures should ever remain in the hands of  the examiner at a time.  Cambridge, March. 1, 1865.  The close of the war happily deprived us of the opportunities  for measuring, by dispersing the citizen soldiery to their homes;  but all means of obtaining the desired data were actively improved,  so that our total number of men measured according to the new  form nearly reaches the number of 15 900. Some of these it has  seemed desirable not to incorporate with our results, but the meas-  urements of 15 781 men seem entitled to full confidence, as hon-  estly, carefully, and intelligently made.  In arranging the stations of the different examiners, and giving  instructions as to the special duties of each, efforts were made to  provide so far as possible that the measurements by each person  should be confined to no one class of men, and that the measure-  ments of no class should be restricted to a single examiner. The  various exigencies of the work, and a proper regard to economy,  prevented entire compliance with this rule ; yet it was never over-  looked, and in those cases where the physical examination of any  class of men was conducted by one person only, the duty was as-  signed to the most experienced and careful person available, and", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-enfm.zb8q~c4yb", "00000000-0000-0000-E729-622CF45BD691", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Images of Andrometer apparatus used for physical measurements of soldiers", "9918573882206676X29", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" In the last year of the war, the USSC had 12 \"andrometers\" built and used them to take a variety of measurements of each soldier. Pages 234-235 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "234  MEAN DIMENSIONS OF BODY-  the proper size for soldiers' clothing, for which he had undertaken  a considerable contract with the military authorities. Ballingall  has given1 some account, as well as a representation of it; and  states that the instrument is deposited in the Museum of the Edin-  burgh University. It enables the total height, breadth of neck,  of shoulders, and of pelvis, the length of legs and height to the  knee to be measured with greater accuracy and rapidity than oth-  erwise would be possible, since when the man to be measured has  taken his position, gauges are quickly set for the measures of all  these dimensions, and the numerical values read off after the man  has left the instrument. Instruments of this kind were con-  structed for the Sanitary  Commission in 1863, at the  office of the U. S. Coast  Survey, under the special  supervision of the late Pro-  fessor Bache, the lamented  Superintendent of the Sur-  vey, and Vice President of  the Commission. These  contained some improve-  ments upon the original in-  strument, especially such  as permitted more accu-  rate adjustment to the per-  son, as well as an addition-  al gauge for measuring the  height of the body proper,  of which the seventh cer-  vical vertebra was taken  as the limit. When in Au-  gust and September, 1864,  the new instruments were  ordered, Dr. Douglas kind-  ly charged himself with the  supervision of the work,  which was executed with  great care and fidelity by  Mr. William Belcher of  New York. In the new  instruments many addi-  1 Outlines of Military Surgery, 1855, pp. 35, 36. MEAN DIMENSIONS OF BODY.  235  tional improvements were introduced, a considerable part of them  being suggested by the experience obtained by the use of the two  former ones, which were themselves correspondingly modified as  soon as they could be spared for the purpose. The annexed figures  will indicate the general construction of the andrometer, and the  manner of use.  The graduations of this instrument, and of all our implements  for linear measure, are in inches and tenths, all danger of error  from the use of divisions not decimal being thus avoided. It is  a source of regret to the author that he did not employ the met-  ric system for all these measurements, not only as attended with less  uncertainty on account of the smaller unit employed when centi-", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rwsg~a9ew_u3vx", "00000000-0000-0000-94C5-C4D1D4E1C27D", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Table showing mean dimensions of white soldiers", "9918573882206676X30", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" This table showed the mean dimensions of the white soldiers measured by the USSC examiners. Pages 276-281 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "6", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "276  MEAN DIMENSIONS OF BODY.  TABLE I.  Mean Dimensions of White Soldiers.  Nativity  Number of Men  Actual Mean Age  4  2  *5  w  Tip of Finger  to Margin of £  Patella  Height to 7th  Cervical Ver- c,  tebra  sw  Height to a  Perineeum  Breadth of  Neck  A. New England States  in.  in.  in.  in.  in.  in.  In usual vigor  1 000  25.36  67.21  4.90  57.25  18.75  31.10  4.18  Others . .  211  27.67  67.15  5.06  57.15  18.73  31.02  4.11  T otal. . .  1 211  25.76  67.20  4.93  57.23  18.75  31.09  4.17  B. N. Y., N. J., and Penn.  In usual vigor  3 177  25.71  67.13  4.92  57.18  18.64  31.06  4.25  Others . .  588  28.71  67.20  4.96  57.16  18.65  31.04  4.11  Total. . .  3 765  26.18  67.14  4.92  57.18  18.64  31.05  4.23  C. Ohio and Indiana  In usual vigor  1443  24.44  67.68  5.37  57.69  18.74  31.43  4.18  Others. . .  219  26.46  68.12  5.37  58.08  18.87  31.64  4.10  Total. . •  1 662  24.70  67.74  5.37  57.74  18.76  31.46  4.17  D. Mich., Wise., and Ill.  In usual vigor  945  24.44  67.22  4.82  57.29  18.06  31.02  4.32  Others. . .  71  23.54  67.60  5.13  57.57  18.44  31.30  4.16  Total. . .  1 016  24.38  67.26  4.84  57.32  18.09  31.05  4.31  E. Coast Slave States  In usual vigor  315  25.89  67.62  5.25  57.62  19.08  31.60  4.18  Others. . .  52  32.80  67.19  5.21  57.28  18.91  31.41  3.99  Total. . .  367  26.88  67.56  5.24  57.57  19.06  31.57  4.15  F. Kentucky and Tenn.  In usual vigor  223  25.19  68.57  6.07  58.68  19.20  31.68  4.16  Others. . .  44  30.13  68.31  5.69  58.35  19.16  31.67  4.13  Total. . .  267  26.00  68.53  6.01  58.63  19.19  31.68  4.15  Gi. W. of Miss. R. - Free .  In usual vigor  10  22.28  67.89  5.83  58.00  18.90  31.32  4.10  Ga. W. of Miss. R. - Slave  In usual vigor  46  24.50  66.29  5.52  56.66  18.88  31.06  4.25  Others. . .  5  25.09  66.56  5.52  56.62  19.06  31.16  3.94  Total. . .  51  24.56  66.32  5.52  56.65  18.90  31.07  4.22  H. Brit. Prov. excl. Canada  In usual vigor  36  27.16  67.31  5.25  57.45  18.74  30.85  4.22  Others . . .  2  23.48  66.40  5.90  56.30  17.70  29.58  4.00  Total. . .  38  26.96  67.26  5.28  57.39  18.69  30.78  4.21  I. Canada  In usual vigor  474  24.91  66.85  4.70  57.05  18.43  30.82  4.30  Others . . .  46  30.64  67.20  5.04  57.02  18.43  30.84  4.18  Total . . .  520  25.43  66.88  4.73  57.04  18.43  30.82  4.29 MEAN DIMENSIONS OF BODY.  277  TABLE I. - ( Continued.)  Mean Dimensions of White Soldiers.  7i  8a  86  9  10a  106  11  1  Hi  s  Circumfer-  o  43  ence of Chest  Nativity  o  « a  Q  2 s.  o  dth of  betwee  rnia  dth of  ilders  i a  CtJ  £  13  6  Brea  ders  Aero  o o  «S CL  Full  spira  After  Expi  tion.  do  | 2  A. New England States  in.  in.  in.  in.  in.  in.  in.  in.  In usual vigor  13.44  12.77  16.28  11.91  36.74  34.06  31.08  36.50  Others . . .  13.42  12.71  16.26  11.76  36.58  34.33  30.98  36.52  Total . .  13.44  12.76  16.28  11.88  36.71  34.11  31.06  36.51  B. N. Y., N. J., and Penn.  In usual vigor  13.63  12.69  16.38  12.02  37.09  34.33  31.42  37.03  Others . . .  13.51  12.69  16.18  11.85  86.88  34.65  31.35  36.91  Total . .  13.61  12.69  16.36  11.99  37.06  34.38  31.41  37.01  C. Ohio and Indiana  In usual vigor  13.70  12.74  16.40  11.90  37.60  34.98  32.01  37.27  Others . . .  13.57  12.61  16.28  11.77  37.07  34.76  31.81  36.95  Total . .  13.68  12.72  16.38  11.88  37.53  34.95  31.98  37.22  D. Mich., Wise., and Ill.  In usual vigor  13.52  12.33  16.21  11.68  37.35  34.01  31.07  36.78  Others . . .  13.50  12.47  16.48  11.80  36.78  34.42  31.20  36.93  Total . .  13.52  12.34  16.23  11.69  37.29  34.04  31.08  36.79  E. Coast Slave States  In usual vigor  13.64  12.75  15.85  11.73  36.68  34.27  31.30  36.67  Others . . .  13.40  12.27  15.64  11.61  36.39  34.00  30.92  36.24  Total . .  13.61  12.68  15.82  11.71  36.64  34.23  31.25  36.61  F. Kentucky and Tenn.  In usual vigor  13.72  13.59  16.70  12.03  37.87  35.31  32.69  37.82  Others . . .  13.83  13.17  16.22  11.80  37.61  35.23  32.31  37.51  Total . .  13.73  13.51  16.65  11.99  37.83  35.30  32.63  37.77  Gi- W. of Miss. R.- Free  In usual vigor  14.01  13.12  17.30  11.84  37.53  34.84  31.83  38.09  Gfr W. of Miss. R. - Slave  In usual vigor  13.32  13.34  15.83  11.65  35.64  33.38  29.89  35.40  Others . . .  13.48  12.38  -  11.47  34.60  32.70  29.32  36.00  Total . .  13.33  13.23  15.83  11.64  35.54  33.31  29.83  35.46  H. Brit. Prov. excl. Canada  In usual vigor  13.87  12.90  16.77  11.84  37.24  34.91  31.25  36.60  Others . . .  13.05  13.00  -  11.50  35.05  33.10  30.50  35.55  Total . -  13.83  12.91  16.77  11.82  37.13  34.81  31.21  36.54  I. Canada  In usual vigor  13.60  12.64  16.30  12.05  37.13  34.30  31.38  37.00  Others . . .  13.57  12.78  15.90  11.79  37.26  34.90  32.17  37.07  Total . .  13.60  12.65  16.29  12.03  37.14  34.35  31.45  37.00 278  MEAN DIMENSIONS OF BODY.  TABLE I. - ( Continued.')  Mean Dimensions of White Soldiers.  12a  124  12c  26*  26c  36a  36*  36c  364  g  \"w  Distance be-  £  A  0  gX  tween Angles  0  Q  g-s  Nativity  a  of Eyes  &amp;R  c5  0  a  0  0 J  co  t! n  -2 p  a s  -  .g  S  C O V  5 S3 g  4  §  Outer  Inner  0  a  gf  otsS  A. New England States  in.  in.  in.  in.  in.  in.  in.  in.  in.  In usual vigor .  29.26  35.09  13.80  3.805  1.224  10.092  9.912  2.53  13.07  Others....  29.08  34.85  13.58  3.760  1.202  9.931  9.749  2.65  13.05  Total . . .  29.23  35.05  13.76  3.797  1.220  10.065  9.883  2.55  13.06  B. N. Y., N. J., Penn.  In usual vigor .  29.10  35.01  13.62  3.765  1.237  10.071  9.878  2.50  13.20  Others....  29.25  35.13  13.62  3.744  1.196  9.970  9.793  2.67  13.16  Total . . .  29.12  35.03  13.62  3.761  1.230  10.055  9.864  2.53  13.20  C. Ohio and Indiana  In usual vigor .  29.50  35.47  13.70  3.744  1.199  10.105  9.918  2.68  13.40  Others....  29.72  35.50  13.83  3.695  1.157  10.112  9.949  2.76  13.30  Total . . .  29.53  35.47  13.72  3.738  1.194  10.106  9.922  2.69  13.39  D. Mich., Wise., and Ill.  In usual vigor .  28.74  34.74  13.37  3.622  1.225  10.036  9.854  2.47  13.21  Others....  29.33  35.26  13.67  3.680  1.213  10.070  9.886  2.69  13.30  Total . . .  28.81  34.77  13.39  3.626  1.224  10.039  9.856  2.49  13.22  E. Coast Slave States  In usual vigor .  29.40  35.08  13.74  3.747  1.188  10.108  9.926  2.67  13.20  Others....  29.42  35.02  13.84  3.679  1.110  9.979  9.798  2.72  13.14  Total . . .  29.40  35.07  13.75  3.737  1.177  10.089  9.908  2.67  13.19  F. Kentucky and Tenn.  In usual vigor .  30.01  36.00  13.61  3.828  1.231  10.270  10.077  2.85  13.68  Others....  30.10  35.94  13.74  3.763  1.188  10.123  9.957  2.80  13.67  Total . . .  30.02  35.99  13.63  3.817  1.224  10.245  10.057  2.84  13.67  Gj. W. of Miss. R.-Free  In usual vigor .  29.19  35.09  13.30  3.860  1.230  10.000  9.840  2.84  13.42  G2. W. of Miss. R.-SI.  In usual vigor .  29.10  34.36  13.43  3.700  1.254  9.891  9.678  2.65  12 90  Others....  29.16  34.66  13.52  3.720  1.180  9.880  9.600  2.64  12.90  Total . . .  29.11  34.39  13.44  3.702  1.247  9.890  9.671  2.65  12.90  H. Brit. Prov. excl. Can.  In usual vigor .  29.28  35.11  13.88  3.800  1.225  10.075  9.908  2.56  13.17  Others....  28.20  33.50  12.80  3.600  1.200  10.150  9.900  2.80  13.80  Total . . .  29.22  35.03  13.82  3.789  1.224  10.079  9.908  2.57  13.20  I. Canada  In usual vigor .  28.93  34.82  13.57  3.926  1.265  10.082  9.889  2.41  13.19  Others....  29.38  35.10  13.90  3.772  1.169  9.989  9.843  2.72  13.21  Total . . .  28.97  34.83  13.60  3.912  1.256  10.074  9.885  2.44  13.19 MEAN DIMENSIONS OF BODY.  279  TABLE I. - ( Continued.)  Mean Dimensions of White Soldiers.  Nativity  Number of  Men  Actual Mean  Age  Height  Tip of Finger  to Margin of  Patella  Height to 7th  Cervical Ver- e*  tebra  5|  is  0 a  Height to m  .Perinaeum  Breadth of  Neck  Ji- England  In usual vigor  261  26.16  in.  66.17  in.  4.90  in.  56.27  in.  18.28  in.  30.39  in.  4.23  Others . . .  45  31.33  66.75  4.84  56.62  18.41  30.76  4.12  Total . . .  306  27.08  66.25  4.90  56.32  18.30  30.45  4.21  J2. Wales &amp; I. of Man  In usual vigor  18  30.10  66.83  5.45  56.78  18.58  30.59  4.19  Others . . .  2  40.49  67.25  5.20  56.95  19.10  30.80  4.00  Total . . .  20  31.14  66.87  5.42  56.80  18.63  30.61  4.17  K. Scotland  In usual vigor  70  28.48  66.83  4.89  56.87  18.34  30.75  4.23  Others . . .  11  31.67  67.59  5.25  57.51  18.52  31.30  4.16  Total . . .  81  28.91  66.94  4.94  56.95  18.36  30.83  4.22  L. Ireland  In usual vigor  648  28.36  66.68  5.08  56.75  18.57  30.71  4.24  Others . . .  179  32.42  66.29  5.07  56.28  18.42  30.51  4.09  Total . . .  827  29.24  66.59  5.08  56.65  18.54  30.67  4.21  M. France, etc.  In usual vigor  84  27.38  65.73  5.01  55.77  18.22  30.24  4.23  Others . . .  16  29.62  65.31  4.97  55.48  18.03  29.99  4.10  Total . . .  100  27.74  65.66  5.00  55.72  18.19  30.20  4.22  N. Germany  In usual vigor  462  28.88  66.22  5.00  56.49  18.54  30.76  4.31  Others . . .  100  33.85  65.96  4.88  56.06  18.44  30.51  4.14  Total . . .  562  29.76  66.17  4.98  56.41  18.52  30.71  4.28  0. Scandinavia  In usual vigor  28  27.92  68.06  5.14  58.20  19.04  31.63  4.34  Others . . .  6  34.99  66.37  5.30  56.40  18.67  30.63  3.98  Total . . .  34  29.17  67.76  5.17  57.88  18.97  31.45  4.27  P. Spain, Portugal, etc.  In usual vigor  6  31.99  65.52  5.70  55.93  18.15  29.72  4.22  Others . . .  1  29.49  63.90  5.30  54.80  17.40  29.50  4.30  Total . . .  7  31.63  65.29  5.64  55.77  18.04  29.69  4.23  Q. Miscellaneous  In usual vigor  25  26.07  67.07  5.15  57.17  18.68  30.82  4.24  Others . . .  7  32.49  66.43  5.73  57.04  18.56  30.64  3.91  Total . . .  32  27.48  66.93  5.27  57.14  18.65  30.78  4.17  All Nativities  In usual vigor  9 271  25.705  67.150  5.028  57.218  18.603  31.069  4.238  Others . . .  1 605  29.165  67.148  5.081  57.131  18.644  31.040  4.108  Total . . .  10 876  26.215  67.149  5.036(57.205  18.609  31.065  4.219 280  MEAN DIMENSIONS OF BODY.  TABLE I. - (Continued.')  Mean Dimensions of White Soldiers.  Nativity  7i  1  0  1  s  Breadth of Shoul-  ders between g&gt;  Acromia  8&amp;  0  0  xa  QQ  O  5  Breadth of „  Pelvis  10a 104  Circumference  of Chest.  Circumference  of Waist *■*  Circumference £  around Hips •*-  Full In-  spiration  After  Expira-  tion  Jj. England  in.  in.  in.  in.  in.  in.  in.  in.  In usual vigor.  13.65  12.80  16 21  11.85  36.92  34.24  31.25  36.72  Others . . .  13.44  12.77  16.17  11.80  36.89  34.69  31.36  36.50  Total . . .  13.62  12.80  16.21  11.84  36.91  34.30  31.26  36.68  J2. Wales &amp; I. of Man  In usual vigor.  13.69  12.42  16.35  11.80  36.42  33.94  31.08  36.60  Others . . .  14.30  14.55  -  12.20  38.25  36.50  33.50  38.50  Total . . .  13.75  12.69  16.35  11.84  36.60  34.19  31.32  36.79  K. Scotland  In usual vigor .  13.61  12.46  16.62  11.70  37.57  34.69  31.24  36.67  Others . . .  13.46  12.47  16.70  11.66  36.74  34.56  31.44  36.80  Total . . .  13.59  12.46  16.64  11.69  37.45  34.67  31.26  36.69  L. Ireland  In usual vigor .  13.76  13.07  16.52  12.05  37.54  35.27  31.67  36.89  Others . . .  13.54  12.71  15.92  11.74  36.87  34.74  31.27  36.41  Total . . .  13.71  12.98  16.47  11.98  37.39  35.15  31.59  36.79  M. France, etc.  In usual vigor.  13.82  12.90  16.70  12.02  36.91  34.37  31.53  36.99  Others . . .  13.59  12.91  16.60  11.69  36.29  33.92  31.39  36.76  Total . . .  13.78  12.90  16.69  11.97  36.81  34.30  31.51  36.96  N. Germany  In usual vigor .  13.83  12.97  16.49  11.98  37.20  34.74  31.67  36.98  Others . . .  13.65  12.78  15.47  11.91  36.74  34.60  31.35  36.65  Total . . .  13.79  12.93  16.44  11.97  37.12  34.72  31.62  36.92  0. Scandinavia  In usual vigor .  14.06  13.19  16.30  11.94  38.44  35.36  32.39  37.74  Others . . .  13.50  12.82  -  12.23  38.17  35.42  32.48  37.15  Total . . .  13.96  13.12  16.30  11.99  38.39  35.37  32.41  37.63  P. Spain, etc.  In usual vigor .  13.83  13.05  -  11.40  35.20  32.93  30.75  36.13  Others . . .  13.00  -  14.80  11.10  36.00  34.20  31.40  35.90  Total . . .  13.71  13.05  14.80  11.36  35.31  33.il  30.84  36.10  Q. Miscellaneous  In usual vigor.  13.90  12.87  16.62  12.11  37.15  34.32  31.59  36.77  Others . . .  13.14  13.30  15.30  11.57  34.39  32.31  29.46  35.56  Total . .  13.73  12.97  16.43  11.99  36.54  33.99  31.12  36.51  All nativities  In usual vigor .  13.633  12.738  16.370  11.936  37.195  34.476  31.483  36.957  Others . . .  13.521  12.693  16.151  11.800  36.846  34.604  31.377  36.770  Total . . .  13.617  12.731  16.350  11.916  37.143  34.494  31.467  36.930 MEAN DIMENSIONS OF BODY.  281  TABLE I. - ( Continued.')  Mean Dimensions of White Soldiers  Nativity  Length of Arm  Middle of Breast-  bone to Tip of to  Finger  Acromion to M  Elbow J?  266 26c  Distance be-  tween Angles  of Eyes  CO  Length of Foot g  Length to Hollow co  above Heel ©  36c  a  a  ■  ■\"3  ja a  Circumf. around „  Heel &amp; Anterior g  Ligament  Outer  Inner  Jb England  in.  in.  in.  in.  in.  in.  in.  in.  in.  In usual vigor  28.62  34.52  13.35  3.720  1.232  10.051  9.841  2.55  13.03  Others . . .  28.84  34.72  13.45  3.744  1.198  9.857  9.700  2.63  12.20  Total . . .  28.66  34.55  13.37  3.724  1.227  10.023  9.821  2.56  13.02  J2. Wales, I. of Man  In usual vigor  29.09  35.09  13.82  3.711  1.183  9.933  9.761  2.48  12.79  Others . . .  28.85  35.10  13.25  3.900  1.050  10.150  9.900  2.70  12.20  Total . . .  29.07  35.09  13.76  3.730  1.170  9.955  9.775  2.50  12.73  K. Scotland  In usual vigor  28.87  34.76  13.48  3.721  1.226  10.066  9.871  2.54  13.15  Others . . .  29.51  35.34  13.86  3.791  1.173  10.000  9.818  2.70  13.60  Total . . .  28.96  34.84  13.53  3.731  1.219  10.057  9.864  2.56  13.21  L. Ireland  In usual vigor  29.03  34.90  13.51  3.771  1.262  9.965  9.789  2.64  13.08  Others . . .  28.84  34.62  13.30  3.783  1.213  9.781  9.621  2.67  13.02  Total . . .  28.99  34.84  13.46  3.774  1.251  9.925  9.752  2.65  13.07  M. France, etc.  In usual vigor  28.58  34.49  13.29  3.764  1.263  10.100  9.898  2.58  13.12  Others . . .  28.21  34.02  12.87  3.687  1.144  10.024  9.812  2.74  13.05  Total . . .  28.52  34.42  13.22  3.752  1.244  10.087  9.884  2.61  13.11  N. Germany  In usual vigor  28.98  34.80  13.53  3.806  1.258  10.087  9.905  2.46  13.15  Others  28.92  34.70  13.56  3.788  1.219  9.978  9.796  2.66  12.72  Total . . .  28.97  34.78  13.54  3.802  1.251  10.068  9.886  2.49  13.07  0. Scandinavia  In usual vigor  30.02  35.94  13.92  3.807  1.239  10.261  10.075  2.75  13.35  Others . . .  29.27  35.22  13.55  3.800  1.050  10.000  9.867  2.82  13.20  Total . . .  29.89  35.81  13.86  3.806  1.235  10.216  10.038  2.76  13.32  P. Spain, etc.  In usual vigor  28.60  34.63  13.57  3.817  1.200  10.067  9.850  2.65  13.47  Others . . .  26.10  32.70  12.10  3.500  1.300  9.300  9.100  2.30  12.40  Total . . .  28.24  34.36  13.36  3.771  1.214  9.957  9.743  2.60  13.31  Q. Miscellaneous  In usual vigor  29.03  34.70  13.50  3.863  1.258  10.124  9.920  2.53  13.19  Others . . .  28.69  34.51  13.10  3.671  1.200  10.043  9.829  2.77  13.07  Total . . .  28.95  34.66  13.41  3.819  1.245  10.106  9.900  2.58  13.16  All Nativities  In usual vigor  29.139  35.040  13.604  3.761  1.231  10.073  9.886  2.552  13.312  Others . . .  29.235  35.055  13.609J3.743  1.191  9.970  9.797  2.689  13.140  Total . . .  29.153  35.042  13.605l3.759  1.225  10.058  9.873  2.572  13.201", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wr2z-hsfm-z5hi", "00000000-0000-0000-5DEB-E7CE4AA46CBB", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Table showing mean dimensions of \"full black\" soldiers", "9918573882206676X31", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" This table showed the mean dimensions of the Black soldiers considered to be \"full Black\" rather than \"mulatto.\" Pages 303 to 305 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "3", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "MEAN DIMENSIONS OF BODY.  303  TABLE V.  Mean Dimensions of Full Blacks.  Class  Number of Men  Actual Mean Age  Height >e.  Tip of Finger  to Margin of  Patella  Height to 7th  Cervical Ver-  tebra  5}  o  .be g  \"S t3  Height to  Perinseum  Breadth of  Neck  Naked, Free States  123  26.08  in.  65.93  in.  3.09  in.  55.85  in.  18.35  in.  32.21  in.  4.12  Slave States  554  24.75  65.80  2.59  56.05  18.45  32.12  4.14  All . .  677  24.993  65.821  2.682  56.012  18.429  32.140  4.137  Half Naked  Free States  2  22.98  66.65  3.00  57.15  19.90  31.20  4.35  Slave States  145  28.18  65.15  2.45  55.98  19.35  30.49  4.23  All . .  147  28.112  65.169  2.456  55.998  19.359  30.501  4.233  Clothed  Free States  101  24.20  66.86  3.55  57.24  19.48  32.40  4.30  Slave States  1095  25.89  66.53  3.01  56.96  19.51  32.26  4.22  All . .  1196  25.750  66.558  3.053  56.984  19.509  32.275  4.227  In usual vigor  Free States  194  24.88  66.35  3.37  56.49  18.91  32.32  4.21  Slave States  1598  25.42  66.22  2.86  56.62  19.18  32.10  4.20  All . .  1792  25.364  66.237  2.914  56.610  19.148  32.123  4.202  Not in usual  Free States  32  27.21  66.39  2.86  56.48  18.63  32.08  4.14  Slave States  196  28.25  65.94  2.62  56.39  19.11  31.89  4.16  All . .  228  28.104  66.003  2.655  56.405  19.043  31.917  4.160  Total born in  Free States  226  25.212  66.354  3.298  56.487  18.870  32.289  4.212  Slave States  1794  25.727  66.192  2.832  56.599  19.169  32.076  4.196  Grand Total . . .  2020  25.668  66.210  2.884  56.587  19.136  32.100  4.197 304  MEAN DIMENSIONS OF BODY.  TABLE V. - ( Continued.')  Mean Dimensions of Full Blacks.  7i  8a  86  9  10a  106  11  Hi  •g  Circumference  , .3  of Chest  g  8  Class  1  o  th of  ders be  Acron  th of  ders  A  o  > a  fl .2  * -  H g  1  1  is.  5  •!S  S § 8  ce 5  o  £  §  £8  it  If  .8 £  0  ffloo-S  ffl on  fa \"  <! S.  Q o  0 cd  in.  in.  in.  in.  in.  in.  in.  in.  Naked, Free States  13.98  14.72  16.33  10.56  36.05  34.18  29.81  34.94  Slave States  13.89  15.06  16.13  10.34  36.28  34.87  29.51  34.53  All . .  13.907  15.003  16.271  10.378  36.240  34.745  29.568  34.606  Half Naked  Free States  13.75  15.00  -  10.85  38.15  36.20  29.75  36.85  Slave Sti :s  13.61  14.00  -  10.77  35.99  34.50  29.81  36.64  All . .  13.615  14.010  -  10.775  36.018  34.524  29.812  36.639  Clothed  Free States  14.07  13.55  16.25  11.44  35.66  33.92  30.91  36.44  Slave States  13.96  13.56  16.44  11.29  35.69  33.98  30.75  35.94  All . .  13.966  13.556  16.378  11.300  35.691  33.979  30.767  35.983  In usual vigor  Free States  14.05  14.12  16.35  10.99  35.80  33.99  30.32  35.64  Slave States  13.92  13.99  16.40  10.97  35.88  34.28  30.32  35.59  All . .  13.933  14.000  16.390  10.969  35.870  34.248  30.320  35.598  Not in usual vigor  Free States  13.85  15.18  15.72  10.75  36.45  34.61  30.20  35.58  Slave States  13.82  14.70  -  10.83  36.07  34.47  30.09  35.31  All . .  13.826  14.759  15.717  10.819  36.123  34.487  30.103  35.346  Total bom in  Free States  14.018  14.276  16.276  10.954  35.893  34.082  30.303  35.630  Slave States  13.909  14.070  16.414  10.952  35.890  34.300  30.295  35.562  Grand Total . . .  13.921  14.089  16.358  10.952  35.899  34.275  30.296  35.569 MEAN DIMENSIONS OF BODY.  305  TABLE V. - (Continued.)  Mean Dimensions of Full Blacks.  Class  Length of Arm to  Middle of Breast-  Bone to Tip of to  | Finger  Acromion to  Elbow J?  264 26c  Distance be-  tween Angles  of Eyes  36a  8  P.  o  5  M  J  Length to Hol- co  low above Heel ©  Thickness at co  Instep S'  Circumf. around  Heel and Anterior g  Ligament \"■  Outer  Inner  Naked, Free States  in.  29.32  in.  35.50  in.  13.14  in.  3.92  in.  1.26  in.  10.44  in.  10.19  in.  2.60  in.  13.54  Slave States  29.20  35.54  12.99  4.03  1.32  10.61  10.27  2.44  13.82  All . .  29.222  35.573  13.014  4.009  1.310  10.583  10.252  2.471  13.766  Half Naked  Free States  28.95  36.45  14.00  3.65  1.25  10.75  10.70  2.65  13.85  Slave States  29.07  35.99  14.30  3.73  1.37  10.53  10.17  2.69  13.80  All . .  29.067  35.997  14.292  3.727  1.366  10.534  10.180  2.695  13.803  Clothed  Free States  30.10  35.54  14.32  4.24  1.41  10.61  10.32  2.90  13.55  Slave States  29.50  35.98  13.27  4.17  1.34  10.62  9.94  2.77  13.56  All . .  29.549  35.939  13.346  4.181  1.350  10.618  9.969  2.783  13.555  In usual vigor  Free States  29.69  35.49  13.62  4.07  1.33  10.52  10.24  2.76  13.54  Slave States  29.32  35.84  13.21  4.10  1.34  10.61  10.03  2.67  13.64  All . .  29.362  35.801  13.247  4.101  1.337  10.596  10.053  2.683  13.631  Not in usual vigor  Free States  29.55  35.77  13.54  4.00  1.32  10.55  10.33  2.57  13.58  Slave States  29.77  35.87  13.76  4.00  1.35  10.65  10.28  2.59  13.76  All . .  29.740  35.861  13.732  4.004  1.345  10.633  10.289  2.586  13.735  Total born in  Free States  29.669  35.525  13.604  4.061  1.329  10.522  10.253  2.734  13.550  Slave States  29.371  35.843  13.267  4.094  1.339  10.610  10.058  2.664  13.655  Grand Total. .  29.405  35.808  13.302  4.090  1.338  10.600  10.079  2.672  13.643", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ipan~i2gx_qtmx", "00000000-0000-0000-7271-4E75F28AB1C5", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Table comparing mean dimensions of different groups of soldiers measured", "9918573882206676X32", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" This table compared the mean dimensions among the different groups of soldiers measured by the USSC examiners. Page 316 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "316  MEAN DIMENSIONS OF BODY.  TABLE IX.  Comparison of Mean Dimensions.  White Soldiers  Sailors  Students  Full  Blacks  Mixed  Races  Indians  Later  Series  Earlier  Series  Number of Men . .  10 876  7 904  1 061  291  2 020  863  517  Mean Age ....  y  26.2  y  25.1  y  26.1  y  21.7  y  25.7  y  26.2  y  30.7  Length Head &amp; Neck  in.  9.944  in.  9.981  in.  10.091  in.  10.098  in.  9.623  in.  9.561  in.  9.547  Length of Body . .  26.140  26.099  24.549  26.109  24.487  24.680  26.870  Knee to Perinaeum .  12.456  -  12.880  12.652  12.964  12.692  12.799  Height to Knee . .  18.609  -  18.498  19.240  19.136  19.318  19.009  Stature  67.149  67.366  66.018  68.099  66.210  66.251  68.225  Acromion to Elbow .  13.605  -  13.171  13.712  13.302  13.856  13.757  Elbow to Finger-tip .  15.548  -  15.367  15.309  16.103  16.415  17.035  Dist. betw. Acromia  12.731  16.359\"  12.879  13.085  14.089  14.742  12.830  Ratio of parts of Arm  1.143  -  1.167  1.116  1.211  1.185  1.238  \" \" Leg  1.494  -  1.436  1.521  1.476  1.522  1.485  Med. line to Finger-tip  35.042  -  33.848  34.920  35.808  35.822  37.198  Acromion \" \"  29.153  29.200 6  28.538  29.021  29.405  30.271  30.792  Height to Perinaeum  31.065  31.286  31.378  31.892  32.100  32.010  31.808  Ratio of Leg to Arm  1.066  1.071  1.100  1.099  1.092  1.058  1.033  Height to Pubes . .  -  -  33.269  -  34.302  34.534  -  Finger-tip to Patella  5.036  -  5.778  6.473  2.884  4.125  3.653  Circumf. of Waist .  31.467  32.089  30.457  31.240  30.296  30.546  34.593  Circumf. of Hips  36.930  -  34.942  36.549  35.569  35.357  38.962  Circumf. of Chest .  35.818  35.353 c  35.124  35.313  35.087  34.966  38.001  Play of Chest. . .  2.65  -  2.08  3.07  1.62  1.57  1.84  Dist. between Nipples  8.136  -  8.304  8.071  7.970  7.891  -  Ratio to circum. Chest  0.226  -  0.236  0.229  0.225  0.227  -  Dist. between Eyes .  2.492  2.606  2.473  2.484  2.714  2.670  2.716  Breadth of Pelvis  11.916  13.153d  11.625  11.187  10.952  11.267  12.889  Length of Foot . .  10.058  -  10.114  9.957  10.600  10.439  10.123  Thickness of Foot .  2.572  -  2.921  2.786  2.672  2.770  2.687  Length of Heele . .  0.48  -  0.49  0.46  0.82  0.57  0.48  a Full breadth of shoulders. b Measured from arm-pit.  c Not the half-sum of circumferences at inspiration and expiration, as the others are.  d Probably the breadth of hips. See page 262.  e These values are obtained by adding 0.3 to the difference between the dimensious 36a  and 366. See page 274.", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-k4mu.zv29-qe8u", "00000000-0000-0000-B47A-8117D2D0537D", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Tables showing average weights of soldiers, by nativity, and by color", "9918573882206676X33", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" Tables I - IV, shown here, showed the average weights in General ;  by nativity (white soldiers), and, separately, for Black soldiers. Pages 402-404 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "3", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "402  WEIGHT AND STRENGTH.  TABLE I.  Average Weight of Men examined.  Class of Men  In usual Vigor  Not in usual Vigor  Total  Number  Pounds  Number  Pounds  Number  Pounds  White Soldiers, Earlier Series  5 936  143.49  2 162  140.99  8 098  142.83  White Soldiers, Later Series .  9 157  142.08  1 600  137.35  10 757  141.38  Sailors  1 144  138.92  -  -  1 144  138.92  Students  288  136.51  -  -  288  136.51  Full Blacks  1 775  143.83  226  142.62  2 001  144.58  Mulattoes  680  145.12  140  143.15  820  144.78  Indians  507  162.82  9  148.01  516  162.56  Assorting the weights according to the nativities of the men, we  find the means to be as given in the next two tables, in which the  results of the earlier and the later series of examinations are kept  distinct from each other.  TABLE II.  Average Weight of White Soldiers by Nativities.  (Earlier Series.)  Nativity  In usual Vigor  Not in usual Vigor  Total  Number  Weight  Number  Weight  Number  Weight  New England ....  589  lbs.  142.60  350  lbs.'  142.89  939  lbs.  142.71  New York  1 521  145.15  546  142.58  2 067  144.47  New Jersey and Penn. .  849  144.64  364  140.32  1 213  143.35  Ohio &amp; other West. States  413  148.73  187  144.26  600  147.34  Slave States  1 659  140.64  375  137.16  2 034  140.00  Canada  135  144.73  50  141.70  185  143.91  England and Scotland .  159  140.96  72  134.82  231  139.04  Ireland  350  142.99  122  141.11  472  142.50  Germany  191  143.77  76  140.39  267  142.81  Miscellaneous ....  70  143.59  20  139.59  90  142.70  Total  5 936  143.49  2 162  140.99  8 098  142.83 WEIGHT AND STRENGTH.  403  TABLE III.  Average Weight of White Soldiers, hy Nativities.  (Later Series.)  Nativity  In usual Vigor  Not in usual Vigor  Total  Number  Weight  Number  Weight  Number  Weight  New England ....  974  lbs.  140.05  211  lbs.  136.11  1 185  lbs.  139.39  N. ¥., N. J., and Penn. .  3 139  141.39  588  137.43  3 727  140.83  Ohio and Indiana . . .  1 442  145.99  218  141.24  1 660  145.37  Mich., Wise., and Illinois  944  141.78  71  139.72  1 015  141.78  Coast Slave States . .  301  142.08  52  134.68  353  140.99  Kentucky and Tennessee  223  150.58  44  146.10  267  149.85  Free Sts. west Miss. River  10  145.09  -  -  10  145.09  SI. Sts. west Miss. River  38  135.76  5  128.59  43  134.95  Br. Am. Pr. excl. Canada  35  143.82  2  139.54  37  143.59  Canada  474  141.26  45  142.28  519  141.35  England  258  138.15  45  134.58  303  137.61  Wales and Isle of Man .  18  138.05  2  148.09  20  139.13  Scotland  70  138.71  11  132.38  81  137.85  Ireland  644  141.08  177  132.26  821  139.18  France, Belgium, etc. . .  80  138.76  16  133.35  96  137.85  Germany  448  141.06  99  137.27  547  140.37  Scandinavia  28  150.28  6  138.12  34  148.14  Spain, etc .  6  138.16  1  109.79  7  134.15  Miscellaneous ....  25  140.31  7  126.58  32  137.31  Total . . ...  9 157  142.08  1 600  137.35  10 757  141.38  The degree of trustworthiness of the mean weights as tested by  the accordance between the actual and theoretical distribution of  the individual weights is very satisfactory, and the range of varia-  tion in all appears analogous to that in the nativities A and C,  which are 1 as follows : -  1 See foot-note to page 275. 404  WEIGHT AND STRENGTH.  Nativity  Mean  Weight  Number of  Men  r  To  New England States  Ohio and Indiana  lbs.  140.06  145.99  953  1 417  lbs.  10.853  11.383  lbs.  0.351  0.302  TABLE IV.  Average Weight of Colored Men.  Class  In usual Vigor  Not in usual Vigor  Total  Number  Weight  Number  Weight  Number  Weight  Full Blacks, Natives of Fr. Sts.  192  lbs.  144.60  32  lbs.  144.93  224  lbs.  144.65  (C Ci « (C g| <<  1 583  144.86  194  142.24  1 777  144.58  Mulattoes, Natives of Free Sts.  125  141.51\"  40  145.04  165  142.37  \" \" \" SI. \"  555  145.93  100  142.40  655  145.39  Total Full Blacks . . .  1 775  144.83  226  142.62  2 001  144.58  Total Mulattoes . . *. .  680  145.12  140  143.15  820  144.78  It is manifest that the variations of the mean weight with the  nativity must he closely commensurate with those of the mean  stature ; and, in order to determine the degree to which these ele-  ments are independent of one another, the Tables V., VI., and VII.  have been prepared, exhibiting for each nativity-group the ratio of  weight to stature, or in other words the weight in pounds corre-  sponding to each inch of stature. These have not been prepared  by dividing the mean weights by the mean heights, but have been  computed for each individual case; and the accuracy of the results  here also tested where the numbers are sufficiently large, by the  character of the distribution of individual weights around their  mean. They apply to men in full vigor, exclusively.  a If we omit the forty-five members of the two Massachusetts colored infantry regiments,  which appear to have been composed of men much lighter than the average of their class,  the mean weight of the remaining eighty men is 143 lbs. The average age of these forty-  five men was a year and a half less than that of the other colored soldiers measured.", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-64gq.qkfw-jqg2", "00000000-0000-0000-6F66-9C35AC9F23FF", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Tables showing the mean weights of soldiers, by age and height", "9918573882206676X34", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" This series of tables showed the mean weights of soldiers examined by age and height, with separate tables for Black and Native American men. Pages 420-434 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "15", "pages", "Text", "English", "The National Library of Medicine believes this to be in the public domain.", "Public domain", null, null, "420  WEIGHT AND STRENGTH.  TABLE XVI.  Mean Weights of White Soldiers, by Age and Height.  (Earlier Series.)  Age  64 Inches  64| Inches  65 Inches  65| Inches  66 Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  1  lbs.  112.8  3  lbs.  119.8  4  lbs.  128.5  2  lbs.  114.8  6  lbs.  121.8  16  10  125.6  8  119.4  5  140.0  8  118.7  6  126.3  17  12  129.0  18  124.0  10  138.6  17  129.9  14  130.5  18  26  122.4  26  133.1  17  126.2  40  132.3  31  133.2  .19  16  126.3  28  134.7  37  129.0  32  129.6  43  137.5  20  15  127.9  30  131.4  28  133.0  40  136.6  29  137.1  21  25  131.9  16  137.0  27  132.9  39  138.9  36  134.4  22  14  129.8  22  131.1  17  133.9  35  136.8  25  139.0  23  12  132.4  19  133.6  23  136.0  20  133 1  35  134.9  24  8  127.7  13  137.7  13  136.3  32  138.8  15  139.2  25  3  126.6  7  133.7  9  137.9  12  141.5  18  141.5  26  8  136.4  6  140.5  4  139.4  11  133.3  16  139.4  27  3  127.0  7  134.7  12  139.5  14  137.6  8  136.5  28  4  138.3  5  148.6  8  140.7  10  138.4  16  141.5  29  1  111.8  8  130.2  6  134.5  8  136.8  10  134.1  30  2  136.8  7  141.6  3  140.3  7  137.0  7  146.9  31  1  144.3  1  145.3  3  130.8  5  134.4  2  137.0  32  -  -  2  118.3  3  120.6  10  139.2  5  133.2  33  2  129.3  1  118.3  2  152.8  4  144.2  4  140.3  34  1  138.8  2  126.8  3  133.3  1  138.8  4  141.6  35  2  118.0  3  130.6  3  143.1  3  149.5  2  153.0  36  1  145.8  4  142.3  3  132.1  7  150.2  4  134.8  37  1  137.8  3  148.5  -  -  3  143.1  1  131.8  38  2  144.5  1  128.8  1  171.3  3  132.0  4  139.0  39  -  -  1  169.3  2  124.8  1  135.3  4  144.3  40  -  <*  1  138.3  -  -  1  111.8  -  -  41  1  117.3  -  -  -  -  -  -  -  -  42  1  152.3  -  -  1  139.8  1  166.8  2  154.0  43  -  -  1  150.3  -  -  -  -  -  44  -  -  1  140.3  1  175.8  1  131.8  -  -  45  4  148.4  2  137.5  -  -  -  -  2  142.5  46  1  140.3  1  167.8  -  -  -  -  -  -  47  -  -  -  -  -  -  -  -  -  -  48  1  142.3  -  -  -  -  -  -  -  -  49  -  -  -  -  -  -  -  -  -  -  50  -  -  1  117.3  1  166.3  1  128.8  -  -  51  -  -  1  114.8  -  -  1  123.8  -  -  52  -  -  -  -  -  -  -  -*  -  -  53  -  -  -  -  1  119.8  -  -  -  -  54  -  -  -  -  -  -  -  -•  -  -  55  •*  -  -  -  -  -  •*  -  - WEIGHT AND STRENGTH.  421  TABLE XVI. - (  Mean Weights of White Soldiers, hy Age and Height.  (Earlier Series.)  Age  66| Inches  67 Inches  671 Inches  68 Inches  68| Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  2  lbs.  119.0  -  lbs.  -  lbs.  1  lbs.  120.3  -  lbs.  16  4  123.4  2  137.3  3  142.1  5  132.0  -  -  17  17  128.4  14  134.4  16  135.6  8  136.5  13  142.8  18  31  131.6  38  137.6  34  142.3  27  138.9  24  143.6  19  42  135.7  38  141.2  53  139.2  37  139.2  32  139.7  20  60  139.8  52  138.7  51  143.5  47  147.1  54  148.6  21  41  142.3  43  143.1  55  141.9  45  145.9  55  147.4  22  39  141.8  42  143 9  51  146.1  40  148.6  35  148.0  23  22  138.4  30  142.7  42  145.3  32  142.9  45  146.5  24  26  137.0  17  148.0  40  149.0  41  147.2  32  151.3  25  26  141.9  15  148.9  22  147.7  24  154.0  29  152.0  26  17  145.5  17  147.4  18  143.6  18  148.5  22  154.8  27  12  140.9  16  141.0  19  152.9  15  148.4  21  150.5  28  14  141.5  12  144.0  9  147.8  16  149.9  19  148.7  29  6  138.6  6  142.3  9  148.5  20  150.3  14  150.3  30  10  155.9  10  147.3  15  151.4  18  146.1  11  162.5  31  4  134.7  7  138.6  5  149.6  8  147.6  7  146.6  32  7  142.3  9  149.1  7  142.4  8  152.6  4  158.2  33  9  158.9  1  151.8  5  141.6  7  147.9  7  150.2  34  4  144.5  5  151.7  7  151.9  7  154.1  7  155.1  35  3  150.0  12  146.5  9  148.1  4  156.8  8  153.0  36  8  139.7  3  153.0  4  141.5  1  149.8  5  155.7  37  3  144.8  3  137.6  6  147.5  3  133.8  5  150.8  38  4  134.3  3  140.8  2  139.0  1  144.3  1  149.8  39  4  136.9  1  126.3  3  146.0  -  -  1  124.3  40  1  160.8  2  171.3  3  173.0  2  147.8  2  154.0  41  4  140.7  -  -  3  148.6  -  -  1  153.3  42  1  147.3  -  -  6  150.9  -  -  2  139.0  43  -  -  1  152.3  1  154.3  3  140.6  4  156.9  44  1  150.8  2  154 3  3  141.6  2  133.5  3  155.8  45  2  153.8  2  161.0  -  -  1  139.8  -  -  46  3  143.5  2  139.3  1  143.8  -  -  2  163.5  47  1  143.3  -  -  -  -  1  158.8  -  -  48  2  150.3  -  -  1  140.8  -  1  164.3  49  1  160.8  -  •*  -  -  -  -  -  -  50  -  -  1  156.8  -  -  1  143.5  -  -  51  1  123.8  1  147.3  -  -  -  -  -  -  52  -  -  -  -  -  -  1  144.8  -  -  53  -  -  -  -  1  144.8  -  -  -  -  54  -  -  -  -  -  -  -  -  -  -  55  -  -  -  -  -  -  -  *  - Ij 422  WEIGHT AND STRENGTH.  TABLE XVI. - ( Continued.')  Mean Weights of White Soldiers, by Age and Height.  (Earlier Series.)  Age  69 Inches  69| Inches  70 Inches  70J Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  -  lbs.  lbs.  -  lbs.  lbs.  16  2  125.5  1  113.3  -  -  -  17  3  142.6  3  135.6  3  150.5  4  140.3  18  27  141.1  13  149.9  12  150.3  10  145.7  19  30  146.1  22  147.0  21  146.6  10  152.8  20  37  150.2  27  144.8  24  153.4  14  152.7  21  37  146.6  34  153.0  29  156.2  26  154.8  22  36  151.5  29  147.6  32  158.3  18  152.9  23  29  150.8  18  146.4  23  153.9  20  150.4  24  23  150.6  20  150.4  19  152.7  19  162.6  25  20  154.3  22  154.3  19  155.5  12  162.8  26  11  151.4  17  153.4  6  156.5  15  158.2  27  16  149.8  16  155.3  10  152.1  8  159.2  28  20  150.6  15  156.5  12  156.4  7  160.4  29  7  147.2  9  156.9  8  159.2  8  166.0  30  7  160.4  5  152.8  8  158.4  2  154.5  31  7  152.3  8  162.2  5  149.1  3  162.5  32  5  156.2  4  148.7  7  163.9  4  174.5  33  3  160.5  6  155.8  1  175.8  5  161.9  34  3  146.3  2  170.0  2  151.5  3  179.1  35  10  143.6  5  139.5  4  141.8  2  161.8  36  4  156.5  -  -  1  161.3  2  160.3  37  5  152.5  4  152.4  1  160.3  -  -  38  3  156.3  2  163.8  3  154.8  1  151.3  39  -  -  1  148.8  4  158.9  4  153.3  40  1  136.8  2  173.0  -  -  2  177.3  41  -  -  1  166.8  1  138.8  2  161.5  42  5  165.7  4  148.8  2  177.0  1  181.8  43  4  143.6  2  161.3  -  -  -  -  44  2  186.0  2  158.5  1  140.8  1  159.8  45  1  152.8  2  146.8  2  154.3  -  -  46  1  160.8  -  -  1  172.8  -  -  47  1  162.8  -  -  -  -  -  -  48  2  154.5  2  158.8  -  -  -  -  49  -  -  -  -  -  -  -  -  50  1  146.8  -  -  -  -  -  -  51  -  -  -  -  1  150.3  -  -  52  -  -  -  -  -  -  -  -  53  -  -  1  150.3  2  164.3  -  -  54  -  -  -  T-  -  -  -  -  55  -  -  -  -  -  - WEIGHT AND STRENGTH.  423  TABLE XVII.  Mean Weights of White Soldiers, by Age and Height.  (Later Series.)  Age  64 Inches  641 Inches  65 Inches  65 £ Inches  66 Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  2  lbs.  116.8  -  lbs.  4  lbs.  127.8  -  lbs.  3  lbs.  117.8  16  9  123.6  9  119.7  9  114.1  10  129.1  7  125.4  17  17  124.5  22  118.4  17  122.7  19  131.2  18  130.7  18  33  122.0  50  127.1  40  127.0  53  128.2  53  130.2  19  36  122.8  37  127.0  43  128.0  50  131.0  41  133.2  20  14  128.4  43  129.7  42  133.6  58  131.8  42  135.9  21  32  131.7  37  130.1  37  134.2  51  134.7  47  136.5  22  23  129.1  44  133.5  46  135.2  67  133.6  38  139.6  23  20  137.7  38  133.1  31  135.6  48  136.8  27  139.6  24  17  132.7  22  133.0  26  133.9  48  137.2  38  141.7  25  19  134.0  20  127.1  24  131.3  22  136.5  28  137.7  26  7  136.9  14  135.4  14  133.1  32  139.7  17  141.6  27  6  126.9  17  134.9  17  134.6  17  135.7  15  144.5  28  8  129.3  14  137.6  13  137.7  18  138.5  19  136.2  29  9  130.4  18  135.3  10  134.7  18  135.7  6  135.9  30  5  139.3  10  130.4  8  130.3  22  136.2  20  141.5  31  7  132.6  7  133.9  7  125.5  15  137.7  7  147.6  32  6  136.0  9  128.8  15  133.8  17  138.0  12  140.6  33  9  132.0  8  130.7  5  138.3  6  143.0  14  150.7  34  3  137.8  7  131.1  6  131.3  15  135.3  6  139.7  35  5  131.4  10  131.8  9  131.3  4  133.9  8  139.0  36  5  141.6  4  146.6  6  130.7  9  141.5  6  137.0  37  -  -  3  131.5  3  138.8  8  136.8  9  138.9  38  6  133.6  7  128.4  5  132.9  7  139.0  5  145.3  39  4  137.2  3  130.5  4  137.9  5  134.2  7  138.7  40  2  127.5  2  120.5  3  129.0  9  141.9  4  148.0  41  1  150.8  -  -  3  127.1  4  153.8  2  125.5  42  2  128.8  3  142.8  3  137.7  4  136.8  6  137.2  43  2  127.3  4  138.5  3  138.8  4  143.0  2  132.3  44  1  140.8  2  137.3  3  145.1  2  139.8  5  141.2  45  3  132.1  1  128.8  2  150.3  6  143.7  2  150.8  46  -  -  1  111.8  -  -  5  135.6  2  143.8  47  1  115.8  -  -  1  118.8  1  138.8  -  -  48  -  -  1  124.8  2  129.3  1  131.8  2  135.8  49  -  -  -  -  -  -  -  -  -  -  50  1  124.8  •  -  -  -  4  136.9  -  -  51  -  -  1  148.8  -  -  2  139.8  -  •-  52  -  -  -  -  -  -  1  142.8  1  137.8  53  -  -  -  -  -  -  -  -  -  -  54  -  -  -  -  -  -  1  160.3  -  -  55  -  ••  -  -  -  -  1  143.8  2  138.8 424  WEIGHT AND STRENGTH.  TABLE XVII. - ( Continued.')  Mean Weights of White Soldiers, hy Age and Height.  (Later Series.)  Age  66} Inches  67 Inches  67} Inches  68 Inches  68} Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  2  lbs.  133.8  1  lbs.  115.8  -  lbs.  -  lbs.  -  lbs.  16  10  128.3  11  130.9  7  129.3  5  132.1  4  124.9  17  18  126.4  20  136.7  10  133.1  11  132.2  5  138.0  18  52  131.0  40  133.2  50  137.9  47  139.4  48  141.7  19  53  136.5  51  136.5  52  141.0  54  143.6  48  145.1  20  87  137.3  69  141.4  77  142.3  64  145.0  55  146.0  21  72  137.9  64  138.7  85  143.3  59  146.2  61  146.7  22  92  137.7  74  141.4  84  142.4  73  143.8  57  146.2  23  50  139.8  48  140.6  61  143.8  59  148.0  51  148.1  24  65  141.8  59  145.2  70  146.9  51  147.5  58  149.4  25  41  143.2  31  148.1  36  145.1  35  148.0  41  148.1  26  32  139.4  39  145.1  37  145.4  24  151.0  37  150.1  27  19  144.3  27  145.2  23  149.6  27  150.3  28  152.7  28  21  137.4  27  144.9  40  147.9  29  143.9  30  153.4  29  25  139.8  20  147.2  20  144.5  18  147.9  23  152.4  30  19  142.2  30  146.1  24  146.1  24  149.2  20  147.3  31  13  140.0  18  143.2  14  142.6  11  147.9  8  150.2  32  20  140.1  11  138.5  19  146.3  17  146.3  19  146.4  33  9  149.0  11  144.1  18  150.3  9  151.6  9  152.6  34  5  147.4  14  146.2  24  146.3  10  147.0  19  148.3  35  20  146.6  16  143.9  17  149.9  7  147.4  18  151.3  36  11  153.8  11  144.6  12  147.6  4  154.4  9  148.7  37  9  135.5  9  142.0  9  151.7  8  148.9  3  145.8  38  17  138.9  8  151.8  9  151.5  6  143.6  6  151.3  39  5  144.9  6  148.0  8  146.6  11  149.1  4  138.4  40  3  161.6  10  146.2  9  148.2  8  143.7  6  150.5  41  4  140.9  6  144.0  2  136.0  5  139.9  2  144.3  42  4  146.0  5  137.6  5  135.4  3  149.1  10  149.3  43  7  149.8  4  151.0  8  141.5  1  122.8  5  150.7  44  7  149.5  7  149.9  4  145.9  4  152.3  1  142.3  45  3  151.5  2  133.5  6  145.9  9  149.2  -  -  46  4  143.8  2  154.5  1  146.3  2  152.0  -  -  47  1  138.3  3  142.8  2  157.8  -  -  -  -  48  2  115.4  4  137.8  2  160.0  1  173.8  1  135.8  49  1  141.8  -  -  1  144.8  -  -  -  -  50  4  132.5  1  150.8  1  156.8  -  -  -  -  51  -  -  1  163.3  -  *  1  127.8  -  -  52  -  -  -  -  1  131.8  -  -  -  -  53  1  133.8  -  ••  1  128.3  2  142.3  1  159.8  54  1  143.8  1  156.8  1  139.8  -  -  1  157.8  55  -  -  -  - WEIGHT AND STRENGTH.  425  TABLE XVII. - (Continued.)  Mean Weights of White Soldiers, by Age and Height.  (Later Series.)  Age  69 Inches  69 j Inches  70 Inches  70 j Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  -  lbs.  1  lbs.  156.8  1  lbs.  146.8  -  lbs.  16  2  167.0  -  -  -  -  -  17  9  136.2  6  138.5  3  134.5  3  147.7  18  43  143.8  23  148.9  20  147.7  8  148.7  19  27  141.8  21  151.3  15  149.5  16  152.5  20  38  148.4  30  149.6  23  150.5  18  156.2  21  43  153.9  51  150.7  35  151.4  30  158.0  22  33  150.5  38  150.9  23  160.8  18  157.7  23  40  150.6  38  155.8  27  151.3  16  154.4  24  32  149.6  42  157.6  22  156.1  15  156.8  25  29  149.9  19  160.9  22  150.8  13  159.5  26  17  154.1  26  150.9  12  161.5  16  157.5  27  24  153.8  22  151.5  18  158.0  14  159.2  28  23  150.4  18  155.8  11  159.0  10  162.0  29  14  146.9  14  152.3  8  158.0  7  156.9  30  14  152.1  12  161.6  14  162.8  9  156.9  31  6  149.8  8  160.1  7  159.5  8  162.5  32  5  164.0  9  152.6  10  152.8  7  162.4  33  7  159.7  14  154.0  5  152.2  6  156.0  34  7  156.2  14  155.0  10  159.4  7  152.9  35  2  157.0  9  148.6  7  148.8  4  154.9  36  6  151.5  2  147.3  6  167.5  2  160.3  37  13  162.6  10  148.0  3  164.4  4  163.8  38  6  155.5  9  154.8  4  154.0  5  161.3  39  2  152.5  1  175.8  5  148.9  7  166.5  40  4  161.8  4  162.2  -  -  1  160.8  41  2  141.5  3  153.5  5  146.2  1  183.3  42  8  145.3  1  146.3  2  163.5  2  193.8  43  2  153.5  2  153.8  3  164.6  2  155.5  44  9  158.8  3  150.5  ■1  177.8  2  140.8  45  2  152.3  2  149.8  -  -  2  151.5  46  -  -  -  -  -  *  1  151.8  47  -  -  -  -  -  -  1  141.3  48  -  -  1  161.8  -  -  -  49  2  146.3  1  155.8  1  164.3  -  -  50  -  -  -  -  -  -  -  -  51  1  174.8  -  -  -  -  -  -  52  1  164.8  -  -  -  -  -  -  53  -  -  -  -  -  -  -  -  54  -  -  2  196.8  -  -  -  -  55  -  -  1  161.3  -  -  -  - 426  WEIGHT AND STRENGTH.  TABLE XVIII.  Mean Weights of White Soldiers, by Age and Height.  {Both Series.)  Age  64 Inches  64J Inches  65 Inches  65.} Inches  66 Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  3  lbs.  115.5  3  lbs.  119.8  8  lbs.  128.2  2  lbs.  114.8  9  lbs.  120.5  16  19  124.7  18  119.7  14  123.4  18  124.5  13  125.8  17  29  126.4  40  121.0  27  128.6  36  130.6  32  130.6  18  59  122.2  77  129.1  58  127.0  93  130.0  86  131.4  19  52  123.9  65  130.3  81  128.4  82  130.4  84  135.4  20  29  128.1  73  130.4  70  133.4  98  133.8  74  136.4  21  57  131.8  54  132.4  67  133.6  92  136.4  85  135.8  22  37  129.4  66  132.7  63  134.9  105  134.8  64  139.6  23  32  135.7  58  133.3  56  135.4  70  135.7  64  137.3  24  25  131.1  35  134.7  42  135.3  81  137.9  56  140.8  25  22  133.0  27  128.8  33  133.1  35  137.7  46  139.2  26  16  136.1  21  137.6  19  134.6  43  138.0  33  140.6  27  10  130.6  24  134.8  29  136.6  31  136.6  23  141.7  28  12  132.3  19  140.5  21  138.9  29  138.5  35  138.6  29  11  128.2  27  133.6  16  134.6  28  136.2  16  134.8  30  7  138.6  17  135.0  11  133.1  29  136.4  27  142.9  31  8  134.0  8  135.4  10  127.1  20  136.8  9  145.3  32  6  136.0  11  126.9  18  131.6  28  139.0  17  138.4  33  11  131.5  9  129.3  7  142.4  10  143.4  18  148.4  34  4  138.0  9  130.1  9  132.0  16  135.6  10  140.5  35  7  127.6  13  131.5  12  134.3  7  140.6  10  141.8  36  6  142.3  8  144.4  10  133.0  16  145.3  10  136.1  37  1  137.8  6  140.0  3  138.8  11  138.5  10  138.2  38  8  136.4  8  128.5  6  139.3  10  136.9  9  142.5  39  4  137.2  4  140.2  6  133.5  6  134.4  12  140.5  40  2  127.5  3  126.5  3  129.0  10  138.9  4  148.0  41  2  134.0  -  -  3  127.1  4  153.8  2  125.5  42  4  136.2  3  142.8  4  138.2  5  142.8  8  141.4  43  2  127.3  5  140.9  3  138.8  4  143.0  2  132.3  44  1  140.8  3  138.5  4  152.8  3  137.1  5  141.2  45  7  141.4  3  134.6  2  150.3  6  143.7  4  146.7  46  1  140.3  2  139.8  -  -  5  135.6  2  143.8'  47  1  115.8  -  -  1  118.8  1  138.8  -  -  48  1  142.3  1  124.8  2  129.3  1  131.8  2  135.8  49  -  -  -  -  -  -  -  -  -  -  50  1  124.8  1  117.3  1  166.3  5  135.3  -  -  51  -  -  2  131.8  -  -  3  134.5  -  -  52  -  -  -  -  -  -  1  142.8  1  137.8  53  -  -  -  -  1  119.8  -  -  -  -  54  -  -  -  -  -  -  1  160.3  -  -  55  -  -  -  -  -  -  1  143.8  2  138.8 WEIGHT AND STRENGTH.  427  TABLE XVIII. -  Mean Weights of White Soldiers, by Age and Height  (Both Series.)  Age  66| Inches  67 Inches  67£ Inches  68 Inches  68j Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  4  lbs.  126.4  1  lbs.  115.8  -  lbs.  1  lbs.  120.3  -  lbs.  16  14  126.9  13  131.9  10  133.2  10  132.0  4  124.9  17  36  127.5  35  135.4  26  134.6  20  134.8  18  141.5  18  83  131.2  78  135.3  86  139.4  75  139.5  72  142.3  19  95  136.1  90  138.6  108  140.0  91  141.8  83  143.1  20  149  138.5  121  140.2  130  142.8  112  146.0  110  147.3  21  116  139.6  110  140.2  141  142.9  108  146.2  120  147.5  22  134  138.9  117  142.3  137  144.1  114  145.5  93  147.0  23  74  139.7  80  141.5  104  144.5  92  146.3  96  147.3  24  91  140.4  78  145.9  112  147.9  96  147.4  91  150.1  25  67  142.7  46  148.4  61  146.2  62  150.4  72  149.8  26  49  141.6  56  145.8  57  145.1  42  149.9  60  152.4  27  31  143.0  44  143.6  43  151.4  42  149.6  50  151.5  28  35  139.0  39  144.6  49  147.9  45  146.1  49  151.5  29  31  139.6  26  146.1  29  145.7  39  149.0  38  151.2  30  29  147.0  41  147.0  39  148.2  42  147.9  31  152.7  31  17  138.7  25  141.9  19  144.5  19  147.8  15  148.5  32  27  140.7  20  143.3  27  145.4  25  148.3  23  148.4  33  18  149.0  12  144.8  23  148.4  17  149.8  16  151.5  34  9  146.1  19  147.7  31  147.6  17  149.9  26  150.1  35  23  147.0  28  145.0  26  149.3  11  150.8  26  151.9  36  19  147.8  14  146.4  16  146.1  5  153.5  14  151.2  37  13  139.8  12  140.9  15  150.0  12  145.0  8  148.9  38  21  138.1  11  148.8  11  149.2  7  143.7  7  151.1  39  9  141.4  7  144.9  11  146.4  11  149.1  5  135.6  40  5  156.9  12  150.4  12  154.4  11  145.8  8  151.4  41  9  142.1  6  144.0  5  143.6  5  139.9  3  147.3  42  5  146.3  5  137.6  12  143.1  3  149.1  12  147.6  43  7  149.8  5  151.3  9  142.9  4  136.2  9  153.5  44  8  149.7  9  150.9  7  144.1  6  146.0  4  152.4  45  5  152.4  4  147.3  6  145.9  10  148.3  -  -  46  7  143.7  4  146.9  2  145.0  2  152.0  2  163.5  47  2  140.8  3  142.8  2  157.8  1  158.8  -  -  48  4  132.8  4  137.8  3  153.6  1  173.8  2  150.0  49  2  151.3  -  -  1  144.8  -  -  -  -  50  4  132.5  2  153.8  1  156.8  1  143.5  -  -  51  1  123.8  2  155.3  -  -  1  127.8  -  52  -  -  -  -  1  131.8  1  144.8  -  53  1  133.8  -  -  2  136.5  2  142.3  1  159.8  54  1  143.8  1  156.8  2  132.3  -  -  1  157.8  55  -  -  -  -  -  -  - 428  WEIGHT AND STRENGTH.  TABLE XVIII. - ( Continued.')  Mean Weights of White Soldiers, by Age and Height.  (Both Series.)  Age  69 Inches  69 J Inches  70 Inches  70j Inches  No.  Weight  No.  Weight  No.  Weight  No.  Weight  15  -  lbs.  1  lbs.  156.8  1  lbs.  146.8  -  lbs.  16  4  146.3  1  113.3  *-  -  -  -  17  12  137.8  9  137.5  6  142.5  7  143.9  18  70  142.7  36  149.3  32  148.7  18  147.1  19  58  144.2  43  149.1  36  147.8  26  152.6  20  77  149.3  58  147.4  47  152.0  33  154.7  21  81  150.8  87  152.0  65  153.8  56  156.5  22  70  150.9  69  149.5  58  159.3  36  155.3  23  72  151.3  56  152.8  52  152.4  38  151.9  24  58  150.0  63  155.4  43  154.8  37  160.0  25  50  151.9  42  157.6  41  153.0  26  161.8  26  29  152.9  45  152.6  18  159.8  31  157.8  27  40  152.2  38  153.1  28  155.9  24  161.9  28  43  150.5  33  156.2  23  157.6  17  161.4  29  21  147.0  25  153.9  16  158.6  15  161.8  30  22  155.3  17  159.0  22  161.2  11  156.5  31  13  151.1  17  161.1  12  155.2  6  162.5  32  10  160.1  13  151.4  17  157.4  11  166.8  33  10  159.9  20  154.6  6  156.1  11  158.7  34  10  153.2  16  156.9  12  158.1  10  160.8  35  12  145.9  14  145.4  11  146.2  6  157.2  36  10  153.5  2  147.3  7  166.7  4  160.3  37  18  159.8  14  149.3  4  163.4  4  163.8  38  9  155.7  11  156.4  7  154.4  6  159.6  39  2  152.5  2  162.3  9  153.3  11  161.7  40  5  156.8  6  165.8  -  -  3  171.8  41  2  141.5  4  156.8  6  145.0  3  168.8  42  13  153.1  5  148.3  4  170.3  3  189.8  43  6  146.9  4  157.5  3  164.6  2  155.5  44  11  163.7  5  153.7  2  159.3  3  147.1  45  3  152.5  4  148.3  2  154.3  2  151.5  46  1  160.8  -  -  1  172.8  1  151.8  47  1  162.8  -  -  -  -  1  141.3  48  2  154.5  3  159.8  -  -  -  -  49  2  146.3  1  155.8  1  164.3  -  -  50  1  146.8  -  -  -  -  -  -  51  1  174.8  -  -  1  150.3  -  -  52  1  164.8  -  -  -  -  -  -  53  -  -  1  150.3  2  164.3  -  -  54  -  -  2  196.8  -  -  -  -  55  -  -  1  161.3  -  -  -  -* 429  WEIGHT AND STRENGTH.  TABLE XIX.  Mean Weights of Sailors, by Age and Height.  Age  64j Inches  65| Inches  66| Inches  67| Inches  68| Inches  69| Inches  70| Inches  No.  wt.  No.  wt.  No.  wt.  No.  Wt.  No.  wt.  No.  Wt.  No.  wt.  16  1  lbs.  116.0  lbs.  lbs.  lbs.  lbs.  lbs.  lbs.  17  1  121.3  -  -  2  116.0  18  4  110.2  2  143.3  2  135.6  3  141.6  2  141.5  1  151.0  -  -  19  8  122.5  10  124.6  5  131.0  2  145.0  1  125.0  2  174.0  -  -  20  11  121.4  12  127.1  11  133.9  10  143.9  5  140.2  5  138.6  2  143.4  21  15  131.4  20  129.1  15  137.6  16  137.7  13  143.9  12  143.4  4  157.0  22  14  132.6  24  142.0  16  135.0  17  143.9  11  158.5  8  147.9  4  159.0  23  5  129.2  11  132.8  12  145.8  13  144.4  7  148.8  5  152.8  1  159.0  24  16  139.5  16  134.6  10  142.1  13  146.3  17  157.8  7  154.3  5  158.3  25  14  135 5  15  142.7  10  147.2  15  138.3  13  154.6  9  156.4  5  156.8  26  9  134.1  11  135.6  2  140.0  12  146.7  7  153.2  5  153.0  3  157.8  27  3  130.7  6  132.7  8  142.7  3  149.3  5  142.3  5  152.6  3  154.0  28  8  132.6  4  139.8  6  146.0  14  151.3  6  151.8  4  154.5  2  161.4  29  8  130.2  11  136.9  9  142.0  4  137.3  3  151.0  2  137.0  1  149.0  30  2  131.0  7  131.4  4  146.1  5  145.2  6  140.7  1  128.0  -  -  31  5  130.8  6  142.5  2  136.9  2  150.9  3  138.3  -  -  2  164.5  32  4  138.8  7  135.8  1  153.0  8  145.9  3  144.3  3  155.3  1  150.0  33  2  139.8  2  133.5  2  142.5  2  142.5  -  -  -  -  1  149.0  34  5  137.8  -  -  3  142.1  2  141.0  2  155.8  1  160.0  -  -  35  2  147.0  3  142.7  4  146.2  2  145.9  2  146.0  4  152.9  -  -  36  3  147.0  5  141.7  -  -  2  143.8  -  -  2  155.0  3  157.3  37  -  -  -  -  1  143.0  2  136.5  1  158.0  -  -  -  -  38  -  -  1  146.8  2  150.1  1  123.0  -  -  1  140.0  -  -  39  2  131.0  1  124.0  2  145.5  2  159.9  1  184.0  -  -  -  -  40  1  137.0  1  133.0  1  135.0  -  -  1  173.8  1  148.0  -  -  41  -  -  1  133.0  -  -  -  -  -  -  1  133.0  -  -  42  -  -  1  148.0  1  129.0  1  168.0  -  -  -  -  -  -  43  1  137.0  -  -  -  -  -  -  1  134.0  -  -  -  -  44  -  -  -  -  -  -  1  164.0  -  -  -  -  -  -  45  2  128.9  -  -  2  144.5  Over 45  2  150.5  1  139.0  1  115.0  2  167.6  -  -  3  149.3 430  WEIGHT AND STRENGTH.  TABLE XX.  Mean Weights of Students, by Age and Height.  Age  64} Inches  65} Inches  66} Inches  67} Inches  68} Inches  69} Inches  70} Inches  No.  wt.  No.  Wt.  No.  wt.  No.  wt.  No.  Wt.  No.  wt.  No.  Wt.  17  -  lbs.  -  Ibs.  1  lbs.  127.8  1  Ibs.  120.8  1  lbs.  133.8  -  lbs.  -  lbs.  18  2  112.8  1  111.8  1  139.8  -  -  -  -  2  127.5  -  -  19  3  132.1  8  124.5  5  121.8  5  124.5  6  130.0  7  145.3  1  150.8  20  1  114.8  7  126.5  7  136.5  8  130.3  13  132.4  15  138.8  8  147.4  21  3  123.0  7  126.4  11  130.1  11  128.5  10  138.5  9  143.3  16  145.0  22  1  113.8  6  121.6  5  131.8  7  131.9  6  136.5  6  147.5  6  152.5  23  1  105.8  -  -  1  142.3  1  133.3  1  131.8  4  142.3  -  -  24  2  125.8  -  -  1  142.8  2  125.8  4  136.2  2  132.3  -  -  25  1  123.3  -  -  3  134.8  1  144.8  2  146.3  1  143.8  1  153.8  26  -  -  1  134.8  2  125.3  -  27  -  -  1  119.8  -  -  -  -  1  139.8  -  -  2  150.3  Over 27  -  -  2  120.6 WEIGHT AND STRENGTH.  431  TABLE XXI.  Mean Weights of Full Blacks, by Age and Height.  Age  64} Inches  65} Inches  66} Inches  67} Inches  68} Inches  69} Inches  70} Inches  No.  wt.  No.  Wt.  No.  wt.  No.  Wt.  No.  wt.  No.  Wt.  No.  wt.  15 &amp; und.  1  lbs.  137.7  1  lbs.  102.8  -  lbs.  1  lbs.  131.0  -  lbs.  -  lbs.  -  lbs.  16  1  145.0  8  128.3  4  120.8  1  129.3  1  146.8  1  153.8  -  -  17  5  128.5  6  131.5  4  133.2  9  128.8  2  143.8  2  151.1  -  -  18  9  126.4  10  130.5  13  134.9  7  136.4  7  144.7  3  152.2  2  148.8  19  12  136.0  7  131.1  11  133.2  12  139.8  11  144.9  6  149.3  3  154.9  20  19  140.7  26  139.1  26  142.8  23  147.4  17  149.2  7  156.3  2  143.5  21  19  139.8  20  143.9  19  142.5  21  146.4  15  155.6  8  152.4  2  160.0  22  23  137.7  24  143.5  28  140.9  20  151.9  18  152.4  11  157.4  5  162.3  23  16  137.1  23  140.9  29  143.5  24  149.7  23  151.6  14  156.5  -  -  24  8  139.9  9  144.0  26  144.1  30  151.9  21  157.5  16  159.0  11  160.4  25  8  131.8  14  143.4  18  145.4  17  151.4  16  156.9  10  150.9  12  166.4  26  8  133.9  9  150.9  10  146.5  20  151.0  9  150.4  4  151.5  4  172.3  27  9  135.8  4  149.0  8  144.7  14  151.6  10  153.9  4  164.5  1  134.0  28  5  138.2  10  142.8  12  147.7  9  145.8  12  155.3  8  156.4  6  164.8  29  3  146.0  3  135.4  8  143.7  7  155.4  4  148.9  7  163.4  1  166.8  30  5  140.5  8  140.4  9  141.1  4  150.1  7  156.6  5  162.4  3  162.1  31  2  145.2  3  137.9  3  131.3  4  154.8  3  155.7  3  150.2  1  173.0  32  2  124.3  4  135.8  5  149.7  3  148.5  1  174.8  3  196.5  2  163.8  33  1  135.0  3  140.7  2  143.4  3  147.2  2  146.9  1  152.5  -  -  34  2  143.3  3  149.9  3  156.0  2  159.8  1  168.3  4  151.3  1  174.8  35  3  141.9  4  135.6  4  144.6  1  170.0  2  153.4  1  161.8  -  -  36  1  137.0  3  151.9  1  152.8  3  151.5  1  140.8  -  -  1  170.8  37  2  151.5  2  129.4  3  142.9  1  165.0  3  159.6  3  162.0  1  170.8  38  1  137.8  4  139.4  1  131.8  2  141.8  1  119.3  -  -  1  159.8  39  2  150.0  2  140.9  -  -  3  139.3  -  -  1  150.0  -  -  40  3  144.9  1  153.0  7  142.2  1  156.0  2  146.1  1  184.8  1  177.8  41  -  -  -  -  -  -  1  174.3  -  -  -  -  -  -  42  -  -  -  -  2  151.4  1  156.8  1  156.8  -  -  2  165.0  43  3  142.9  -  -  -  -  1  134.8  1  126.8  -  -  1  145.4  44  -  -  -  -  -  -  -  -  1  154.8  -  -  -  -  45  1  132.4  -  -  -  -  -  -  -  -  3  160.7  -  -  46  1  146.8  -  -  1  153.4  -  -  -  -  1  179.8  -  -  47  -", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7krh-unu7-zq6i", "00000000-0000-0000-2092-0748CAA39503", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Discussion and illustration of dynamometer apparatus for measuring strength", "9918573882206676X35", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" To measure physical strength, the examiners used dynamometers like that shown on these pages. Pages 458-459 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "458  WEIGHT AND STRENGTH.  4. Determinations of Muscular Strength.  The dynamometers employed were devised for measuring the  strength in pulling upward, and are represented in the annexed  figures, which will render detailed verbal description needless.  One of them represents the general aspect of the instrument, and  the other shows the internal arrangement as disclosed by the re-  moval of the dial-plate. The man stands upon the movable lid of  the wooden packing box, to which the apparatus is firmly attached,  and grasps with both hands the rounded extremities of a wooden  bar, of convenient shape and adjustable in height. Although this  apparatus is less compact and portable than the well known dyna-  mometer of Regnier,1 and lacks  the incontestable advantage of  testing the force of pressure as  well as that of traction, yet the  form of construction here em-  ployed seems to avoid the objec-  tions urged2 against that instru-  ment, and to be well fitted for  practical use. The handle is  conveniently shaped for firm and  easy grasp, its height well suited  for application of the full mus-  cular power, and the mechanism  such as to afford results which  are to all appearance very trust-  worthy.  The first two of our instru-  ments were made by Mr. Thom-  as, of New York, under the di-  rection of Messrs. Olmsted and  Elliott; the subsequent ones by Mr. Thomas Morton.  Any comparison of our results with those of the renal [lifting]  force, as determined by others, is unsatisfactory, without a careful  comparison of the structure of the instruments employed and the  manner of their use. Very few sets of such measurements are on  record, and these generally comprise too few individual cases to  afford results at all satisfactory.  Regnier, in the memoir already cited, gives8 as the result of his  1 Journal de I'Hcole Polytechnique, II. 160. 2 Quetelet, Bur P Homme, II. 64, 68, 73.  8 Page 168. WEIGHT AND STRENGTH.  459  experiments, 130 kilograms (287 lbs.) for the weight which a man  of from 25 to 30 years can generally lift with both hands, and says  that this degree of strength continues until about the age of 50  years.  was the first1 to carry a dynamometer as part of the  apparatus of a scientific expedition, and to attempt its employment  for ethnological purposes. Although he evidently took much pains  with his observations, the results proved quite discordant from  those of other observers, until the source of the error was de-  tected 2 by Mr. Freycinet, his companion on the Southern Explor-  ing Expedition, who after  death edited the  second volume of his narra-  tive. The dynamometer had  been provided with two grad-  uated scales, one for showing  the force of pressure, the  other for the force of trac-  tion ; and its indications had  been transcribed from the  wrong scale. This discov-  ery rendered it easy to re-  produce the true values,  which Mr. Freycinet has  given.8  The measures of  thus afford the following  mean results for the lifting,  or renal, force: -  No.  Kilograms  Lbs.  Savage natives of New Holland above 18 years old  13  102  225  Malays of the Island of Timor, from 18 to 20 years  4  96  212  20 to 30  15  118  260  30 to 40  7  119  262  40 to 50  8  106  234  50 to 60  4  109  240  French members of the Expl. Exp., from 20 to 50  17  152  335  English residents of Port Jackson, from 20 to 50  14  163  359  His dynamometer was left4 with the government physician at  Mauritius, Mr. Chapotin, in the hope that extensive observations  might be made upon the strength of men of different races.  1 Voyage, aux ferret Australes, I. 447. 2 Ibid. II. 461.  8 Ibid. II. 463, 464. 4 Ibid. I. 457.", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xnma.sbmm_esdu", "00000000-0000-0000-6E2C-0942E9EB1F95", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Table showing the average strength of soldiers examined", "9918573882206676X36", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" The table displayed the average strength of the soldiers examined, as measured with dynamometers. Page 460 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "460  WEIGHT AND STRENGTH.  Mr. Ransonnet, also a member of the same expedition, and  whose determinations of the renal strength of sailors at Havre led  to the discovery of the error in Boron's records, found the average  lifting power of 345 French sailors to be 142 kilograms, or 313  lbs.1  Quetelet's measures in Brussels, gave 2 the mean values for men  at different ages as follows, the number of individuals in each  group being not less than ten; but he regarded his values as  probably less than the truth.3  The mean lifting strength for the various classes of men exam-  ined during the present investigations is shown in the appended  table.  TABLE XLII.  Average Lifting Strength of Men examined.  Class of Men  In usual Vigor  Not in usual Vigor  Total  No.  Strength  No.  Strength  No.  Strength  White Soldiers, Earlier Series.  5 776  lbs.  314.46  2 082  lbs.  266.25  7 858  lbs.  301.69  \" \" Later Series .  6 381  343.20  1025  280.89  7 406  334.58  Sailors  1 141  307.36  -  -  1 141  307.36  Students  208  308.41  -  -  208  308.41  Full Blacks  1 600  323.51  195  276.15  1 795  318.36  Mulattoes  704  348.90  128  293.69  832  340.41  Indians  503  419.31  5  290.00  508  418.04  1 Voyage aux terres Australes, II. 461; Quetelet, Bur I'Homme, II. 66.  2 Bur I'Homme, II. 70. 8 Ibid. II. 74.", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-a6px-8pdq~vq5e", "00000000-0000-0000-3CEB-F8659D9B3720", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Discussion and illustration of spirometer used to measure lung capacity", "9918573882206676X37", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" These pages showed the spirometer used by examiners to measure lung capacity, also called pulmonary or \"vital\" capacity. Pages 468-469 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts", null, "Data Collecting during the U.S. Civil War, 1861-1865", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "CHAPTER XII.  PULMONARY CAPACITY.  1. Preliminary.  The Spirometers employed are simply dry meters, agreeing in  their general construction with the most approved form of those  used for illuminating gas, and were made for the Sanitary Commis-  sion by the American Meter Company, of Philadelphia. Their  structure and general appearance are shown in the accompanying  figures. Those metallic portions which are exposed to the breath  are of copper, or some alloy which does not corrode by moisture at  ordinary temperatures ; and they are provided with special contri-  vances for removing the vapor as it condenses. They were tested  from time to time, and so far as experience warrants a judgement PULMONARY CAPACITY.  469  they appear far superior to the cumbrous and complicated appa-  ratus hitherto employed for the same purpose. It must not be  forgotten that our aim was not to introduce such apparatus as would  permit the highest degree of precision absolutely, but such as  would, under the circumstances of the case, afford the best re-  sults. For instruments which are to undergo the rough usage  inseparable from transportation by army trains or on military rail-  roads, which are in danger of being handled roughly at some un-  guarded moment by rude men, and which must be employed at  posts remote from facilities for repairing injuries or maladjustments,  the conditions to be consulted are widely different from those which  would be imposed under other circumstances. And although there  are of course many respects in which the experience now obtained  would indicate important modifications of method, inquiries, and  precautions, were this work to be repeated or continued, yet the  instruments employed have given entire satisfaction and very few  points have suggested themselves in which the apparatus could  clearly be changed for the better. The spirometers are graduated  to indicate cubic inches (although cubic centimeters would be  preferable for any future occasions), and are furnished with a  mouthpiece of convenient form, connected with the instrument  by flexible tubing.", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bnn9.qp33.nbek", "00000000-0000-0000-9C9E-AC0D545DF844", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Table showing average lung capacities of soldiers examined", "9918573882206676X38", null, "1869", "1869", "The United States Sanitary Commission (USSC), a civilian organization, sent physicians and others to inspect U.S. Army camps and hospitals during the Civil War, gathering data on camp and hospital conditions, health of the troops, and supplies needed. The USSC also gathered a wide variety of anthropological and sociological data on thousands of Union soldiers, hoping to determine \"important facts relative to the moral and physical characteristics and capacities of our soldiers and of men in general.\" This table showed the average lung capacity of soldiers examined, using the spirometer instrument. Page 471 of Benjamin A. Gould, Investigations in the Military and Anthropological Statistics of American Soldiers. New York: Arno Press, 1979. (Reprint of Vol. 2 of the 1869 Edition of the United States Sanitary Commission's Sanitary Memoirs of the War of the Rebellion, published for the commission by Hurd and Houghton, New York.)", "Monographs, Excerpts, Charts (graphic documents)", null, "Data Collecting during the U.S. Civil War, 1861-1865", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "PULMONARY CAPACITY.  471  TABLE I.  Average Capacity of Lungs.  In usual Vigor  Not in usual Vigor  Total  No.  Men  Cubic  Inches  No.  Men  Cubic  Inches  No.  Men  Cubic  Inches  White Soldiers, Earlier Series .  4 837  175.655  1 915  155.699  6 752  169.995  White Soldiers, Later Series . .  8 895  187.868  1 541  166.321  10 436  184.686  Sailors  1 104  179.217  -  -  1 104  179.217  Students  288  204.382  -  -  288  204.382  Full Blacks  1 631  165.319  221  149.697  1 852  163.455  Mulattoes •. .  671  161.635  138  145.428  809  158.870  Indians  504  185.058  7  179.286  511  184.978  The extreme values recorded for any individual in the several  classes were, in cubic inches: -  In usual Vigor  Not in usual Vigor  Greatest  Smallest  Greatest  Smallest  White Soldiers, Earlier Series .  360  50  353  10  White Soldiers, Later Series. .  358  40  325  36  Sailors  387  50  -  -  Students  312  100  -  -  Full Blacks  360  70  246  55  Mulattoes  359  43  262  33  Indians  310  60  283  110  The great difference of the mean volume found for the black  race from that which seems to belong to the whites, cannot fail  to attract attention at the first glance. Its bearings are perhaps  better manifested by the more detailed tabulations which will fol-  low.  The volume of air expelled from the lungs, as related to the size  and mobility of the thorax, and to the other physical dimensions of  the individuals, has been made the subject of careful and extensive  study by many able men. The present discussion aims only at the  proper presentation and classification of the results, obtained at the  same time as the physical dimensions in our examinations. The  tabulation has been arranged with a view to the acquisition of evi-", "Gould, Benjamin Apthorp, 1824-1896 ; United States Sanitary Commission", null, null, "Arno Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7n3f~tg37~2w38", "00000000-0000-0000-3A34-0FDD1DCE84CA", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Instructions for New York City Sanitary Survey Inspectors", "9918573882206676X39", null, "1865", "1865", "In 1864, the New York Citizens Association, a coalition of civic and sanitary reformers, organized a sanitary survey of New York City. The data collected by 31 medical inspectors were condensed into a report published in 1865, which also included background information on the project, such as this list of instructions for inspectors. Pages xxiv-xxxi of Report of the Council of Hygiene and Public Health of the Citizens Association of New York upon the Sanitary Condition of the City. New York: D. Appleton and Co., 1865.", "Monographs, Excerpts", null, "Public Health Data Collecting, 1860-1900", "8", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Xxiv              REPORT  OF  EXECUTIVE COMMITTEE.   TWENTY-FIRST DISTRICT—Comprising the northern  half of the Twentieth Ward;       bounded north, by Fortieth Street; east, by Sixth Avenue; south, by Thirty-third       Street;  west, by the North River.  Assigned to Dr. James L. Little, 268 West For-       ty-second Street.   TWENTY-SECOND DISTRICT—Comprising the southern half of the Twenty-first Ward;       bounded north, by Thirty-third Street; east, by the East River; south, by Twenty-       sixth Street; west, by the Sixth Avenue.   Assigned to Dr. R. L. Parsons, 263 Fourth       Avenue.   TWENTY-THIRD DISTRICT—Consisting of the Twenty-first Ward, north of Thirty-third       Street;  bounded north, by Fortieth Street; east, by the East River ; south, by Thirty-       third Street; west, by Sixth Avenue.  Assigned to Dr. Ellsworth Eliot, 48 West       Thirty-sixth Street.   TWENTY-FOURTH DISTRICT—Comprising the southern  half of the  Twenty-second       Ward;  bounded north, by Fiftieth Street; east, by Sixth Avenue; south, by Fortieth       Street;  west, by the Hudson River.  Assigned to Dr. Robert  Stewart, 141 West       Forty-fifth Street.  TWENTY-FIFTH DISTRICT—Comprising the northern half of the Twenty-second Ward;       bounded north, by  Eighty-sixth Street; east, by  Sixth  Avenue;  south, by Fiftieth       Street;  west, by the Hudson River.  Assigned to Dr. J. Lewis Smith, 147 West For-       ty-ninth Street,  TWENTY-SIXTH DISTRICT—Consisting of the southern half of the  Nineteenth Ward;       bounded north, by Fifty-ninth Street; east, by the East River; south, by Fortieth       Street; west, by Sixth Avenue.   Assigned to Dr. H. Mortimer  Brush, 1 West For-       ty-sixth  Street.  TWENTY-SEVENTH DISTRICT—Consisting of the northern half of the Nineteenth Ward;      bounded north, by Eighty-sixth Street; east, by the East River;  south, by Fifty-ninth      Street;  we3t, by Sixth  Avenue.   Assigned to Dr.  Alexander Hadden,  118 East      Fifty-first Street.  TWENTY-EIGHTH  DISTRICT—Comprising the western section of the Twelfth Ward;      bounded north, by Harlem River and Spuyten Duyvel Creek; east, by Sixth Avenue;      south, by Eighty-sixth Street; west, by North River.   Assigned  to Dr. L. A.  Roden-       stein, Manhattanville.  TWENTY-NINTH DISTRICT—Comprising the eastern section  of  the Twelfth  Ward;       bounded north,  by Harlem  River; east, by the  East  River; south, by  Eighty-sixth      Street; west, by Sixth  Avenue.  Assigned to Dr. J. 0.  Farrington, 128 EasSt One      Hundred and Twenty-eighth Street, Harlem.            INSTRUCTIONS  TO  INSPECTORS.-^*™*.)                 THE LEADING POINTS OF SANITARY INQUIRY.     \" [The main object of the inquiries that are recorded in the Record-book is to procure  and preserve accurate information concerning the sanitary condition of every portion of the  city.   Each inspector is expected to record his observations daily, &c.  &c.~|     *       *       **         *         *         *        *         »         *  •         ******„,                            \"the inquiry will relate to                                        A.     \" The Block or Square  Visited.—Its boundaries;  nature of  the ground;  drainageREPORT OF  EXECUTIVE  COMMITTEE.                XXV   natural and artificial;  nuisances and any sources of insalubrity; the prevailing character   of the population inhabiting the block; the general condition and uses of the buildings, etc.;                                            B.      \" The Streets,  Courts, and Alleys.—The sewerage; the local drainage; the pavement;   gutters; garbage;  cleanliness, etc.;                                            C.                 \"the inspection op the house or houses  in the block.      \"The frontage, north, south, etc.; stories and height  of each story;  condition and   material of buildings; dimensions of habitations,  and rate of population to area and to air-   space; water supply;  house drainage, whether ample, independent, and in good keeping;   cesspools;  location and character of water  closets;  care  of garbage,  etc.; ventilation,   external and internal; cellars and basements, and cellar population;                                            D.                                \"sickness and mortality.      \" Inquire concerning the prevalence and character of diseases in house and in neighbor-   hood, and state whatever may be ascertained respecting sickness and death-rates.        *         *******        \" On the first page of the Book of Records it is requested that each inspector draw a   map of his district, and on the corners of each square  place figures, indicating the number   of the street; also that a red line be drawn through those streets in which sewers exist.      \" The Records of Inspection begin on the second page.  On the top of the page should   be written distinctly the names of the streets between which  lies  the belt of squares first   to be examined.  Underneath should be written the names of the streets intersecting these   and forming the different squares, as will be seen  in the form of heading which accom-   panies these instructions.   On the margin should be placed numbers corresponding to the   several points to  be reported upon, as contained  in  the printed list.   [See Schedule of   ' Subjects for Inquiry;'   *     *    *    *    *]      \" The Inspection.—This should be pursued in accordance with the suggestions relating   to the scope and leading points of Inquiry, as presented upon the first page of the Record-   book.      \" It is recommended that each Inspector begin bis examination  at one corner of his dis-   trict, and inspect a square at a time, the word square being here used, in a general sense,   for any collection of houses bounded  by three or more streets.  This is considered prefer-   able to following  the  line of any single  street,  and inspecting each side,  because each   square  constitutes  in itself a small sanitary district, and should be considered a distinct   entity.  Having completed the square, take  the next one between the same streets, and  so   on, until tlfe  Inspector has  reached  the  limit of his district, or,  the  termination of the   streets.  Having  completed this belt of squares, let him take the next series of squares   lying parallel to it,  and thu3 proceed until  his district is completed.  But it will be the   duty of the Inspector, whenever he has information of the existence of fever, small-pox,  or   any special source of pestilence within his  district, immediately to make a thorough inspec-  tion of the locality, in accordance with the forms  provided for the  Inspection of  Insalubri- 9  ous Quarters, and without delay to render said report to  the Council of Hygiene; but the  entry of the regular notes of inspection of such localities should not be made in the Book  of Records until such square is reached in its proper place in  the series.   The proper  memoranda of such inspections should be preserved in a separate book or on labelled slips  until required for entry in the Record.     \" When the whole or greater part of a square is composed of tenant-houses,  it will  generally be found that many of them are alike;  and in  such cases, while it will be nece3-XXvi                REPORT  OF  EXECUTIVE  COMMITTEE.  sary to make, at least, a general Inspection of each house, it will be necessary to describe,  in detail, the  condition of only one or two, stating the fact (which is always to be verified  by actual Inspection), that the adjoining houses, for a given  distance, present the same  general features; or, if different, state the essential points of difference.  When a square  or block is composed mainly or entirely of houses occupied by one or two families, the  Inspection can generally be made in a very short time, and the record may be brief.      \" Reports and  Requests.—It is expected that each Inspector will be  present at the  weekly meeting, and that he will present, in writing, a condensed report or summary of the  week's labors. It is also expected that each Inspector will write a full report of his whole  district as soon as its Inspection shall have been completed, such report to contain a  sum-  mary of all the points contained in each special report, and  at  the same time present a  concise and faithful account of the sanitary condition  of the district.      \" Special and  very minute reports should be made of  tenements and localities in which  fevers and other preventable diseases shall be found to prevail, which need not be entered  in full upon the Book of Records, but are to be reported upon separately, and  may be  referred to by note.      \" The extensive prevalence and diffusion of communicable and preventable diseases in  this city render it desirable that each Inspector report such maladies  as frequently and as  fully as practicable.   The Council expects to receive at least four such reports weekly from  each Inspector.      \" It is particularly desirable and necessary that each  Inspector should so lay out his  work, and prosecute his  daily inquiries, as to complete the survey of his  district within the  period specified, and care should be taken not  to expend time needlessly on points  that are  not essential.     \" [For suggestions respecting further details of domiciliary inspection, see the forms for Special  Inspection, Reports of Insalubrious Quarters, and Pestilential Diseases'],     \" [Paper for maps, blank forms, and books for reports and records, and any facilities that the Council  of Hygiene or the Citizens'1 Association  can supply for the furtherance of this work, will be furnished  upon application at their office, S13 Broadway.     \" Any Inspector who requires the presence or aid of the police in the prosecution of his inquiry, will  obtain such aid  by applying to the Superintendent of Metropolitan Police, or to Captain Lord, at the  Police Headquarters.     \"Insalubrious quarters that urgently demand immediate reform, should* be promptly reported with  specific statements, to the Council of Hygiene.  And in the whole course of these inquiries, the Inspector  will bear in mind that the improvement of the health and condition of the people, and the acquisition  of accurate knowledge of prevalent diseases and their causes,  is the main object of this sanitary survey  of the city .J\"                               SYSTEM OF DAILY  RECORDS.                          Eighth Waed.—Broadway to Mercer.    ^                      1st Square.     Canal to  Grand St.  i  ^ 1.  &c.  For this schedule see next page.REPORT  OF  EXECUTIVE  COMMITTEE.              XXVU      Tho inspectors were directed to make this  inspection, as far as possible, a  house-to-house visitation, and so thorough as to leave no cause of unhealthiness  and no existing disease undiscovered and  uninvestigated.  The inquiry was to  proceed systematically, commencing at a given limit of the district and pro-  gressing by contiguous squares until the work was completed.   The following  arrangement of the leading subjects of inquiry was issued for the purposes of  facilitating the inspections and insuring uniformity in all the records and reports.  Inspectors were, however,  directed to develop each head by such subordinate  questions  as they might deem desirable :                         SUBJECTS  FOR SANITARY INQUIRY.       1. Nature of the ground.       2. Drainage and sewerage.       3. Number of houses in the square.       4. Vacant lots and their sanitary condition.       5. Courts and alleys.       6. Rear buildings.       1. Number of tenant-houses.       8. Description of a single tenement [of a family],       9. Description of a single tenant-house.      10. Description of a row of tenements.  These descriptions should state—           a. Condition and material of buildings.           b. Number of stories and their height.           c.  Number of families intended to be accommodated, and space allotted to each.           d. ^Yater supply and house drainage.           e.  Location and character of water-closets.           /. Disposal of garbage and house-slops.           g. Ventilation, external and internal.           h. Cellars and basements, and their population.           i.  Condition of halls and passages.           j.  Frontage on street, court, alley—N.,  E., S. or W.           k. Miscellaneous items.           I.  Statement of sickness and mortality [according to the  printed formula for                insalubrious quarters].      11. Drinking shops, brothels, gambling saloons, etc.      12. Stores and markets.      13. Factories, schools, crowded buildings.      14. Slaughter-houses [describe particularly].      15. Bone and offal nuisances.      16. Stables, etc.      17. Churches and school edifices,      18. Prevailing  character of the population.      19. Prevailing  sickness and mortality.      20. Sources of preventable disease and mortality.      21. Condition of streets and pavements.      22. Miscellaneous information.      [Each of these subjects to be developed by such subordinate questions as the Inspector deemsXXViii            REPORT  OF  EXECUTIVE  COMMITTEE.      desirable.  The special points to which attention is directed under the \\\\0th head, suffi-      ciently illustrates the method of elaborating such questions.']       To each  sanitary inspector a commission of appointment was issued in the  following terms:            The Council of Hygiene and Public Health, reposing special   confidence in your skill and ability to pursue sanitary inquiries and  make   reports thereon, hereby commissions you for such duty, under direction of its   Executive Committee.                  (Signed)          Joseph M. Smith, M. D., President.      Elisha Harris, M. D., Secretary.         The attention of inspectors was especially called to the importance of a   thorough and exhaustive  inquiry into the condition  of insalubrious quarters   and localities where  pestilential diseases were found  to be prevalent, and  re-   specting the diseases  themselves, particularly as relates to fever, infantile mor-   tality, small-pox, etc.  A form of special  reports was accordingly issued to fa-   cilitate this branch of inquiry.  The following is a partial abstract of the sched-   ule of points requiring replies in such reports:          \"REPORT  OF PESTILENTIAL DISEASES AND INSALUBRIOUS QUARTERS.     \" Name of Hie disease reported----------------------.................-----------------.     \" Dales of the visits and inspection........................................-------------..............................._                       \"THE  PLAN OP  INQUIRY AND RECORD.      I. To trace and record the Medical history of the sick person.  *  n. To ascertain and record facts relating to the Family and other persons exposed to the            patients and to the  causes of the malady.    in. To report the Sanitary condition of the Domicile.    IV. To report the Statistics  and Sanitary condition of the Population of that domicile.     V. To report upon the Sanitary condition of the Locality or Neighborhood and its Popu-            lation.    VI. To preserve and make returns of these Records.   VH. To prepare on the spot  the necessary outlines or data for the sketching of a Map or            Descriptive Chart of the Domicile, Block, or Locality.\"REPORT OF EXECUTIVE COMMITTEE.                       \"I.  1. Name,...................................................................                [If there are several patients                 here, insert all the names]........................................  2. Residence, Street and No.....................................................  S. Age and Nativity..........................................................              6. Period of residence in this city.....................................  i. Social condition;  [married ? unmarried ?        General condition of family ? etc.].........................................              b. Occupation.....................................................              c. Grade  of intelligence;  [igno-                rant, educated, depraved, etc.]......................................              d. Income and means of subsist-                ence ...........................................................  Date when this patient or place was first seen     by Inspector...............................................................              b. When  this  case of  sickness                commenced ?....................................................  History of the case or cases of disease..........................................              6. What the principal cause ?........................................              c. How and  when exposed to do. ?.....................................              d. What  relation to  do. had this                patient's  personal  and  domi-                ciliary habits ?...................................................              e. What other prediposing causes                are known ? \"...................................................                        \"II.  Who, and how many persons, have been and    now are exposed to  the immediate causes    of the malady ?...........................................................  How many cases of this malady have already    occurred  in this family,  or among these    persons ?..................................................................  What  has  been  the  constant  sickness-rate    among the persons over  ten  years of age    here during the last six months ? \" *..........................................              [And eight other questions.']                        \"in.  What number of persons in this house ?.........................................              b. Pro rata of  ground area  to                each occupant ? [in sq. ft. ?].......................................              c. Average  cubical space in the                apartments to each person ? \"......................................              [And thirty-six other questions.]       * \"State what proportion or percentage, on an average, upon such testimony as can bo obtained, is  constantly sick: |th, jth, or 20per cent, &c.\"TYY  REPORT  OF EXECUTIVE  COMMITTEE.                    \"IV.  Natural drainage.............................................................  Sewerage...................................................................  Width and peculiarities of Streets...............................................             b. Width  and  peculiarities  of               Courts and Alleys................................................  Special Nuisances in the locality \"..............................................             [And twenty-three other questions.]                            \" APPENDED STATEMENTS.     \" Other Prevalent Diseases in the same Locality.—Particular attention is to be directed to the  extent and causes of any other malady that is found to be especially prevalent in the family, the house,  block, or district that is being canvassed with reference to the disease that is recorded in the preced-  ing pages.  After filling out  the following registry, relating to Infant mortality, make a brief record  of any important facts that may be ascertained respecting the existence of Pulmonary Phthisis, Scrof-  ulous diseases—Marasmus, Ophthalmia, etc.—Erysipelas, Peurperal Fever, Shll-lwths, etc., etc.  Let the range of such inquiries extend to all diseases that are ascertained to be  peculiarly prevalent  and fatal in the domicile or the locality reported.\"      [The foregoing form of Record fills eight pages.]        These special reports enable us to preserve in permanent form the valuable  records they contain, and at the same time  they serve as the basis for summary  tabulations, specimens of which are herewith  submitted for publication in the  General Eeport.  The statistics, distribution, and grouping of such pestilential  diseases  as typhus, small-pox,  and cholera  infantum, have been systematically  studied, and the practical results of this labor will aid very materially in  work-  ing out  lasting benefits to the community.   Yet both the  plans and the result  thus far attained by them mu st be regarded at present as simply the beginning  of a system of Sanitary Inquiry and Sickness Registration that  should be  ex-  tended and sustained in such manner as to  reach all those economical  and  humane results which an enlightened people have a right to expect and demand  from sanitary science.      While making this sanitary survey the Inspectors have constantly endea-  vored to render their labors immediately beneficial to the inhabitants of insalu-  brious quarters by means of personal counsel during the inspection, also by com-  municating information  to the  Metropolitan Police;  the Inspectors  being fur-  nished with the following blank-forms  for the latter purpose:                         Zlt <£iti?ms'  glsjsotiattott of Ntfaj  goife.                                 Office,  No. 813 Broadway, .__________________ 1864.     To the Captain of the Sanitary Company,  Central Department of Metropolitan Police :        Sir :  I have the honor to inform you that  the following complaint is made against  an insalubrious place:  Nature of the Complaint, -.......................-........................................______________    Number  and Street,--------------------------------..___________________   Disease Prevailing or Threatened----...................---------------------------  M. D., Sanitary Inspector for the Council of Hygiene.REPORT  OF  EXECUTIVE  COMMITTEE.  xxxi      On Saturday evening of each week the  physicians thus  engaged convened  for mutual counsel, the comparison of results, and for advice from a committee  of the Council of Hygiene.   Tho following extracts from circulars issued by the  Executive Committee show the nature of the advice given:        \" The Committee advises that the records  and reports  of current work of Sanitary  Inquiry be fully written up at the close of each  week and  each month, so as to insure  such recording while the  facts are yet freshly  in mind.  And  in pursuing a work that  promises to be of such great and lasting importance, it is desirable that each physician  engaged in it should, in  his own field, strive to give the most scientific and thoroughly  practical bearings to his hygienic inquiries and  study.      \" The Committee requests that the summary or review which each  Inspector  is ex-  pected to present, in writing, at the weekly meeting, comprise  a brief statement of the  progress and  the specially interesting points made in the work.   These summary state-  ments  are designed to interest and profit all who  hear them read, and then to guide the  Council in its  own duties from week to week.      \" It is desirable that each Inspector should observe a systematic method of recording in  accordance with the schedule of subjects as numbered, and  that each of the subjects for  inquiry be expanded, according  to the Inspector's views of  utility, at each new point of  observation.   It is especially desired that the rate of crowding in Tenements upon limited  areas be accurately noted, and that such crowded and insalubrious quarters be occasionally  revisited.  ***********      \" What have been the prevailing diseases during the past season, and to what causes  and conditions are they in general attributable ?      \" 1. Specify  particular  localities in which  small-pox, typhus, typhoid,  malarial and  exanthematous  fevers, cholera  infantum,  diphtheria, diarrhceal and  puerperal diseases,  erysipelas, and kindred affections, have  been unusually prevalent, and  give in full and  minutely the local conditions influencing the  rise, progress,  and mortality from these  diseases.      \" 2. What has been the constant sickness-rate in insalubrious quarters ?      \"3. Have  you  found insalubrious quarters  where the constant sickness-rate and  mortality were slight; and if so, how do you explain the fact ?      \" 4. Have  you found  diseases emanating from immigrants ?  If so, specify the class of  diseases, the extent of their prevalence, the circumstances under which they occur; with  examples of their contagiousness.  ***********      \" The Council fully appreciate the fact that  the labors which have been undertaken by  the  gentlemen who are engaged in this  work of Sanitary Inquiry, are inspired by noble  professional and philanthropic zeal and purposes of public utility, and  that these must  be the leading incentives to that thoroughness of research which is to be the most essential  condition of success in the effort to make these  labors contribute to the public weuare and  to the  progress and usefulness of sanitary science.\"        The work of sanitary inspection was  prosecuted uninterruptedly until the  month of December (1864), and since that time the Inspectors have carried for-  ward  specific  inspections relating to small-pox, typhus, and the sickness and  death-rates in  particular localities.   This class of inspections is still in progress,  and the results are most instructive  and practically important.", "Citizens' Association of New York. Council of Hygiene and Public Health", null, null, "D. Appleton and Company", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bute_g864.zkz8", "00000000-0000-0000-4E1A-2F63E1C85DED", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Report of the Third Sanitary Inspection District", "9918573882206676X40", null, "1865", "1865", "In 1864, the New York Citizens Association, a coalition of civic and sanitary reformers, organized a sanitary survey of New York City. The data collected by 31 medical inspectors were condensed into a report published in 1865, including this account of the third district inspection. Page 19-32 of Report of the Council of Hygiene and Public Health of the Citizens Association of New York upon the Sanitary Condition of the City. New York: D. Appleton and Co., 1865.", "Monographs, Excerpts", null, "Public Health Data Collecting, 1860-1900", "14", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "REPORT                                OF TEE    THIRD  SANITARY  INSPECTION  DISTRICT.   [Section A.]  HAMPTON  HARRIOT,  M. D.,            Sanitary Inspector.      Boundaries.—North  by Canal  Street,  east by Broadway, south by   Reade Street, west by the North River.  This District (Third, Section A)   comprises the whole of the Fifth Ward.        Topography.—For completeness and convenience,  the  topography   of the entire area of the Third District (Sections A and B) wUl  be de-   scribed under one head.      The topography of this district is somewhat peculiar, consisting of a   depressed section of ground, originaUy  swamp land, bordered  by high,   sandy hills.  The original  formation of  the surface was easUy  ascer-   tained, several maps of that section of the  island as originaUy constituted   having been preserved and republished in various works.   The accompa-   nying map shows the original formation of the surface  of the district,   and the ground reclaimed from the river, with the relative position of the   present streets to the same.  The rocky formations, of which the island   is principaUy composed, are here  depressed  to  a depth of from forty to   eighty feet below the surface, thus  forming a basin, which, where  not   occupied  by high sand-hUls,  formed deep depressions, fiUed with more or   less soft  quagmire.   These swamps were  indeed so unsubstantial  in   places as to yield under the weight of the dirt  dumped in to make firm  ground for building purposes ; necessitating, in some places, the makin^  of newT ground to the depth of forty feet.   This  wras ascertained to be the  depth  of  made ground before reaching the  remains of the  original deposit  of mud, at the corner of Wooster and Grand Streets, where a weU was  sunk  some years ago, an  additional  depth of thirty feet  of the original  deposits being passed through before the rock was reached.20            REPORT  OF THE THIRD  SANITARY  DISTRICT.  SANITARY  AND TOFOGRAPHICAL  MAP  OF  THE THIRD INSPECTION                                DISTRICT.                   THE FIFTH, AND A PART OF THE EIGHTH WARD.  Sandy   gravelly                         EXPLANATION  OP FIGURES, ETC.     The numbers at the intersections of the streets indicate the present elevation at those  points above tide level.       The wavy line in streets and sections of streets indicates sewers into which the high-  tide water flows.       The dotted contour line-------indicates the line of demarcation between the section  that is dramed by sewers through which tide-water flows, and that section of the district  in which the bottoms of the sewers are above tide-level.     The crests of the highest of the original  hills are shown upon the right-hand margin  of the MaD.  076OLD  SWAMPS.—PRESENT DRAINAGE.  21     On the map the part unshaded represents the ground reclaimed from the  river, being the greater part of the  district outside of Greenwich Street.  The  surface represented in the marshy shading shows the  extent  of  swampy ground, formerly called the Lispenard Meadows.  This  ground  was very soft and boggy in its nature, being overflowed by water  at high  tides, and untU filled in it was almost impassable.  The amount of mate-  rial required to fiU in this swamp, and the enormous expense attending it,  induced the authorities of that day to make the grade as low as possible,  consistent with mere surface drainage.  Portions of it have  since been  filled in and other parts  wiU probably be soon  raised in grade, thereby im-  proving the drainage ;  but yet leaving a very large surface of low and  badly-drained ground, especially near and north of Canal Street.     This  swampy land  was intersected by a sluggish  stream of water,  running from the pond, called the CoUect,  situated in the present neigh-  borhood of Centre and Leonard Streets, and covering several squares of  ground.  This outlet foUowed nearly the line of Canal Street from Broad-  way, where it was crossed by a stone bridge, to the Hudson River, empty-  ing therein.  A branch of this stream on  the line of  West Broadway,  drained the meadows in that direction.     The shaded portion of the map represents the solid ground, which  was  formerly somewhat irregular and picturesque in appearance, being  high and hUly, rising on Broadway and Leonard Street to a height of  near one hundred feet above the river.  These high grounds  have  since  been much lowered by grading down  for buUding purposes ; and the sur-  plus dirt was used for filling in the swamp lands and the section reclaimed  from the river.   Part of one of the original hills can  yet be seen in the  grounds of the New York Hospital, which have not yet been reduced to  the street grade.  The hUl that existed in the  neighborhood of Broadway  and  Broome Street was also so high as to be cut down some forty or fifty  feet, being below the bottom of some of the weUs in the vicinity.  The  tongue of land between the Lispenard Meadows and the Hudson River  was  of less elevation, but was also more or less disturbed by the necessary  grading of that part of the city.     Drainage and Sewerage.—The original sewerage of this  district  had  a direct  relation to the natural formations of the  surface.   As the  meadows were fiUed in the original streams  were carried through open  box  sewers, buUt of timber, foUowing the lines of  Canal Street and West  Broadway.   In the course of time, and from the growth of the city around  them, these sewers  became so offensive and dangerous that it became ne-  cessary to  enclose them.   This was done by  building very large  sewers,  with comparatively flat bottoms, which remain to this day a continual22           REPORT OF THE THIRD SANITARY DISTRICT.  source of expense and nuisance.  The bottoms of these sewers were laid  with flat stones sloping to the centre ; the sides were built of brick, perpen-  dicular in direction, and some four feet in height; and from these side walls  a low arch was thrown enclosing the sewer ; the whole being covered with  the street and pavement.   Owing to this plan of building, the bottoms of  these  sewers are badly broken  and cannot be thoroughly cleaned.  AU  the other  sewers of the district  are buUt of brick or tile, being oval in  shape and  weU adapted  to their purpose ;  some of the smaUer branch  sewers being of earthen ware, moulded and  burnt in  sections.   The dis-  trict is pretty weU sewered, and the streets  needing it are being rapidly  supplied.      Street  Elevations.—On the  map I have  given  in numbers the  elevation, in feet, of each street  intersection above high-water mark, and  have  also indicated by wavy lines such  streets in which the sewers, if  built, would be  entered by the tide  at high water.  This has, from the  nature of the case, been arrived at by estimating the depth  of the bottom of  the sewer from the level of the surface of the street.   Officers of the Croton  Aqueduct Department, to wliom I am indebted for much of the  informa-  tion contained in this portion of my report, inform me that  my calcula-  tions are nearly correct.  They informed me that in all cases where pos-  sible the bottom of each sewer is laid at a depth of thirteen feet below the  level of the  street.  To this there are two exceptions: First. To drain a  basin lying behind a ridge of land, it sometimes becomes necessary to  build  a sewer at a greater depth  through the intervening  ridge.  Second.  On approaching the rivers, and where the ground lies too low,  as in a large  part of the Third District, the sewer  cannot be built at so great a depth.  I have taken the estimate of thirteen feet as the proper depth of the bot-  tom of the  sewers, and have represented all the sewers in the  streets hav-  ing a less elevation  above high water-mark, as being reached  by the river  water at high tide.  When the river is above this level, it is well known  that several of these sewers are entirely fuU of water, and unable to carry  off the surface water in case of heavy rains ; and consequently the  streets  and ceUars in such localities  are frequently overflowed.   On several of  these  streets it has for the same reason been necessary to place traps, &c,  on the sewer connections to prevent the flow of water back into the houses  or ceUars.  By referring to the  map it will be seen that tide-water runs  up the sewer in Canal Street as far as Broadway, spreading north through  Thompson and Laurens Streets as far as Spring Street, and  through the  other streets to a somewh^ less extent;  also running south through West  Broadway as far as Thomas Street, there meeting the tide coming directly  from the river.   Adding  to these facts  the flow of tide-water  into theDRAINAGE.--INFLUENCES ON HEALTH.  23  branch sewers, and  in  all the sewers within two to four squares of the  river, it wUl be seen that two-thirds of the sewer surface of this district is  reached by salt water.   AU the sewers of the Third District empty into  the slips between the wharfs along the North River, with the single ex-  ception of the Canal Street sewer and its connections, which empty at the  head  of the long  pier at the foot  of Canal Street, and  in deep water.  This  system of emptying the sewage  into the slips is a  prolific source  of offence to the senses and of disease to the district, and  also keeps the  city at continual and great expense for the necessary dredging to keep them  navigable and prevent their filhng up.   The Croton Aqueduct Department,  however, is desirous of correcting this, by building sewers along the river  front, to cut off the  present sewers, and carry their discharge to  certain  points, as at the Battery, &c, where their contents can be at once thrown  into free currents of deep water, thus avoiding these difficulties.   This sys-  tem regards the  sewage as  of no value, but the time will probably soon  arrive when some plan wUl be  desirable to save it and make it valuable.      Influence of Topographical  Characteristics of this District on Health.—  The influence of the peculiar formation of this  district upon the pubhc  health has been marked.  Those portions reclaimed from the  swamp and  river being undesirable for a good  class of population  have  been conse-  quently buUt upon by an inferior class of buUdings, and occupied by people  too ignorant or indifferent as to consequences to seek to avoid them.  The  observations  of physicians and sanitary  officers, however, have shown  that such sections  of the city are, in case of epidemic diseases, always the  first to be attacked, and that they invariably present the  greater number  and more severe class of cases.  During the various epidemics of fevers,  cholera,  influenza, &c,  that have  visited  this city,  these sections  have  furnished a very large proportion of the  patients, and  a stUl larger pro-  portion of the fatal cases.  During the recent and  present prevalence of  typhus and typhoid fevers, I have noticed that the number and severity of  cases have borne a direct relation to the nature of the ground upon which  they occurred.   Our hygrometers, which have been in constant use in the  worst  sections of this district, indicate more  than twice  the amount of  moisture in the atmosphere given by the standard instrument kept at the  Eastern  Dispensary.  Such diseases  as  cholera infantum,  fevers, &c,  especiaUy of chUdren, always prevaU most extensively in such moist local-  ities, not to speak  of rheumatism, scrofula, and pulmonary diseases, which  are generaUy  recognized as  being  speciaUy dependent upon exposure to  cold and wet.      Streets.—The streets of the Third District are laid out, more or less,  at right angles with each other—running north and south, or east and                                                                      a24           REPORT OF  THE THIRD  SANITARY DISTRICT.    west.  They vary in width from forty to eighty feet, except Canal Street,  which is much wider.  The greater number of the streets are paved with  cobble stones ; and, at the time of this survey, were found in pretty good  repair, except West Street and its immediate neighborhood, which is con-  siderably lower than the proper grade, and is  in very bad order.  Those  sections of the different streets, however, where there  are tenant-houses,  are always wet and filthy from garbage and slops that are thrown into the  street.   Broadway, Washington, Greenwich, and Worth Streets, in their  fuU length, in this  district, have been repaved with the trap-block pave-  ment ; and portions of Desbrosses, Reade, Duane, North Moore, and Jay  Streets, have been similarly paved.  It is to be hoped that this kind of  pavement will, as soon as possible, replace the cobble-stone pavement, as  it has proved to be not only by far the most durable, but it is much more  easily kept clean, and does not accumulate in the interstices such a quan-  tity of the foul matters of the streets.  The  connection  of civic cleanli-  ness with  civic health is too evident to be questioned at this day.  Under  my own observation in this district,* the widening and repaving of various  streets have been foUowed by the most marked improvement in the health  of not only those streets, but of the whole immediate  neighborhood.  It  is true that the population of this section was  greatly diminished by these  improvements, but those that remained have not shown half the propor-  tional amount  of sickness,  although of a class more  subject  to disease  than the original  residents.      Squares.—The number of squares in the Third District is 74.  Of  these 32  are in  a  good,  25 in a mixed, and 17 in a bad condition, as  regards sanitary considerations.   I report as bad those squares in which,  from the character of the  soil, buildings, and population, more than the  average amount of disease prevaUs.  Those squares are regarded as mixed  in which the same causes prevail to a partial extent, or where, from the en-  croachments of business property, or from other causes, the population is  mostly removed.  All others are regarded as being in a good sanitary state.      Inhabitants.—The population of the Third District has undergone,  and is now undergoing, great and rapid changes.  OriginaUy settled by a  native-born population, the  greater numbers of residents are now of for-  eign birth, or children of foreign  parentage.   The colored population,  formerly  so numerous in  the  Fifth Ward,  has almost  disappeared,  a few scattered tenant-buildings—not  more than twenty-five in number—  containing aU that are left.  The large number of houses of prostitution  west of Broadway, for which this district was, not long ago, so notorious,     * The Inspector has been sixteen years connected with the New York Dispensary, and  daily engaged in professional duty in this part  of the city.—Editor.  ITHE INHABITANTS.—TENANT-HOUSES.  25  is also rapidly disappearing from this  section of the city, their former  inhabitants being  replaced by foreigners, and they, again, being  soon  crowded out by the encroachments of mercantUe business.     Tenant-Houses.—Nearly aU the houses remaining in the district have  been  altered  for tenant-houses, or are now occupied as boarding-houses,  or for business purposes.   Large tenant-houses, which accommodate more  than six or eight famihes each, are comparatively scarce, there only being  about forty of such character.  All others reported as tenant-houses, are  smaU in size, the  greater number being altered from private residences,  or occupied by several  famihes, without alteration.  Most of the large  tenant-houses are located in the neighborhood of West Broadway, from  Leonard to Thomas Streets, or near Greenwich Street; the others being  scattered throughout the district.     I have reported 450 houses as being tenant-houses ; being such houses  as contained three or more  families.  With the  exceptions noted, these  were formerly occupied as private residences, or by two or three famihes ;  but, with the  changes of population, the greater number of them  have  been  fiUed with  as many families as  there  are rooms to  accommodate  them, many of the famihes having no  other  sleeping-apartment than that  occupied as the kitchen and living-room.   In some of these houses the  Croton-water and waste-pipes have been introduced, but in* most of them  the Croton-water is introduced only to  the court-yard, or area.   In other  respects these houses remain as built long ago for private families.   As a  consequence, ventUation is very imperfect, from the doors between rooms  being always  closed; water-closets are too few  in number, and filthy,  from being used by so many persons ;  and the  houses become  more and  more dilapidated, untU many of them,  in the neighborhoods where busi-  ness  is encroaching, are in part or entirely unfit for human habitation.     Number and  Classification of the  Houses.—Including the 450  noted as tenant-houses, there are 1,244 dweUing-houses in the district,  95  of them being rear buUdings.  The larger number of those not noted as  tenant-houses are occupied for boarding-houses, there being comparatively  few which can properly be  reported as private  houses.   Many of the  objections relating to tenant-houses also apply to the boarding-houses,  except that the latter are  kept  in a more cleanly condition.   Still, they  are necessarily overcrowded and deficient in most of the accommodations  needed for a perfect state of health in the occupants.     There are also many buildings occupied for stores,  offices, &c, in  which a famUy is living who take charge of the buUding, keep the passages  clean, &c, which  I have not reported as dwelling-houses.  There are  also  several buUdings occupied by fire companies, in which some of the26           REPORT OF THE THIRD SANITARY DISTRICT.  members always lodge, which are not  included as dwelhngs.  The actual  number of the population of the district I made  no effort to ascertain, as  the time allowed wras too limited, and  I had  none of the necessary facili-  ties for  such investigation.   The figures  given in the last United States  Census  would be very incorrect, as the changes produced  since that was  taken, by the encroachments  of business property, &c, have been  very  rapid and  extensive.   There is no doubt,  however, that the average num-  ber of  inhabitants to each house would  be high, as would be expected  from my statements in regard to the character of the population.  From  the same reasons, no investigation has been  made in regard to the num-  ber of people occupying basements and ceUars—a  subject  needing inves-  tigation, and which would repay fuU and  careful  inquiry.      Liquor Stores.—There are 341 places in the Third District in which  liquor is retaUed, including  groceries, restaurants, and hotels and dram-  shops.  This is a very large  number in proportion to the  population,  being one  to less  than four  (1 to 3'65) dwellings.  However, it must be  remembered that, owing to the situation of the district, large numbers of  their patrons are from  outside the district.  Thus a great many of the  liquor-saloons are located along or near the river, and are  almost entirely  supported  by boatmen and persons employed along the docks, &c.; many  other saloons are  located on the hne of Broadway, Canal  Street, and  other thoroughfares, which derive a large  support from the  transient popu-  lation and persons employed in the stores in the neighborhood.      Brothels.—I report 81  as the number of buildings occupied exclu-  sively as houses of prostitution in this  district.  This number is probably  below the  truth, from the difficulty of  ascertaining, with the means at my  disposal, which houses were  thus occupied.   I have  also  not included in  the number a large number of houses occupied only in part for such pur-  poses.   It is a well-known fact that in many of the tenant-houses of this  district  such persons  occupy suites of  apartments interspersed with those  of the respectable laboring-classes, and frequently difficult to be distin-  guished  from them, except upon a more searching investigation than this  survey could give.   During the  last few years, a large proportion of the  more notorious brothels  have been removed from this district to the upper  parts of the city, the houses being now occupied by the laboring-classes,  or for business purposes.      Stores and Commercial Warehouses.—There are  283  buildings  in this district occupied  exclusively for commercial purposes.   There are  also 294 smaU stores, of various kinds, occupying parts of buUdings.   Of  these 29 are meat and vegetable markets  exclusively, there being no  large  market-buUding in the district; although Clinton Market, at the foot of  %COMMERCE.—NUISANCES,  ETC.                  27   Canal Street, is just north of this southern section (A) of the Third Dis-   trict, and Washington  Market, at the foot of Fulton Street, is not far   removed, both of them being largely patronized by the  residents  of this   district.    There are also 87 groceries, of Avhich nearly aU sell liquor, and   many deal in meat and vegetables.      Factories.—There are 151 manufactories and workshops of various   kinds in  the district, many of them occupying only parts of buildings,   used otherwise for trade or residences.      Of these  6 are sugar-refineries, &c.;  11 boiler and machine shops ; 26   carpenters, joiners, and box manufactories ; 2 large coffee and spice mUls ;   2  distilleries; 1 brewery;   3 oU and lard works; 12 blacksmith and   wheelwright shops ; 2 tobacco and snuff manufactories ; 5 smoking and   provision establishments ; 14 artificial flower, feather, and leaf manufac-   tories ;  and  the  remaining  67  are manufactories of various kinds, too   numerous to be particularized.      Stables.—There are 108 stables, or ranges of single-stall stables, in   this district.   Of these 68 contain each  less than 5 horses, or 202 horses   in the aggregate ; and 40 contain each  5  or more horses, or 383 in the   aggregate.  These stables are scattered pretty generaUy over the district,   there being few squares without one or more of them.  There are certain   neighborhoods, however, which contain  a large number upon a  smaU   area.  Such  is the neighborhood  of Greeawich Street from Desbrosses   to Vestry Streets, where 3 squares contain 16 ranges of stables, accom-   modating 117 horses.   Also, in  the neighborhood  of'West Broadway   from Beach to Worth Streets, wThere  6  squares contain  19 stables, with   163 horses.   Most of the larger stables are kept in a quite cleanly and   comfortable condition, but the greater number of small stables are crowded   together, and their surroundings are frequently neglected and uncleanly.   In regard to  the influence of stables upon the pubhc health, I think that   I have, in many  cases, traced to their influence (especiaUy when occupied   by sick and disabled horses) an earlier invasion and increased prevalence   of such diseases  as scarlatina and diphtheria.   However, further investi-   gation and much more extended  observation over a  larger field, is neces-   sary to decisively settle this point.      Nuisances.—There  are no  slaughter  houses, gas manufactories, or  bone and fat-boiling estabhshments in or near the immediate neighborhood  of this district, in  any manner  or degree influencing its health.   Such  causes as do produce such  influence are connected  with the topography,   character of  the population,  and condition  of  the piers, slips, and sewers,  which we have already noted.      Public Buildings.—Of public buUdings  there  are  but few.  There   is  one Police Station-house on  Leonard Street, badly arranged for the28  REPORT OF  THE THIRD SANITARY DISTRICT.  health of its occupants, both as regards its own internal  arrangements  and its immediate surroundings.   There is a large hospital with  several  buildings, known as the New York Hospital, on the  square bounded by  Broadway, Worth,  Church, and  Duane Streets.  These buildings are  finely situated, both as  regards the location of the ground and their in-  ternal arrangements for the comfort and health of the inmates.   A de-  tailed report is  unnecessary in  this place. There  are only two church  edifices in this district; both front on St. John's Park, and both are well  located for light and ventUation.   There are  also  two Mission Churches  occupying lofts of buildings otherwise used for  business purposes ; and a  floating chapel for seamen and others  at the foot of Laight Street.   As  regards School BuUdings, there is one large Ward School on North Moore  and Varick Streets, with an extension running through to West Broadway.  This  school is of the first class, accommodating nearly two thousand  pupUs.  There are two Public Primary Schools, one occupying a building  erected for that  purpose on Greenwich Street between  Desbrosses and  Watts Streets ; the other occupying a building altered from a private resi-  dence in Varick Street near Canal. Both these buildings are badly arranged  for ventilation and light, having windows, &c, only on  front and rear.  There is  one Primary School for colored children on  Franklin Street, be-  tween West Broadway and Hudson Street, occupying a smaU two-story  buUding erected for a dwelling-house, and necessarUy badly arranged and  ventilated for  such a purpose.   St. John's Episcopal Church has also  connected with it a Parish School, occupying a buUding in the rear of the  church.  Two Mission and Industrial Schools occupying lofts of stores,  badly arranged but occupied only on certain days, complete the hst of free  schools in the district.   There are 19 Hotels in the district, varying in  size  and  capable of accommodating from 25 to 250 lodgers each at one  time.  Many of these being altered froth private houses are badly ven-  tilated and miserably arranged for hotel purposes.     Parks.—The Third District cannot be said to  have any public parks.  St. John's Park, bounded by Hudson, Laight, Varick, and Beach  Streets,  is a private square not belonging to the city, and kept closed to all except  the occupants of the surrounding houses, &c.  This  park,  however, in  connection with  the grounds  surrounding  St. John's Church, and those  belonging to the New York Hospital,  are  kept in the cleanest and best  manner, and are valuable breathing spots for this section of the city.      Piers  and   Slips.—The  piers,  wharves, &c, fronting the  Third  District, are generally in a dilapidated and neglected condition, though  a  few occupied by steamship  hnes are kept  in very good  order, being en-  closed with sheds and gateways.  AU the piers, however, being  buUt of            FAULTY WHARVES.—FEVER.—SMALL-POX.             29    wood, necessarUy furnish  a large amount of decaying  vegetable matter,  which  is known to be a prolific source of disease.  The slips, in  conse-  quence of receiving the sewage of the district and surrounding parts of  the city,  are generaUy foul, and the undoubted source  of much sickness.  I have seen a number of  cases of severe disease, such as fevers, conges-  tions of the brain, liver, &c, induced by bathing or getting  overboard in  the shps  of this district.      Prevailing Diseases.—The prevailing diseases of the Third Dis-  trict during the past year  have been  as foUows : Small-pox has prevailed  more extensively than for many years back.  One centre of this disease was  in the  neighborhood of West  Broadway, Thomas, and Leonard Streets ;  and another centre at Greenwich, Watts, and Desbrosses Streets; with  scattering cases over the whole western part of the district.  Typhus and  typhoid  fevers have been prevalent over the whole  district, but more  particularly in the neighborhood of the lower half of West  BroadwTay,  and between Greenwich Street and  the  Hudson River.   The remitting  fever of chUdren  has been as usual almost  universal  in the tenant-  houses of the  district, more  especiaUy during the  Spring and FaU.  Diarrhoeal diseases, including cholera morbus, &c, commenced early in  the season in the immediate  neighborhood of the Hudson River,  gradu-  aUy spreading  thence over the whole district.   These diseases affected  nearly every house and famUy coming under my observation  as Visiting  Physician for the New York Dispensary.  This curious fact of this class  of disease,  commencing  on the  western border of the  city  and  thence  graduaUy spreading toward the  eastern side, has been noticed in other  parts of the city.   Scarlatina, erysipelas, and kindred diseases, were  prevalent early in the season, but disappeared as warm weather advanced.  These diseases were  more or less prevalent in tenant-houses in propor-  tion to the crowding of their population, cleanliness, &c.   The amount of  sickness in  different houses of the same neighborhood is very various, in  some cases  being not more than  twenty per cent., and in others reaching  as high as two hundred per cent, per annum.  It is difficult to account for  this on a superficial  examination, but the character, occupation, temper-  ance, cleanliness, &c, of the inhabitants  of neighboring  houses vary  greatly,  and are a pretty close indication of the amount and kind of pre-  vailing sickness to  be found in any particular house.  Different  houses  vary greatly also as to their faculties for ventilation ;  the number of in-  habitants in basements and on first floors ; the location and care of water-  closets, sinks,  cesspools,  &c.;  their  connection  with  smoking  and pro-  vision estabhshments, and other  kinds of business locations ; the width,  cleanliness,  and kind of pavement in the streets; the proximity of the30  REPORT OF  THE THIRD SANITARY DISTRICT.  river;  the character of material and state of repair of the building itself  and its accessories ; the arrangement of the rooms, bedrooms, and halls ;  and various other causes  of interference with health.   A thorough inves-  tigation into aU such particulars would be necessary to arrive at any just  or valuable conclusions in regard to the causes of disease and death.  A  large number of such searching investigations have been made and are  now making under the direction of  the CouncU  of Hygien", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-37aa~kqam~yjwr", "00000000-0000-0000-0A86-D616878E0116", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Report of the Fourth Sanitary Inspection District", "9918573882206676X41", null, "1865", "1865", "In 1864, the New York Citizens Association, a coalition of civic and sanitary reformers, organized a sanitary survey of New York City. The data collected by 31 medical inspectors were condensed into a report published in 1865, including this account of the fourth district inspection. Pages 43-65 of Report of the Council of Hygiene and Public Health of the Citizens Association of New York upon the Sanitary Condition of the City. New York: D. Appleton and Co., 1865.", "Monographs, Excerpts", null, "Public Health Data Collecting, 1860-1900", "24", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "i   *~>i   I. Itemjafa.   r FtTJttififs              v whm Ty/j/i/ts.orTypfo'u/ /hrr/t,/sf-n-f//7rdt//frifit///it/HfSf)hX    ■\"  <^w/irrr S'maff Pro-Jiastm/rirt/ timing/firpast hrtr    °' % KabnBwrttiuq Ihust    -' Gnxmrs.   ^ItyttvSttinv, or/Jmtir»t/P/t/trs   — MmtSfMrs.    Ffyamm Sinrff.ftmitHjK\\\\ ttit/mttf Ktemi/m aMfefftt/h Hti/erJfart-                     .  vxvy  »\"l!4'\"-i-;#/                  C IT Y <dj' j, N E W-Y O R K                ~\"  ./(' li'h'\"\"v \" lt',f>iirl    s—       fo'\"                    °f//    „___^  \\\\          •^  -Made lo Hit- ( iiiiuril <ir lly.ju         ■•Utfe^LKiTiSJ A^> ■SMISjXXJ. IAxi               —    -\"\"      \\\\\\\\x           .—  :-               J   U  I'l'LUSti. M. 1).^                         Assisted by                      f J RANDALL.                 EEPOET                           OF THE    FOURTH  SANITARY INSPECTION DISTRICT.  EZRA R.  PULLING, M.D.,         Sanitary Inspector.      Boundarees.—The Fourth District, comprising the  Fourth Ward, is   bounded by Chatham, Catharine, and South Street, Peck Slip, Ferry, and   Spruce Streets.  Its average length and breadth are respectively about 1,900   and 1,600 feet.        Topography.—Deducting the surface  occupied by streets,  &c, a   superficies remains of about 2,240,000 square feet, or about 8,227 square   rods, equal  to 896 building lots  25x100  feet.  The soil is sandy and   porous.   About one-fifth of the entire area is artificial, having  been   filled in at a remote period.  It includes a depressed space near its west-   ern border, formerly known as Beekman's  Swamp, which contains about   100,000 square feet, and  still retains its paludal designation among the   leather dealers by whose places of business it is now chiefly occupied.      The northeast and the northwest corners are the most elevated points,   each being about thirty-six feet above high-water mark.  From the former,   the ground slopes rapidly south and west.  From the latter, the slope is by   a somewhat abrupt declivity, south and east.  The average elevation of the   district above high-water mark is about sixteen feet.  Its natural drain-   age is good, as the ground, except  in the vicinity of the river, is generally   sloping, the  declivity  being  steepest in those streets which run from   Chatham to South.      The following named streets and parts of streets  have no sewers with-  in the boundaries of this ward: East Broadway, Henry, Hague, Chest-  nut, New Chambers, South,  Front, \"Water, Cherry from Catharine  to  Roosevelt, Pearl from \"William to Bowery, Madison from Pearl to Roose-  velt, Oliver  from Chatham to Madison, Frankfort from Cliff to Bowery.4:4           REPORT OF THE FOURTH SANITARY DISTRICT.  All the  sewers empty into the East River below high-water mark;  for  about  one-half their  entire length they  are  swept out by the refluent  tide.*      Of the 714 buildings classed as tenant-houses, less than one-half'were  found to  have a  waste-pipe  or  drain connected directly with the  sewer.  \"Where  this is wanting, liquid refuse is emptied on the sidewalk or into  the street, or in some instances into sinks in the domiciles communicating  with a common pipe which discharges its  contents into the open gutter to  run perhaps hundreds of feet, giving forth the most noisome exhalations,  and uniting its fetid streams with numerous others from similar sources,  before reaching its subterranean destination.      Slops  from  rear buildings of such premises are usually emptied into  a  shallow gutter  cut  in  the  flagging  and extending from  the yard, or  space between  front  and rear buildings, to the street.  This is often  clogged up by semi-fluid filth, so that the alley and those  parts  of  the  yard  through which  it runs are  not unfrequently overflown and sub-  merged to the depth of several inches.      There are more than four hundred families in this district whose homes  can only be reached by wading through a disgusting deposit of filthy  refuse.  In  some  instances, a  staging  of  plank, elevated a  few inches  above  the  surface, is constructed through the alleys.   This affords to  the residents  the advantage of a dry walk, but in a sanitary point of view its influence  is  scarcely favorable, since it prevents the removal of the offensive mat-  ters beneath.      I cannot report favorably on the condition of the thoroughfares in my  district.   Belgian  pavement has  been laid in  Chatham, New Bowery,  New Chambers, and South Streets, and in part of Pearl Street.  All the  other  streets have the cobble-stone pavement,  which in most instances is  in bad condition, and in all is very difficult to keep clean.  As a sanitary  measure alone, an improved pavement is  greatly needed  in all these  densely-populated  streets.   It is hardly necessary to add that a thorough  cleansing  is equally required.      I have indicated on the large map which accompanies this report, the  localities in which accumulations of street-filth were observed at the time  of inspection.   From this it appears that less than  one-third of  the  entire street surface was  in a tolerably cleanly condition,  while  of  the  cobble-stone pavement less  than one-sixth part was clean.   Much  of  the  latter, in fact, never is clean, for the  filth lodging in the interstices of its       * The original water-line is copied by permission  from a new topographical  map by  Gen. E. L. Viele, whose courtesy I take this opportunity to acknowledge.TENANT  AND CROWDED HOUSES.  45  surface is never wholly removed.  Besides, it is often depressed or worn  into deep ruts, which furnish receptacles for both solid and fluid matters  of the most offensive character.      I think there is no  more efficient means of cleansing  such streets  than by directing a stream of water over successive portions  of their sur-  face, thus washing the filthy deposit into the gutters and thence into the  sewers.      Courts and alleys are numerous, the latter generally narrow, averag-  ing  less than three feet in width, where they form conduits for the drain-  age from rear houses ; they are uniformly in bad condition.      The entire  number of buildings in this district is 1,507.             Classified according to the purposes for which they are used:    770  are occupied chiefly or wholly as residences.  * 682       \"        \"       \"      for business purposes.      8  are Churches and Schools.     47 are Stables.         Classified according to the material of which they are constructed:  1,394 are built of brick, stone, or iron.    113      »      wood.                        Classified according to position:  1,342 front on  the street.    165 are rear houses.         Of the  165 rear buildings,    108 are Tenant-houses.     20 are occupied for business purposes.     37 are Stables.        Tenant and Crowded Houses.—Under this head I have included:  1st.  All tenant-houses built as such;  and, 2d. All  those used chiefly or  wholly as residences in which  the occupied space gives a pro rata of less  than 800 cubic feet to each inhabitant, without reference to  the  number  of families or the population, or to the original  design or construction of  the buildings.  The total of these is  714, of which 656 are brick and  58 are wood.      Of these 242 were built as tenant-houses and divided into domiciles.      Of these 472 were originally designed for occupancy by a single fam-  ily or for business purposes.  * Many of these have one or more famihes residing in them.46          REPORT  OF THE FOURTH SANITARY DISTRICT.     The first class  is sub-divided as follows in accordance with the num-  ber of domiciles:                  Of  those having 2 domiciles there are 8                                                    16                                                    33                                                    14                                                    44                                                    15                                                    28                                                    11                                                    25                                                     3                                                    10                                                     2                                                     4                                                     1                                                    12                                                     1                                                     2                                                     5                                                     6                                                     1                                                     1       The whole number of domiciles in houses of this class is 2,119.     The average number  of domiciles  in each house of  this  class is  about 8|.     Both classes together are subdivided in accordance with the number  of families occupying each house at the time of inspection:  t( 3  tt 4  tt 5  tt 6  tt 7  u 8  tt 9  IC 10  u 11  tt 12  tt 13  tt 14  tt 15  tt 16  tt 17  tt 18  tt 20  20 to 50   50 to 100   over 100  those occupied \tby 1 family there were 24  tt tt\t2 « « 66  tt tt 3 \" «\t75  tt tt\t4 « tt 69  tc tt\t5 \" « 49  tt tt 6 \" « 80  tt tt\t7 tt tt 42  tt tt 8 » «\t40  tt tt 9 « « 17  tt tt\t10 \" « 29  tt tt 11 \" « 4  DESCRIPTION OF A TENANT-HOUSE.  47  Of those occupied by 12 fan lilies there were 12   tt tt 13\ttt tt 2  tt tt 14\ttc a 4  tt tt 15 tt tt 1  tt tt 16\ttt tt 12  tt tt 17\ttt tt 1  tt tt 18\ttt tt 1  tt tt 19\ttt tc 1  20 and less than 50 .t tt 6  50 and less than 100 \ttt tt 1  over 100\ttt tt 1  not ascertained \ttt tt 177  The average population of this class of houses is \t\tabout 28      Description of an ordinary Tenant-House.—As an example of an   ordinary tenant-house, I select one from James Street for description.   It   is a brick building five stories high.   A  door of entrance and a liquor   store occupy the front of the first story.  Entering a hall 4% feet wide, we   grope our way up a steep  stair-case 2^- feet wide,  which is perfectly dark,   and reach the second-story landing, from which open four doors communi-   cating with the same number of domiciles.      Calling at the first of these we enter a room 14 X12 feet with ceiling  8 feet high, having on one side two moderate-sized windows.  The small  fire-place  is closed, and a stove exhausts  rapidly the scanty atmospheric  supply which  finds  its way into the apartment through crevices  of  the  door and windows.   We observe that a pungent odor of coal-gas pervades  the apartment.      Opening into this room is another, having an area of 9 X12 feet, with  the same height of  ceiling as the former.   It has no other opening than  the door of communication, and of course possesses no means whatever  of efficient ventilation.  Looking into this we see two beds beside a quan-  tity of bedding on the floor between them,  indicating that this is the dor-  mitory of half a dozen persons.   A  sickening  and stifling  odor, most  offensive to the unaccustomed senses, pervades this apartment and poisons  the atmosphere inhaled by the residents.      The simple fact that this is the abode of six persons might be a suffi-  cient explanation of the latter phenomenon ;  but when we recollect that they  belong to a class who attribute most of their physical ills to a cause,  the  exact reverse of that to which they are generally due, viz., to exposure  to the  external  atmosphere, and whose   sanitary  creed   teaches  them  to exclude it  from  their  apartments  as  far as  possible, we  can only48          REPORT  OF THE FOURTH SANITARY DISTRICT.  wonder that the mephitic  gases  generated and concentrated in these  abodes  do not destroy health and life  even more speedily than they ap-  pear to  do.      \"We find in  this domicile a pro rata of about 370 cubic feet to each  occupant.  At the  time  of visit, the  mother and two small children are  the only members of the family present.   The latter we find to be types  of  a  class.   Although  they  have  no  form of  active  disease pres-  ent, they are  strumous, debilitated, and  lacking in  muscular develop-  ment.   We notice  that  the  conjunctiva is inflamed, and learn without  surprise that every member of the family has been affected with ophthal-  mia.  The  mucous membrane of the eyes as well as of the air-passages  resents the constant irritation of smoke and dust.      The remaining  domiciles are counterparts of the first as to arrange-  ment and condition, and almost  so as regards their occupants.  The halls  are practically destitute  of  ventilation.   The  occasional opening  of the  door of entrance below, or of those of the domiciles above, scarcely has  any favorable  influence on  the condition of the atmosphere.  From the  latter sources, indeed, the  halls are  constantly filled with  noisome  and  fetid exhalations.   Their floors are washed occasionally though  rarely,  but  the walls frequently remain for years without white-washing  or  cleansing.   \"Wherever  the hand comes in contact with them they impart  a sticky or pasty sensation ; and when scraped, an actual  deposit of filth is  brought away.      Pursuing  our investigations,  we next  examine the  rear of  the  premises:      Through a narrow alley, we enter a small court-yard which the lofty  buildings  in front and rear keep in almost perpetual shade.  Entering it  from the street on a sunny day the atmosphere seems like that of a well.  The yard, which is about  25 feet square, is filled with recently-washed  clothing suspended to dry.  In the  centre of this space are the privies  used by the population of both front and rear-houses.  Their presence is  quite as perceptible to the smell as to  the sight.      Making  our  way through this enclosure, and descending four or five  steps, we find ourselves  in the basement of the rear-building.  \"We enter  a room whose low ceiling  is blackened  with smoke, and  its walls dis-  colored  with damp.  In  front, opening  on a narrow area  covered with  green mould, two small windows, their tops scarcely level with the court-  yard, afford  at  noonday  a twilight-illumination  to the apartment.  Through their broken panes they admit the damp air laden with effluvia  which  constitutes the vital atmosphere  inhaled by all who are immured  in this dismal  abode.TENANT-HOUSE IN  GOTHAM COURT.  49      A door at the back of this room communicates with another which is  entirely dark, and has but  this one opening.  Both rooms together have  an  area about eighteen  feet  square, and these apartments are the home  of six persons.  The father of the family, a day laborer, is absent.   The  mother, a wrinkled  crone at thirty, sits rocking in her arms an infant  whose pasty and pallid features  tell that decay  and death are usurping  the place of health and life.   Two older children are in the street, which  is their only playground, and  the only place where they can go to breathe  an atmosphere that is even comparatively pure.  A fourth child, emaciated  to a skeleton, and with that ghastly and unearthly look which marasmus  impresses on  its victims, has  reared its feeble frame on a rickety chair  against the window  sill, and  is  striving to  get a glimpse at the smiling  heavens whose light  is so  seldom permitted to gladden its longing eyes.  Its youth has battled nobly against the terribly morbid and devitalizing  agents which  have  oppressed its childish  life—the poisonous  air, the  darkness, and the damp ; but  the battle is nearly over, it is easy to decide  where the victory will be.      My district contains one tenant-house which has become rather noto-  rious in consequence of having been the subject of several special reports,  one of which I made about three years since.  As this estabtishment is  very extensive, and possesses some  peculiar characteristics, and as the  description of these  premises  and their population which I gave in that  report  is equally  applicable now, I quote from it here : *      \" The building known as No. — and No. — Cherry Street forms a part  of what has heretofore been known as ' Gotham Court.'  As measured, it  is 34 feet 4 inches wide in front and rear, is 234 feet long and 5 stories high.  On the north it  is contiguous  to a large tenant-house fronting on Roose-  velt Street.   On the west an  alley 9 feet wide separates it from a similar  structure forming a part of the ' Court.'  On the  east  another  alley, 7  feet wide, divides it  from the rear of a number of houses on Roosevelt  Street.      \" In the basement of this building are the privies,  through which the  Croton-water is permitted to run for  a short time occasionally ;  but this is  evidently insufficient to cleanse them, for their emanations render the first  story exceedingly offensive,  and may be perceived  as  a distinct odor as  high as the third floor.      \" The contents of the privies are discharged into subterranean drains  or sewers, which run through  each alley and communicate with the exter-       *The Inspector of the Fourth District prepared the special report here referred to when  he was Visiting Physician to  tbe New York City Dispensary in the same district in the  year 1859-60—Editor.                    450          REPORT OF  THE FOURTH SANITARY  DISTRICT.  O   CO          Another Kow of Tenant-houses.  C*52          REPORT  OF THE FOURTH SANITARY;  DISTRICT.  nal atmosphere by a series of grated openings,~ through which feted exha-  lations are continually arising.   These openings receive the drainage of  the .buildings, besides the  refuse matter which is  not too bulky to pass  through the gratings, a bordering of disgusting filth  frequently surround-  ing them.      \" This structure contains  twelve principal divisions, each having a  common staircase communicating with  10 domiciles, making  120 tene-  ments in all.  Each  tenement consists of two rooms, the largest of which  is 14 feet 8 inches long, 9  feet  6 inches wide, and 8 feet 4 inches  high.  The smaller, having the same length and height, is 8  feet 6 inches wide.  The two apartments together contain 1,955^ cubic feet.  Each room has  one small window.  The doors leading from the  landings are contiguous  to the wall in which these windows are  situated, so that it is impossible  for a current of air to pass through the rooms under any circumstances.      \" At the time of visit 49 of the  tenements were either vacant or the  occupants absent. In the remaining 71  there were reported as residing  504 persons, averaging  a little more than 7 persons to  each occupied  domicSe.  The entire amount of space in the rooms  occupied is 138,840  cubic feet, which would be equal to a single  room 118 feet  square, and  about 10 feet high, giving each individual an average of about 275 cubic  feet, equal to a closet 5 feet square  and 11 feet high.  It must be recol-  lected  that the above total space contains  not only its 504 inhabitants,  but their furniture, bedding, and household utensils, besides no small por-  tion of their excretions, as is painfully evident to  every one who, in these  regions,  has  the  misfortune to  possess an acute sense of smell.  Of the  entire  number of tenements, four  only  were found in a condition ap-  proaching cleanliness.  It need scarcely  be  said that the entire establish-  ment swarms with vermin.      \" In seven  of the tenements tailoring was carried on.  In five out of  seven the articles manufactured were for the use of the army.  In two  of these  rooms patients were found sick of contagious diseases.   One was  a case of typhus fever, the other of measles.      \" It  was admitted that 19  persons were unvaccinated.  These were  chiefly children, but it is probable that a much larger  number  are unpro-  tected from variola, for in several instances those who asserted that the  operation had been  successfully performed,  failed, on examination, to ex-  hibit a vaccine scar.      \" The  average length  of time  that  the residents have occupied the  premises is reported to be  about two years and eight months.  There have  been 138 births,  including 12 still-born, in these families during their term  of residence in the building.   Of these only 77 are now living, showing an                                                    K1NNERSLEY. -JOHNSON.                             TKANSVEESE SECTIONAL ELEVATION OP THE GOTHAM KOOKEKY.         CO Cellar.                                       PP  Piivies.                                       SS  Sewer.    [The plan illustrates how the domiciles are superimposed, one above another; and how the loathsome cellar and privies are reached.]  w54          REPORT OF THE FOURTH SANITARY DISTRICT.      infant mortality of over 44 per cent, in two years and eight months ; but      as by far the greater number of these deaths occur during the first year, it      may be safely assumed that 30 per cent, of those born here do not survive      a twelvemonth.   The total number of deaths reported as occurring in the      families now occupying the premises during their term of residence there,      is 98, or about 191 per cent, of the population for that period.         \" Of the 504 inmates, 146, or about 29 per cent., were found to be suf-      fering from diseases of a more  or less serious character, among which      were four cases  of small-pox (three of them unvaccinated), eight cases of      typhus fever, seven cases of scarlatina, and four of measles in the eruptive      stage, twenty-seven  cases of infantile marasmus, twelve cases of phthisis      pulmonalis, five cases of dysentery, three cases  of chronic diarrhoea, and      a large number of slight cases of diarrhoea and of cutaneous eruptions.         \" It is difficult to form a satisfactory estimate of the comparative fre-      quency of the different diseases heretofore prevailing, the inmates being,      in a great proportion of cases, ignorant of their character.  It is, of course,      equally difficult to arrive at the causes of death, but it is pretty well ascer-      tained that  at least twenty cases of small-pox occurred  during the past      year, of which six were fatal.  Scarlatina is assigned as the cause of six-      teen deaths occurring during the above period.   Typhus fever undoubt-      edly claimed numerous victims, as it has  been  quite prevalent.  To the      unaccustomed eye it is a sad and striking spectacle to witness the atten-      uated forms, the sunken eyes, the pinched and withered faces of the little  /\"  patients,  young in years but old in suffering, who are the prey of infan-  t   tile marasmus.  A  glance  is  sufficient to designate this as one of the  >   ghostly janitors, ever ready to open wide the gate which leads  to early      death.         \" A description  of  these premises  would be  incomplete  without, at     least, a passing notice  of  two establishments occupying the  front por-     tion of the first story.   One is  termed a grocery, the other a liquor store.     Both  are apparently pretty well patronized.  At the former are retailed a     variety of articles of food, including partially-decayed vegetables, rather      suspicious looking solids, bearing respectively  the names of butter  and      cheese, and a decidedly suspicious fluid bearing the name of milk.  Beer      and alcoholic compounds are also  dispensed.  At the adjoining shop the      staple commodities  are those indescribable compounds of sundry known      and unknown  ingredients, which are sold as ' pure  imported wines  and      liquors.'   I believe from what I could ascertain that these liquors are used     to a considerable extent by almost every family on the premises,  a fact, in-      deed, which might be expected, for in such apartments as they occupy the      poisonous air begets a deadly lassitude, and generates an inordinate desireSUBMARINE  DWELLINGS.  55  for stimulants.   To the effect of these unwholesome viands and poisonous  beverages may probably be traced much of the diarrhoea which prevails  here even at this season, and which is vastly increased in amount during  the summer months.     \" On the whole, perhaps, this section of Gotham Court presents about  an average specimen  of tenant-houses in the lower part of the city in  respect to salubrity.  There are some which are more roomy, have better  means of ventilation, and are kept cleaner ; but there are many which are  in far worse condition, and exhibit a much higher rate of mortality  than  this.\"      The number of inhabited basements and  cellars is 224, occupied by  268  families, or about 1,400 persons.  Their depth below the  \"curb\" or  street level varies from 2 to 8 feet, averaging about 4^ feet.                         are below high-water mark.                        \"  less than 10 feet above high-water mark.                        \"  from 10 to 20     \"         \"                        \"    \"  20 to 30     \"         \"                        \"  over 30         \"         \"       In the sub-tidal basements 19  families, or 110 persons, live beneath the  level of the sea.     This  submarine region is not only excessively damp but  is liable to  sudden inroads from the domains of  Neptune.   At high tide  the water  often wells up through the floors, submerging them to a considerable depth.  The  constant repetition of this aquatic episode in domestic life, has led to  the abandonment, as residences, of several  of these basements, the number  now occupied being much smaller than it was formerly.     They  are all damp, those in the least elevated localities, of course,  being most so.   In very many cases the vaults of  privies are situated on  the same or a higher level, and their contents frequently ooze  through  walls into the occupied apartments beside them.  Fully one-fourth of  these  subterranean domiciles are  pervaded by a most offensive odor from this  source,  and rendered  exceedingly  unwholesome as  human habitations.  These are the places in which we most frequently meet with typhoid  fever  and  dysentery  during the summer months.  I estimate the  amount of  sickness of all  kinds affecting the residents of basements and cellars  com-  pared with that occurring among an equal number of the inhabitants of  floors above ground, as being about in the ratio of  3 to 2.     Privies.—Of the privies in this district less than 30 per cent, are con-  nected with drains or sewers.  About 15  per cent, are situated in houses  The floors of 16        \"      91        \"      84        \"      28        \"       556           report of the fourth  sanitary' DISTRICT.  occupied as dwellings, generally in the basement.   At least 10 per cent.  were found  in a very offensive condition.  The proximity of the latter to  the fever localities is shown by the large  map.  There is a section of my  district  embracing at least nine blocks, in every part of which the pecu-  liar  odor arising  from privies  is  always distinctly perceptible  during the  summer months.   From this  region fever is never absent.   I refer to  typhus and typhoid, for  intermittent and remittent fever do not prevail in  this  neighborhood even in the low tract adjoining the river.   Such a  gen-  tle fiend as paludal miasma flies  affrighted from the terrific phantoms of  disease that reign supreme in this domain of pestilence.      Rents.—In regular tenant-houses the rent of each domicile at present  averages $9 per month, or $108 per year ; the entire rent of each of these  houses thus averaging $950 per annum.      Excessive  Crowiding  of Houses upon Lots.—In some cases front and  rear buildings are situated on lots less  than 80 feet deep.  They are  gen-  erally crowded into the smallest possible -space, and are constructed in the  cheapest manner.      They are, in  many  instances, owned by large  capitalists, by whom  they are farmed out to  a class of factors who make this  their especial  business.*   These men pay to the  owners of the property a sum which is       * The diagram on the opposite page represents an area eighty yards long and fifty yards  wide, including the cul-de-sac  at the termination  of Cliff Street.  It illustrates the  prox-  imity to crowded habitations of offensive and dangerous nuisances, often observed in the  lower part of the city.  The diagram  presents  an accurate ground plan of each tenant-  house which it embraces.  Within this  space are 20 dwellings occupied by  111 families,  and having a population of 538 persons.  A soap-and-candle  factory, a tannery, and five  stables, in which are  kept not less than 30 horses, are also  wholly or partially included  within its limits.     A, B, C, D, E, are tenant-houses fronting on Vandewater Street.  An alley four feet  wide  running through C forms the  sole  communication with  the five tenant-houses  F, G,  H, I, J, which open into the small  court E, in which stands their common privy, /, situ-  ated within three feet of  the hall door of one of the houses, which is constantly pervaded  by its noisome odor; c, d, e, are privies situated immediately under the windows of the  houses F, G, H;  a and b are  privies belonging to the tenant-houses A and B;  K, L, M,  N, are  tenant-houses standing back to back with two of  those in the  court  above  mentioned and with three stables to which access  is had from Vandewater  Street.   The  position of two  stables fronting on  Cliff Street  will also  be observed.  The soap-and-  candle factory, whose frontage is shown in the cut, is a very extensive one, and its emana-  tions vitiate the atmosphere for a considerable space around.     T, T, T, represent a series of tan vats, in the  rear  of a leather factory on Frankfort  Street, which generally contain a large number of green hides in a very offensive condi-  tion.   The pecuhar stench from this source is usually quite perceptible through the entire  area shown in the engraving.     This locality lies on the borders of a former marsh known as  \" Beekman's Swamp.\"   TheNESTS FOR FEVER AND CHOLERA.  57  CEOWDED AREAS AND AN INSALUBEIOUS QUAKTEK.58           REPORT OF THE", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ug3k.7b5c-qaiq", "00000000-0000-0000-373B-DFBB7899453B", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Report of the Twenty-fifth Sanitary Inspection District", "9918573882206676X42", null, "1865", "1865", "In 1864, the New York Citizens Association, a coalition of civic and sanitary reformers, organized a sanitary survey of New York City. The data collected by 31 medical inspectors were condensed into a report published in 1865, including this account of the twenty-fifth district inspection. Pages 298-324 of Report of the Council of Hygiene and Public Health of the Citizens Association of New York upon the Sanitary Condition of the City. New York: D. Appleton and Co., 1865.", "Monographs, Excerpts", null, "Public Health Data Collecting, 1860-1900", "27", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "EEPOET                             OF TH    TWENTY-FIFTH SANITARY  INSPECTION DISTRICT.  J. LEWIS  SMITH, M.D.,        Sanitary Inspector.     Boundaries.—The Twenty-fifth District comprises that portion of the  Twenty-second Ward bounded north by the Central Park and Eighty-sixth  Street, east by the Sixth Avenue and  the  Central  Park, south by Fiftieth  Street, and west by the Hudson River.       Topography.—The surface of this  district, in its natural state,  is  very uneven, consisting\" of Ioav, marshy ground,  alternating  with  hills,  usuaUy of smaU size, and produced by the  elevation or outcropping of  rock from the bed of gneiss which underlies the whole island.   Some of  these hills consist of bare rock; in other places, soil and vegetation  cover  the rock.  There is a general inclination toward the Hudson of that por-  tion of the district which lies west of Ninth Avenue, whUe that part east  of Ninth Avenue inchnes toward the East River.   In the primitive state  of the district the marshy ground gave rise  to seven distinct streams,  five  of which (A, D, E, F, and  G)  emptied  into the  Hudson, and the  remaining two (B, B, and C, C) crossing what is now the Central Park,  and  along the bed of the skating ponds, finally emptied  into  the East  River.  These  streams, as far as they lay within this district, were aU  smaU, and some of them nearly  or quite disappeared in protracted dry  weather.     Streets.—Their Condition.—There are in the district  5 avenues  paraUel with and equidistant from each other,  and when graded there wiU  be 35 paraUel and equidistant streets, crossing the avenues at right angles.  The district is also crossed diagonally by Broadway, or, as its  suburban  portion is termed, the Bloomingdale Road ;  the old highway of the island.STREETS.—THEIR CONDITION.  299  MAP OP TWENTY-FIFTH DISTRICT.  Above Fifty-ninth no  street has  been  entirely  sixth.  This  enters  the district on  the  south, near Seventh Avenue, and  emerges at Eighty-sixth Street,  west  of Tenth  Avenue.  Sixth,  Seventh, and Eighth Avenues are  graded  and paved  so far as they  he within the  district.   Ninth  Avenue is graded as far as  Sixty-  third Street, and partially  above  this street.  It is paved to Fifty-  fourth  Street.   Tenth Avenue is  graded as far  as  SeArety-fourth  Street, and is open  but not graded  to Eighty-third  Street.   Eleventh  Avenue is graded to Fifty-ninth  Street, and above that is not open.  These avenues all contain railroad  tracks  below Fifty-ninth   Street,  and Eighth Avenue a track in its  entire extent through the district.  The  pavement  used is, in part,  cobble  stone, and in part the trap-  block.   Broadway is   graded to   Seventieth  Street,  paved  with  block  pavement  to Fifty-ninth   Street, and  macadamized from   Fifty-ninth  to  Seventieth  Street.   The cross streets, from  Fiftieth to               Fifty-ninth, are   all               open, and graded with               the exception  of  Fif-               ty-first  Street   be-               tween Sixth and Sev-               enth  Avenues,   and               portions   of  Fifty-               third,    Fifty-fourth,               and     Fifty - eighth               Streets,   near   the               Hudson    River.             opened  except  Eighty-  jfa'6—siizf_l.....  \" i300      REPORT OF  THE  TWENTY-FIFTH  SANITARY DISTRICT.       Seaverage.—This is entirely Avanting in  the  northern or upper tAvo-  thirds of the Twenty-fifth District, and in the lower third, or the part be-  low Sixtieth Street, it is incomplete.  The streets entirely sewered beloAv  Sixtieth Street, are Seventh and Eighth Avenues, and Fifty-ninth Street.  The other streets either contain no seAver, like Broadway, or are sewered  only for limited distances.     Domiciles.—The domiciles of the  Twenty-fifth District may be  di-  vided  into four  classes:   1st.  Shanties.   2d. Wooden tenements.  3d.  Brick or stone tenant-houses.  4th. Good dAvelling-houses, whatever the  material.     Shanties.—The shanty is the cheapest and simplest domicile construct-  ed in civilized communities.  The typical  shanty is built of rough boards,  which form the floor, the  sides, and the  roof.   It is\" built either on the  ground, or but little  raised above it.   It is from six to ten feet high, and  its ground area varies much in different cases ; but is always of moderate  extent.  It contains no fireplace or chimney, but a stove, the pipe from  which passes through a hole in the  roof.   It has from one  to three  or  four Avindows, with single sash, each containing from four to  six panes of  small  size.   Some shanties have but one room; others an additional  small apartment, used as a bedroom.  The better  shanties are lathed and  plastered.   It is evident that, to the  occupants of the shanty, domiciliary  and personal cleanliness  is almost  impossible.  In  one small room are  found the family, chairs, usually dirty and broken, cooking utensils, stove,  often a bed, a dog  or cat, and sometimes more or  less  poultry.   On the  outside, by the door, in many cases  are  pigs and goats, and additional  poultry.   There is no sink or drainage, and the slops  are throAvn upon  the ground.  The water used is sometimes the Croton, which is brought  to the shanties in pails, usuaUy from one of the avenues.   In  other places,  where the Croton hydrants are too far away, and the ground is marshy,  the Avater  is  obtained from holes dug  a  little below the  surface.  This  water often has a  roiled appearance,  and  an  unpleasant flavor.  Shanties  are usually buUt promiscuously over the ground, without the least regard  to order.   Famihes living in them are largely squatters,  and such people  of course  select for residences localities of which no profitable use can be  made  by the  proprietors.  Therefore, shanties in  this district  are built  mainly on rocky, elevated ground, or on lots  sunken and too wet for  til-  lage.  There  are in the district 552 shanties ; and as a shanty accommo-  dates but one family, this is the number of famUies  living in this kind of  domicUe.      Wooden  Tenements.—Next to shanties, in the classification of domi-SHANTIES DESCRIBED.  301  cUes,  come wooden tenements.  In determining Avhat buildings  should  be placed in this class, we have regarded more the appearance  and gen-  eral character of the houses, than the number of families Avhich they ac-  commodate.  The separation of Avooden tenements from shanties on the one  side, and the better class of dwelhngs on the other, is in a measure arbitrary.  Some Avooden tenements are but little removed from shanties, as regards  both size and  mode of construction ; Avhile  others might, Avithout much  impropriety, be placed in the group of ■good private  residences.  Ordina-  rily, howeA*er, there is little danger of error  in their  classification.  The  wooden tenement in  the Twenty-fifth  District has usually tAvo stories,  but some have  only one, some three, and a feAV four.  It is built without  ceUar. and but little raised above  the ground.   It has a mortised frame,  clap-boarded sides, a chimney, and shingled roof.  It has no sewer con-  nection or other drainage, and no gas or Croton  pipes.  The privies are  in the rear, or  in front, and  also Avithout drainage.  The water used by  the occupants is, in some locaUties, the Croton;  in others, spring  or well  water.   The house is heated by a stove, and the fluel used is  coal, fre-  quently partially burnt, and sU'ted  from ashes obtained from hotels and  private residences in the city.  This is also the fluel used in shanties, and  the shanties and wooden tenements are lighted by kerosene oil.      The  ground area  of the Avooden tenement, like that of the shanty, is  nearly square ;  sometimes the AA-idth, sometimes the deptli excelling.  The  width and depth vary  generally from twelve  to tAventy-five feet.      The number of  famihes in the wooden tenement varies from one to as  many as  seven or eight, according to the size of the house.  In 132 of  these domiciles taken  Avithout selection, I find 312 families, which  is prob-  ably not far from the  average number.   There  are in the  entire  district  861 wooden tenements, containing, therefore, according to the above cal-  culation,  2,035 famUies.      Brick Tenant-houses.—There are in the Twenty-fifth District 147 domi-  cUes, buUt, with one exception, of brick, which, from their mode of construc-  tion, may be properly considered tenant-houses.  In the exceptional case,  the material is stone.   There are some dAveUing-houses which are built in  part of brick and in part of Avood.   Whether  these are classed with the  brick or wooden tenements, depends upon which, material is in excess, or  whether  the building resembles most in its appearance the one or  the  other kind of domicile.      Of these 147 tenant-houses 94 have four stories, 32  three  stories,  19 two stories, and  2  one story.      The foUowing table exhibits the water supply of these houses :302      REPORT OF THE TWENTY-FIFTH SANITARY DISTRICT.                                                                 No. of                                                                 houses.  Croton obtained from hydrants in yard (front or rear),    .       .  24     \"       \"           \"        ceUar or basement,   .       .     11     \"       \"   on the floors,      .        .       .       .       .85     \"       \"   elsewhere; no water on the lot,       .       .     15  Spring water used,  .       .       .        .       .       .       .3  Water-supply not stated in the records, ....      9  147                             House drainage.  None, or into gutter or to surface of street,        .       .        .44  Drained into sewer,     ......     80     \"     \"  contiguous sunken lots,       .       .       .        .14  Not ascertained,        .       .       .       .       .       .     ' 9  147                                PRIVIES.              Location.                          Drainage.  In front of house,     .       .   16  None,        .        .       .61   \" rear    \"     .        .     116  Drained into sewer,      .      77   \" ceUar   \"         .       .    1      »     «  sunken lots,     .    2  None (water-closets in house),    7  Placed over the Hudson, .       3  Not ascertained,      .       .    7  Not ascertained,      .       .    4                                  147                              ~147                                   Cellars.  None,      ......                  27  CeUars dry,    ......              86     \"   wet?        .....                  11  Not ascertained,        .                                           23                                                                   147     The number of families occupying brick tenant-houses I have ascer-  tained to be 634, as foUows :      Houses four stories high, 94, containing 460 famihes, average about 5       \"   three      \"     32,     \"     132       \"         «      4       \"   two       \"     20,     \"      40       \"         «      2       \"   one       \"      2,     \"        2       \"         \"1     WhUe there is great uniformity in the shanties, and most of the wooden  tenements, as regards their sanitary condition, there is a wide diversity in  the different brick tenant-houses.   Some of these latter, on account of theCLASSES  AND CHARACTER OF THE POPULATION.       303   lack of house and privy drainage, and of cellars, or the presence of wet   cellars, possess aU the causes of insalubrity A\\\\diich are found in the wooden   tenements ;  while others, though a small minority, are nearly as weU con-   structed to insure comfort and health as the better class of dwellings.  In   general, the oldest brick tenant-houses, and those buUt on unfrequented or   unimproved streets, are the most objectionable.       There are, then, living in shanties, in tenements, and in brick tenant-   houses, in the Twenty-fifth District, 3,221 famUies.   It may be safely as-   sumed that 3,000 families occupy domiciles Avhich, from fault in their con-   struction, or in their surroundings, are decidedly insalubrious.  The exact   character of the insalubrity wUl be pointed out hereafter.  These families   are mostly small, in consequence of the great mortality among infants and   children, probably not exceeding, in the average, five.  This gives a total   of 16,105 individuals in the Twenty-fifth District, who reside in shanties,   wooden tenements, and tenant-houses.       Good Dwellings.—The number of good dwellings in the Twenty-fifth   District is 287, containing probably about 400 famUies.  Below Fifty-ninth   Street  these dweUings  are mostly of  brick;  above Fifty-ninth  Street   chiefly wooden ;  and above Seventieth Street, with  three  exceptions, en-   tirely such.      Population  and Occupations.—Those who reside in  shanties are,   Avith feAV exceptions, Irish and Germans; the Germans predominating.   They are engaged in humble occupations.  Many are  day laborers, em-   ployed by contractors in various kinds of work, as in  grading, paving,   and sewering streets, and in  the removal of rock, or in excavating for   buUding purposes.  Some  are employed  in the stables of the city rail-   roads and stage  companies, or in the Central Park, and not a few  are in   the  army.  In  addition  to these occupations, and occasionaUy as  a sole   means of support, many families keep cows or pigs, with poultry.  In the   brick and  wooden tenements reside  the  large  class of  mechanics,  as   taUors  and shoemakers, the drivers and conductors on the various city   railroads, and many of those engaged in retail business  on the avenues.   In  the  wooden  tenements  the German famihes preponderate;  in the   brick, the American.  Those residing in the  better class of houses are   mostly engaged in business down town, as merchants, clerks, real  estate   agents, brokers, bankers, etc.     Preventable Causes of Disease and Death.—(a.) Stagnant Water.  —Much of the insalubrity in the  Twenty-fifth District is, no doubt, trace-  able to the stagnant water.   The surface of this district has been stated  to consist of an alternation of hills and vaUeys, the latter having been once  the source of several streams.  These streams, with the exception of two, 304     RETORT OF the twenty-fifth sanitary  district.     have been obstructed by the grading of the streets.  Sewerage has in some   places partiaUy obviated the bad effects of this obstruction, but at present   there is stagnant water  along the bed  of  four of these Avater-courses.   There are five ponds of considerable magnitude produced in this Avay, be-   sides smaUer collections of Avater.  Much of the ground  above Sixtieth   Street, between  Eighth and  Ninth  AArenues,  is permanently so wet, in   consequence  of  obstruction to the  natural  drainage, as to be unfit for   tillage ; and the  sunken andundrained  lots become, to a certain extent, the   receptacle of decaying  substances, especially dead animals, and the gar-   bage from adjacent houses.  From the amount of wet ground which the   Twenty-fifth District contains, it *has always been the habitat of malarious   affections.   These affections are less prevalent in the  southern part than   formerly, owing to the greater amount  of seAverage ; Avhile in the upper or   northern part, Avhere there is no  sewerage,  and the course of streams is   interrupted, they are believed to be more frequent. This was the opinion   of the late Dr. Williams, an intelligent and accurate observer, who, for   nearly a third of a century, Avas almost the only  physician living in   Bloomingdale.   Moreover, in  the vicinity  of  the stagnant  Avater,  and   probably, in  part, in consequence of it, the  continued feA'ers, diphtheria,   and  cholera  infantum prevail  to  a greater or less extent.  The ponds or   pools of stagnant Avater generally have a roiled or dirty appearance ; but   in the Avarmest weather the hue  is green, from the  presence of minute   organisms, chiefly animal, as I have  several times observed by the micro-   scope.   During the Avarmest weather many of  the smaller collections dry   away,  and they all become reduced in size.     There is also in the district  considerable standing water in excaATa-   tions, made  in grading the streets or  for building purposes.  Even on  eleA'ated ground these  excavations are occasionally met with, containing  water coUected either from rains, or from adjoining springs ; but they are   oftenest observed in the valleys, along  and near the site of the old water-   courses, even where there is no stagnant water  on the surface.     (b.) Sewerage and House Drainage.—Insufficient sewerage and house  drainage constitute,  also, one  of  the  chief  causes of insalubrity.   The ■  shanties, the  tenements, and a  considerable  portion of the brick tenant-  houses, have no connection with the sewers,  and no drainage, unless into  the gutters.   Generally a connection with the sewer is impossible, as there  is so Uttle sewerage in the district;  but even in sewered streets, shanties  and wooden tenements  have no sewer connection on account of the small  value of these domicUes, and the cost of constructing drains.   This  is less  objectionable as regards shanties, on account of their being built over the  squares, without order and with interspaces ;  but the Avooden, and in part        NEGLECT OF LOCAL DRAINAGE.—FAULTY SEWERAGE.     305    also the brick tenant-houses, being built in rows along the  streets, the  slops  from  them are thrown into the gutters, where they form long Unes  of stagnant, or if the ground inclines, slowly-running Avater, of the most  insalubrious character, and in many places continuing the year  round.  Wherever there are rows of  wooden  tenements this condition of the gut-  ters generaUy  obtains; and  as there  is such  a large number of families  living in these houses, the noxious exhalations from the gutters must be a  prolific source of disease.  In one locality the Avater of the gutters forms  a network with  occasional  breaks,  extending  the   distance of several  squares.  This nuisance might be partially obviated in sewered streets by  the construction of more culverts, which are entirely too few for localities  where the domiciles have no drainage, or  else drainage to the surface of  the street.   That this description of the gutters  is not exaggerated, will  be apparent from a brief statement of the condition of Ninth Avenue and  the cross streets, from Fiftieth to Fifty-fifth, along which  streets there is  a dense German population, Uving in wooden tenant-houses.  All the gut-  ters in this locality contain more  or less water ; and in the  more crowded  streets the  quantity  is ordinarily so great that there is a flow or current  tOAvard the river, as  the ground inclines in that direction.   Thus in Fifty-  second Street  there is a  current in each  gutter from Ninth Avenue to  Eleventh, where it reaches the first culvert, which it enters, although this  street is sewered from near Tenth Avenue to the river.  In Fifty-third  Street, the stream on  either side, from Ninth Avenue, enters the vacant  lots Avest of Tenth Avenue, where there is  a pond of stagnant water.  In  Fifty-fourth Street the  two streams from  Ninth meet at Tenth Avenue,  and thence flow to Eleventh; thence along Eleventh Avenue to Fifty-fifth  Street;  and finally down Fifty-fifth Street to the Hudson, a distance from  the commencement  of probably half a mile.  AU gutter streams pro-  duced by slops  and  house drainage  are of course small, so that in hot  weather they frequently dry aAvay, leaving stagnant water in crevices and  sunken places.      (c.)  The  Condition  of  Domiciles.—It  has been  stated  above that  elevated rocky locahties on the one hand,  and marshy  and sunken ground  on the  other,  are to a great  extent peopled by poor  famihes, who select  these places for the erection  of their cheap dweUings,  either because  they pay no  rent, or but little.  Elevations of rock are  probably not  objectionable  to the sanitarian  as a  place  of  residence, but swampy  oxound  obviously is.   There  are many shanties and  wooden tenements  constructed near the bed of streams, upon  ground which is permanently  wet  so  as to render the apartments  Uable to  dampness.   CeUars in  these locaUties  are  apt to contain water, in some instances through  all                   20306      REPORT OF THE  TWENTY-FIFTH.SANITARY DISTRICT.    the seasons,  as  for  example  a cellar in Fifty-third Street,  where, al-  though  remote from the river, fish have  lived  many  months.  In  ad-  dition to the  objectionable  nature of the ground on Avhich  shanties and  A*t)oden tenements often stand, the  salubrity of these domiciles is greatly  impaired by their mode of construction.  They are generaUy built with  very little elevation of the  floors above the  ground, and sometimes after  a period there is  none in  consequence of the accumulation of dirt and  ashes outside.  A large proportion  of  the Avooden  tenements, and  many  of the shanties,  are Avarm  and comfortable in cold weather; but others  not being properly lathed and plastered, contain apertures through Avhicli  the cold penetrates.  The inmates of such domiciles suffer especially from  those diseases which  occur in consequence  of  sudden changes in  the  weather, as croup, bronchitis,  pneumonia,  and  dysentery.   The  inade-  quate Avater-supply of  both shanties and wooden tenements, by prevent-  ing proper personal  and domiciliary cleanliness, is  doubtless a source of  disease.     Some of the  brick tenant-houses are constructed with a proper regard  for the health as AveU as comfort of  the occupants.  With others the case  is  different.  In a large number of them the bedrooms  have a central  position on each floor,  so that proper ventilation  is impossible, the supply  of air being through the  door only, or a door and a small half Avindow.  Another fault of construction in some houses of this class, is  the  small  size of the rooms, so as to cause crowding or  packing of families, and  thereby promoting contagious  and pestilential diseases.   As an example  may be mentioned four houses, built hoAvever as one,  at the corner of  Broadway and Fifty-third Street.  These houses are four stories high, but  are not deep, and are buUt with the greatest economy of space, containing  forty-five famUies.     Into one  of these families,  a few months since, typhus fever was in-  troduced by an  emigrant from Ireland.  There were  in the family no  children, but six adults, including the emigrant,  and only  one escaped the  disease.   This family occupied a  main apartment, measuring  10 x 12  feet, and two bedrooms each 7X 61 feet.  It wUl be obvious to those fami-  liar with typhus, that  the  spread of this disease was due, mainly,  to the  small size of these rooms, and the consequent crowding.   In this class of  buUdings the pestilential and contagious diseases, when epidemic, prevail  extensively and fatally.  It has been seen that some of the brick tenant-  houses  have no drainage,  and therefore, as regards salubrity, differ but  Uttle from those constructed of wood.     The better class of dwelhngs in the Twenty-fifth District, have been  for the most part constructed with reference to health as  weU as comfortFAULTY DRAINS  AND  POISONOUS EXHALATIONS.  307  of the occupants.   The older houses of this class are, ordinarily, built of  wood, and isolated ; those more recently constructed, of brick, and in rows.  The point ol chief interest to the sanitarian in reference to these dwellings  relates  to  tlieir drainage.   Some  of  the streets, Avhere  rows  of brick  houses  have been  erected, are  not seAvered, and  proper  drainage  under  these circumstances is  hardly to be expected.  It is necessary, in such  streets, to connect the buildings  Avith some distant sewer ;  and as the con-  struction  of houses in this  district is, usually, in the hands of specu-  lators and contractors, the drain to the seAver is apt to be  built in a care-  less  manner, and  of poor materials, so  that obstructions  and leakages  after a whUe occur.  The longest row of houses in the district affords a  striking example in corroboration of  the truth of  this remark.   Fiftieth  Street, betAveen Eighth and Ninth Avenues, is not sewered, except a short  distance near Ninth Avenue.  A row of thirty  first-class houses was  erected a few years ago in this street, and they aU connect Avith the sewer  in Ninth Avenue, by a single drain, Avhich runs under the houses from  No. 1 to 30.  The occupants of  several of these houses have, to  my  knowledge, complained of an offensive odor arising from the cellars, due  doubtless  to cracks in the drain  or a separation of the joints.   In one of  these buildings, where a  child was  sick  last summer with cholera infan-  tum, the parents stated to me that the  odor was sometimes noticed on the  second floor, and it may have been one of the causes of the disease. Two  or three years since an eminent  physician of this city, well known  to the  public  as  a sanitarian,  stated  in  my hearing that he  had visited  two  chUdren in a house in this row, who seemed to be suffering from blood-  poisoning, and one died.  They were  both  much prostrated, and their  tongues presented a singular dark appearance.  In investigating the cause,  he and the attending physician were  shown to the cellar, the ground of  which was wet, and exhaling an offensive odor.  These physicians appear  to have been ignorant of the peculiarity in the drainage of these houses,  though satisfied that they had ascertained the cause of the disease.      The connection  of a house with the sewer necessitates  the payment of  the sum of ten dollars, in addition to the cost  of constructing the  drain,  which, if the ground is rocky, is considerable.  On this account it is cus-  tomary in building  rows of houses to connect several houses, perhaps five  or six Avith the sewer by a single drain ; and if this is done with as little  labor as possible, and as cheap materials as the terms of the contract will  alloAv, it is readily seen that the drainage of the so-caUed first-class houses,  even  in sewered streets, may be, and  often is,  very defective.   In conse-  quence, in many of these houses there is complaint of an offensive odor  from the sewer, especially in certain states of the Aveather308      REPORT OF THE TWENTY-FIFTH SANITARY DISTRICT.      (d.)  The Disposition of Garbage.—One of the principal sources of im-  pure air in the Twenty-fifth District is the garbage.  It is removed pretty  regularly from the better class of dAveUings in a few of the streets, in the  lower part of the district, either by boys sent out from shanties to collect  it for domestic animals, or by the City Inspector's ash and garbage-carts.  From this class of houses, located in the central and upper or northern  part of the district, and  from the tenant-houses and shanties  generally,  there is  no regular removal of garbage.  There are only tAventy-eight  garbage-boxes in the entire district, and some of these are so dilapidated  as to be almost useless.  These boxes are used by the occupants of the  tenant-houses not only as receptacles for garbage but also for ashes ;  and  being so  few and  so irregularly emptied,  they are practically  of  little  consequence.  As there  are about  3,221  famUies in the district  Avho  occupy  tenant-houses and  shanties, it may  be safely said that there are  3,000 families  who  throw their entire garbage  on the ground, where  it  decays.   This disposition  of the  garbage  adds most to  the insalu-  brity of  those  squares  which  contain wooden  tenements,  for  these  houses  are generally built  compactly in  rows, and  the garbage  from  them is  thrown into  the gutters, where there is usually more or less  stagnant water.  This Avater then  becomes  impregnated with  organic  matter undergoing decay, and  to  add to the  insalubrity these streets  are  seldom cleaned.   For example, Fifty-second Street, between  Sixth  and Seventh Avenues, contains a dense population on  either side, Uv-  ing  in  wooden  tenements.  In  the gutters of this  street there is  al-  ways garbage and  stagnant  water.   The street was opened about  eight  years ago, and one family informed me that it had never been cleaned;  another  that it was   cleaned  once about  four years  since.  Garbage  from the shanties  probably does not add much to the insalubrity of  the  district, as  the quantity of it is not great, and it is consumed to a con-  siderable extent by the domestic animals.      (e.)  The Condition of Privies.—Owing  to the unsewered state of the  streets, and the character of  the domicile, there are numerous badly-con-  structed and undrained privies in the  Twenty-fifth District.  Most of  the brick better-class dweUings, and five of the brick tenant-houses, are con-  structed with water-closets inside, and therefore without privies.  AU the  other brick  tenant-houses (142) and  aU the wooden  domicUes, whether  tenement or belonging to the better class, have privies.      Privies  attached  to wooden  tenements  and  to  shanties  have no  drainage, and it has been seen that of those  connected with the  brick  tenant-houses there is no drainage to sixty-one.  Privies belonging to brick  houses, whether drained or not, are  ordinarily well  constructed.   They          LEATTTCAL  LAWS OF PERSONAL  HYGIENE NEEDED.       309    are of large size, Avith suitable doors and covers, and deep vaults.   Those  not drained are cleaned at intervals, so that the privies of such domiciles  cannot be considered a cause of insalubrity.  The case is different with  those belonging to wooden tenements and to shanties.   These are gener-  ally very small, built mainly of rough boards.  Many of them are located on  rocky ground, and Avithout vaults ; others, and the majority, have  shallow  vaults, Avhich are soon fiUed.  It is evident that privies constructed in this Av", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-axb7-xvbu_tbpz", "00000000-0000-0000-FB5A-825F3A2F8CED", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Comparative Stature and Other Measurements--White and Colored Soldiers", "9918573882206676X43", null, "1921", "1921", "One of many statistical graphs derived from U.S. Army anthropological studies of soldiers during World War I, this graph included a male figure with measurement points indicated. Page 41 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "COMPARATIVE  STATURE  AND OTHER MEASUREMENTS            WHITE (93.185) COLORED (6.264) SOLDIERS  DEMOBILIZATION MEASUREMENTS IN INCHES  I  SPAN      2 STATURE      3 STERNAL NOTCH      4 SITTING HEIGHT      5 PUBIS      6 ARM      7 LEG      B KNEE HT.      9 FOREARM      10 CHEST      H WAIST      12 THIGH      13 SUPER-PATELLA      14 KNEE-PATELLA      IS NECK      16 CALF  0   5   10   15   20  25  30  35   40  45  50  55   60  65  70  ...........!......' --\"—]-■  -;—-~f-  ----------------------------.—•————,  ■-  t:.........;........-1  ; —~—'r~~~T.~~\"~?.  m  17 SHOULDER      18 HIP      19 CHEST TRANS.      20 CHEST ANT.-POST!  -~^~-^^~~-~~r^~^-~—--tt-™]                         1                                                       ^^^^^_                                                                                                                                                          'Mmàmm                                        WHITE ^^m        PLATE I.  AV. WEIGHT  WHITE   144 67 LB.  COLORED 149.53 LB.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tz3d~pyfk-rjt4", "00000000-0000-0000-3284-02207E335D91", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Measurement Card for Clothing Patterns", "9918573882206676X44", null, "1921", "1921", "Although the Army Medical Department used these physical dimension measurements for its anthropological studies, the data collection work was authorized for collecting data to improve uniform manufacture. Page 61 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "HISTORY OF THE  WORK--VETERANS.  61  MEASUREMENT  CARD FOR  CLOTHING  PATTERNS                            DEMOBILIZATION-1919  Namc.  John  Doe  Army Sériai No.  278659               Home State-lîïï-York_____  IWhite__.H  i black_n  i black____________g  Negro _Q  iblack_D  Indian....._____  _Q  Chinese n  Japanese Q  Other_____________□  Place of observation..  Camo  Dix  Place of birth of—     „ ,,      United  States  Country.  Date of observation  S ej j'.;....1..?.»_ jjjj..  Initiais of officer in charge AlMjL.S..-              State or Province.                City or Toum.              New York                 _ New York City  Fathcr.......l!rî.i.t.eÉ...S.t*.t.e.E....................                   .^L-Y.?.**                  New  York City  United States  New  York  New  York City  Nationality of father's fathcr... .^r.r.T.Ç8.!!..............................  Nationality of mothcr's father.......Amer iç_8n_..............................  Nationality of father's mother..*?.6.1!.1.0*?}...............................  Nationality of mother's mother......*??.r.Ï5??.....................................  Native language of mother.....J;BË.1.}-.S.<X..................................................                                                                       I Flaxen......................D Dark brown......E                                                                         Light brown...............□ Clear red.........Q                                                                         Médium brown..........□ Red and black-0  Other noteworthy racial traits....................................______.....................                                                                        (Clear blue..................□ Light brown ....Q                                                                         Blue with brown 6pots.D Dark brown ....g  MEASUREMENT  CARD  FOR  CLOTHING  PATTERNS  0. height   1. Height, standing (stature)_________......__...............-...........   2. Span (maximum, botween flnger and tips of outstretehed arms'........   3. Height,sitting......................-.......-......-....................   i. Height of sternai notch----.....—........................-............   5. Height of pubis________......................----.....................------.....   6. Transverse dlam. of shoulders at level ofhead of humer!...................._......   7. Transverse dlam. of hips, levol of crests ofilla----------------....................   S. Transverse dlam. of chest at level of nipples, arms elevated and Ûe.xed-------.....-   B. Ant.-post. dlam. chest; level of nipples-------------------------..........— -  10. Second dorsal vertèbre to styloid procors of ulna (elbow beat, horizontal)--------  11. Circumference of neck, level of laryngeal prominence, p::pendicular to axis of necfc  12. Circumference of chest, level of nipples----------—------------------------.....  13. Circumference of waist, lerel of umbillcus—.......—.......-......................  14. Circumference of thigh (maximum)—.....---------------.................-.....  15. Circumference of leg, Just above patella----........................................  18. Circumference of knee, level of patella...........---.........----..........-......  17. Circumference of leg, Just below level of tuberosity of tibia.........................  18. Circumference of calf (maximum)...................-.......-........... ...........  1». Inslde length of leg, from gluteal fold to tip of internai malleolus of tibia-----  SO. Slieofshoewomif fltted aince Jury 15,1»10, onder par. 14,  S. R. 2S---------  MEASUREMENTS-ALL METRIC          ----145.0   160.1   Ï6Ï.0   \"86Ï0   132.0    76Ï8~    42.7    29Ï8'    28.8    21.0    68.0    38.0    88.7    83.0 .    55.2  ....?.?„• .9...    35..0 .    31.?_    34.7    64.0  ..-.&£_  Axtr**®   s—sms             Number ofmeesurer----r—-.....--------         . _  :  21. Height  of knee..........................f,3.4  22. Length  of forearm..................... ••• dô.V    DOTTED LINES INDICATE DIAMETERS   FULL LINES INDICATE CIRCUMFERENCES  NOS. 2. 3. AND 10 NOT  SHOWN ON FIGURE  PLATE II.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dbrn-7qbe-xnw9", "00000000-0000-0000-67C6-D90089736660", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Statistical Perforated Cards", "9918573882206676X45", null, "1921", "1921", "Three examples of the Hollerith punch cards to which the Army's anthropological data were entered for processing during and after World War I. Page 63 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "HISTORY OF THE WORK--VETKRAN;  63  STATISTICAL  PERFORATED  CARDS  m   r-                              . //                                                                    I       rie. 1                                                                                                                    ^IVwr.l                                                                                                                                     L'C     3». •»«                                                                                 ♦2        V'  -1 .Nu. hr.No.                                  Mil If                  ..L'    \\\\vt    ut.             0. In.          ■ .1.x L.1J     A.B.....b       It'hï.Ue:.                              ). ir.,                                                                  N\".        8       00249                           X       05              X       1       52      65 34                           37 14   4 S     S26                     2       _ll     4rt                                             11 S 10 20 l Me Yr. rj 1 21 ? 4 E2Î| 3 23î|       0   iCtll[>. 000    • •• Occup. 1 1 1 2 2 2 3 3 3           01 Nat 1 1 2 2 3 3      >• AS 1 2 3     • 0 0 l:<l. No. 1 1 1 2 2 2 3 3 3                       • 00000000 000 0 0 #0 M^n Number Cl Ser. No. . sua.-1 •• 1 1111111111 11 22222222 222^2 222 33333 03 313333333                                                                          0 0 0 0 0 ?..c \\\\vt. nt. • 1111 2 2^22 3 3 3 3 3                                0 0 U. In 1 1 2 2 •3    0 0 0 0 C. ExlB U 1111 2 2 2 2 |3 3 3           0 0 00 ï-B Phy. Def. 1111 2 2 02 3 03 3                           1 1 Nul. 00                                                                                                                                                                                                                                   s*  A i/o 0                                                                                                                                                                                                                                   \"3         4 4 4           4 4     4       4 4 4                   444*44444444 #4 4                                                                       4       4       4 4             4 4     4 4 4           4 44 4 4 4 4                            4       »!&  B 9      o 555                   5 C     5       5 5 5                   555555555555555                                                                 «       5       • 5             5i5 5           5       5 5 5   15 5 5                  5       25Î?          7 9     6 6 6           6 e     6       6 6 6                   66666666                                                        3 6 6 6 6 6 6           6       6       6 6             •6      6 6     6       B 6 6   6 6 6 1                 6       26 »                  7 7 7           7 7     7       707                     77777777                                                        7 7 7 7 7 7 7           7       7       7 7             7 7     7 7     7       f 7 7   7 7 7 7                 7       17 c,                8 8 8           & E     8       8 8 8                   388B|e|8                                                        3 8 8 8 8 8 8           8       8       8 8             8 8     8 8     8 8 8 8         8 8 8 8                 8       0             9 9 9           9 £     919 9 0                         9 9 9 9 9 9 9 1                                                 3 9 9 9 9 |9 9                  9       9 9             9 9     9 9     9       9 9 9   9 9 9 9                 9       19  W ivi^lassIrÇ&l2». Ililëlttl                                                                                                              1 J£l|.         ,?J                                                                                                                                                                                                                          Camp t |                I 1148389 NI 00| 000 1 00| 079 lui N». III Ace | Orra?. | «it. | Bi No.                                                                                                   1   nn.\\\\   ,n S&crair                     ento #2 Vir                                                                                             ,„ Doe, John L.                                                                                                       >  0 6        Age 1 1 2 2     ¥jf0 1 1 1 2 2 2                01 su. 1 1 2 2  k«o 0 El Ho. 1 1 1 2 2 2                        0 u.c. 1 2      0o< Suis 1 1 2 2                ) 0 1 2 «       0 0 El 1 1 2 2          0 0 C Et. 1 1 2 2               D 0 0 0 Cln. LB 1. 1111 l 2 2 2 0 0 0 0 MB Phj. M 1111 2 2|2            #0 0 Phj. Bet 1 1 1 2 2 2                       0 0 0 1 L. 4 T. V 1111          3 0 J. Tr. 1 12 000000000 z. Camp Man No. n • 1 •• 1 1 1 1 1 s3 222222222 le                                      .Vu.\"                                                                                                                                                                              COiiiT • i                                                                S2  3 3 4 4 3 3 3 4 4 4             3 C 4x4 3 3 3 4 4 4                     3 4     3 3 4 4 -               3 3 1 4 3 3             • 3 (           13 3 3  3|3 3           3 3 3                   • 3             3       3 3 3   3 3 393 3^                                » Ht. fi 5                                                                                          4 4             4«'             M||     4 4 4 4         4 4 4                   4 4 4 4         4       4 4 4 4 04 4 4 4ls                                            5 5     5 5 5           5 E     5 5 5                   5       5-II            15      5 €1            5 5             5 5 5 5 i 5 5 5         5 05                    5 5 5 E         5       5 • 5   5 5 5 5 5 5 §3                            C.Ex 3 4    6 6     6 6 6           6 e     66 6                    6       6 6             5 6     • 6             6 6             5 6 G 6 6 6 6 i         6 6 1                   6 6 6 6         6       6 6 6   6 6 6 6 6 6 5                                 7 7     7 7 7           7 7     7 «7                    7       7 7             77      7 7             7 7             ri??    7 7 7 7         7 7 7                   7 7 7 7         7       7 7 7   .777777 3                                 (la 3       8 8     8 8 8           8 e     8 8 8                   8       8 8             3 8     8 8             8 8             3 8 8 8 8 8 8 8         8 8 8                   8 8 te          1       B8 88 8|8|3 m                                     7   9.9     9 9 9           9 S     9.9 1                   9       9 9             3 91    9 9             9 9             3 9 9 9 9'9 9 9         9 9 9                   9 9 9 £         9       999999990                                         ■r                                                                                                                          fig. :                                                                                                            V   23 I68 I 38 I 89 83 55 7 5 X 10A I 10B IllNCClJ 12CH.C I 13 WT. C 1 14.TIWM . |i5LL6k||6C                                                                                                               64 4 1 19 LEG WI | S            32 1 TATE COL   01 Roce         3 HAIftC                9 ETE Ct        42 4 43 4 404 41 3      2 40 ii 0 30 te iec 1 31        60 1 HCHT '< ) 0 0 C 19. Léo w 1 1 1    1 61 1 25FAN    86 45 3S. H I3AKH                             r..,^ 278659 |camp Dix                                                                                                                                 ) 0 0 K 12CH C 1 1              0 0 1 1         0 0 1 1                         0 0 1 1 0 0     00 10           J! 0 % 1 ïi  32       1 1     0 0 2 SPAN 10   0 0 ( 3 S. H 3 1 1              ) 0 1   0 0 A SI, Ne 1 1               0 0 5h)«B l 1           O «S» 1 0 00 Et» 7 Mire 1 1 1           0 0 8CH. T» 1 1         0       000c 1111       0 0 ( 1 1                                                                                                         77  2 2     2 2     2 2 .           > 2     2 0             2 2             2       2 2 2           • 2             •       2 • 2 2 2 2 :   1 2 2           2 2             2. ?    323     2 32    2 2 2   20      2       i2              2II  S-       3 3     3 3     3 3 •           30      03              3 3             3       • •3            3 3             3       33lc    30.     i 3 3           30              3 3     33 3    3 33    3 3 3   03      3       3 3             •1  213       4 4     4 4     4 4<            M       4 4             4 4             •       4 44            4 4             4       4 4 4 4 4 4 <   J- 4 4          4 4             .4 4    343     4 34    401     |4 4    4       4 4             4 s1  ,,,'r.       5 G     5 5     5 5 !           3 5     5 5             5 5             5       5 5 5           5 5             5       5 5 5 E 5 5 !   j 5 £           5 5             • 1     351     l»35    5 5 E   5 5     5       5 5             s-r  AO       • 6     • 6     R0<             3 6     6 6             6 6             6       6 6 6           6 6             6       6 6 6l  >6 6 (  5 6 6           6 6             6 6     36 3    5 3G    06 6    6 6     6       6 6             le -|-  ,.   7 7     7 7     7 7 '           r 7     7 7             ••              7       7 7 7^          7 7             7       7 7 7 7 7 7 \"  7 7 7           7 7             7 7     • 3     7 37    7 7 7   7 7     7       7 /             7 *s si  i>9  8 8     8 8     • 8 i           i 8     8 8             8 8             8       8 8 8           8 8             8       8 8 8 £ • 84    10e             • 8             8 8     383     8 38    8 8 6   8 8     8       8 8             al3  21'      9 9     9 9     9 9 ï           3 9     9 9             9 9             9       9 9 9           •               9       9 9 9 £ 9 9     3 90            9 9             9 9     39b     9 39    9 9 £   9 9     9       9 9             0  PLATE III.    Fig. 1. Statistical card used for tabulating the statistics of the flrst million draft recruits (Pi).    Fig. 2. The same for the second million draft recruits (P2).    Fig. 3. The same for the spécial measurements of one hundred thousand vétérans, 1919.    ;:nu:;<;°— 21-----5", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2rt2-vvah.2ix9", "00000000-0000-0000-DFB3-AC43AA9AD29E", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Results of the Standard Army Physical Measurements--Age of Recruits", "9918573882206676X46", null, "1921", "1921", "This table compared the age of recruits examined for service in World War I with data on the ages of soldiers in the Union Army during the Civil War. Page 64 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "C.  RESULTS  OF  THE  STANDARD  ARMY  PHYSICAL  MEASUREMENTS.                                      I. AGE  OF RECRUITS.       Table 2, préparée! from material  published in Gould 2  and from material fur-  nished by  the  War Risk Bureau, gives  the  relative frequency  of  the  various  âges of officers and men serving in the Civil and World Wars.   It  is  apparent  that the great  majority  of the men measured  for  the data in this book were  between the âges of 18 and 31, inclusive.    (See Plate IV.)            Table 2.—Ages of soldiers (officers and men) serving in the Civil a and  World Wars.b  Age.  Civil War.  World War.  Number.  ^SoT  Number\"  50 and over.     127     330     771   2,763   6,430  133,653   90,624   71,745   98,766   75,230   61,818   54,329   48,787   42,357   36,254   37,383   26,269   30,196   19,383   23,580   19,401   17,064   20,414   15,278   12,851   11,379   10,409   14,869   7,992   11,585   10, 825   16,668   7,490   1,184     896     S74     59')   2,889  T°tal............................................................  1,019,456    0.12    .31    .74    2.63    6.13  127.35   86.36   68.36   94.11   71.69   61.76   51.76   46.49   40.36   31.55   35.62   25.03   28.78   18.47   22.47   18.49   16.26   19.45   14.56   12.25   13.70    9.92   14.17    7.62   11.01   10.32   15.88    7.14    1.13    . sô    ,S3    .56    2.75      16     140     935   12,846   62,849  122,977  152,635  293,161  506,426  440,581  381,321  328,185  283,276  235,901  214,133  187,010  160,735  117,316   47,890   20,967   16,407   13,318   10,992    9,356    9,086    8,039    6,747    5,165    4,067    3,438    3,077    2,560    2,050    1,680    1,543    1,237    5,038  1,000.01   3,673.133  Proportion   per 1,000.   0.00    .04    .25   3.50   17.11   33.48   41.56   79.81  137.87  119.94  103.81   89.34   77.11   64.22   58.30   50.91   43.76   31.94   13.04   5.71   4.47   3.63   2.99   2.55   2.47   2.19   1.84   1.41   1.11    .94    .84    .70    .56    .46    .42    .34   1.37  999.99  Average âge, Civil War, Gould's figures. 25.54, volunteer officers and enlisted men  Average âge, «vil War, Baxter's figures, 27.307 (Baxter, p. 51), drafted recruits, sùbstitutes, and late volunteers.    ^Gmdd?lg869™3ilnd 57             '       ^ men'  °ther data in this study fo* draft> enusted men only.  b Estimâted from âges furnished by 3,683,134 applicants for War Risk Insurance,  64", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-uqs9_jabg~vq2i", "00000000-0000-0000-12DF-C7802CD52BB1", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Age Distribution, Civil War Volumes and World War Troops", "9918573882206676X47", null, "1921", "1921", "Page 65 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "AC.i;S OF RECRUITS CIVIL  ANU WORLD  WAR.  65  AGE DISTRIBUTION  CIVIL WAR VOLS.. AND  WORLD WAR TROOPS.                          OFFICERS AND  ENLISTED  MEN  RATIOS PER 1000  NUMBCRS   140   135   130   125   120   115   110   105   100    95    90    85    80    75    70    65    60    55    50    45    40    35    30    25    20    15    10     5                                                               r       \\\\                                                                                                                                                                                                                                                                            \\\\      \\\\                                                              !                                                                                                                                                                                     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                                                                                                                                                    NUMBER 0 CIVIL WAR, WORLD WAR.                         F MEN                                                                                           1                                                                                                                                                                                     3 673.133                                                                                               ^~T^~\\\\ /                                                                                                                                                     Il III                                                                                                                                          Li '                                                                                                                          _                                                                                                                                       ~r-             h       _                               ■^TT    ^À^                               13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38. 39 40 41 42 43 44 45 46 47 48 49                           CIVIL -------     WORLD -------                  AVERAGE AGE.  CIVIL AND WORLD WAR  0 1  2 3 4 5  6  7  8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  CIVIL WAR (GOULD)    (VOLUMTEERSl  CIVIL WAR 'BAXTER |   (DRAFTED RECRUITS)  WORLD WAR    (ALL TROOPS  25.54    27.307    24.89  PLATE IV      (iould stated that appareiltly many who were under 18 or 21 gave their âges as such that they might be ableto  cnlist at the minimum âge of 18 (with consent) or at the minimum légal âge of 21.      BuxtorV drafted recruits iucluded ail troops raised during the draft period, that is draftees, substitutes, and late  volunteers.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-byqe.quww~7jhb", "00000000-0000-0000-0220-1DD1470D557C", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Mean stature by States, first million draft recruits", "9918573882206676X48", null, "1921", "1921", "This series of data showed the variation in soldiers' stature according to state of origin. Page 75 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "STATURE.  75  at the head of the list, while the States of the  Northeast, especially those en-    gaged in manufacturing, lie at the bottom of the  list (Rhode Island, Connec-    ticut,  Pennsylvania,  Xew York, Massachusetts, and New Jersey).   The high    stature of the men of the Southern States is due,  as indicated, in part to the    absence of récent immigration from  southeastern  Europe, and also in part to    the average tall stature of  Negroes.   The short stature of the population  of the    manufacturing  and  maritime States  of  the Northeast is  due in part to the    présence in them of members of the shortest European races.   In  the  upper    half of the table one finds also States like Kansas, Idaho, Oregon, Nebraska,  South Dakota, Iowa, and Minnesota, which are  populated largely by Nordics.    Table 13.—Mean stature by States, first million draft recruits; States arranged in order of standing        vith proportional weight and chest circumference at (expiration) for each inch of stature.  State.   Texas...............   Oklahoma...........   Mississippi..........   Tennessee...........   Arkansas...........   Kansas.............   Alaska.............   Colorado............   North Carolina......   Arizona.............   Idaho...............   Oregon..............   Nebraska............   South Dakota.......   Iowa................   Minnesota...........   Kentucky...........   Alabama............   Montana............   Georgia.............   Washington.........   Missouri.............   North Dakota.......   West Virginia.......   Utah................   Nevada.............   Virginia.............   Wyoming...........   Indiana.............   California...........   South Carolina......   District of Columbia   Louisiana...........   Wisconsin...........   Florida..............   New Mexico.........   Illinois..............   Ohio................   Maine...............   Michigan............   Delaware............   Vermont............   Maryland...........   New Hampshire.....   New Jersey..........   Massachusetts.......   New York...........   Pennsylvania.......  Connecticut.........   Rhode Island.......  Number of men measured.        Mean    height. Mean weight. Mean height.       Mean chest. Mean height.      Inches. Centimeters.    Pounds. Inches.  34,531      68.40   173. 74 2.079   0.483  19,429        68.28   173.43  2.084   .485  8,543  68.27   173.41  2.10    .485  14,426 68.27   173.41  2.052   .483  10,111 68.20   173.23  2.071   .486  9,571  68.20   173.23  2.107   .487  106     68.15   173.10  2.208   .493  6,635  68.15   173.10  2.069   .485  14,668 68.15   173.10  2.076   .487  3,850  68.13   173.05  2.099   .488  4,031  68.10   172.97  2.133   .495  2,748  68.09   172.95  2.150   .492  10,774 68.08   172.92  2.126   .488  3,892  68.05   172.85  2.159   .493  19,537 68.04   172.82  2. 126  .491  27,341 68.04   172.82  2.15    .494  15,502 68.02   172.77  2.058   .484  15,988 68.01   172.75  2.077   .485  11,648 68.01   172.75  2.151   .492  20,305 67.99   172.69  2.071   .488  13,316 67.96   172.62  2.140   .492  24,964 67.95   172.59  2.081   .486  6,444  67.92   172.52  2.163   .497  12,367 67.87   172.39  2.085   .490  4,568  67.85   172.34  2.109   .488  1,441  67.83   172.29  2.143   .497  17,616 67.80   172.21  2.070   .489  1,927  67.79   172.19  2.13    .492  23,194 67.75   172.09  2.090   .489  35,461 67.67   171.88  2.127   .493  9,343  67.64   171.81  2.077   .489  4,486  67.63   171.78  2.077   .482  12,356 67.60   171.70  2.065   .489  18,433 67.60   171.70  2.137   .496  5,895  67.58   171.65  2.061   .489  2,690  67.50   171.45  2.051   .491  69,491 67.40   171.20  2.103   .493  52,814 67.38   171.15  2.098   .491  3,315  67.28   170.89  2.10    .497  41,872 67.23   170.76  2.11    .496  1,891  67.19   170.66  2.075   .492  2,077  67.12   170.48  2.091   .498  9,192  67.08   170.38  2.09    .494  2,240  66.97   170.10  2.095   .495  29,958 66.77   169.60  2.079   .498  29,534 66.76   169.57  2.07    .496  87,818 66.72   169.47  2.091   .497  77,186 66.72   169.47  2.094   .496  13,585 66.71   169.44  2.095   .501  3,928  66.40   168.66  2.06    .494       (b)  Demobilized men.—Table  14 gives  thc distribution  of  mean  stature  of men  at demobilization, by  States.   In  this  table the States  are arranged  in order of mean stature of men,  the States with the tallest men being placed    at the top of the table.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-a7w8.6i32.pqx2", "00000000-0000-0000-1A0E-A308394A972F", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Mean stature, by states, of soldiers at demobilization (1919), and increase in stature of soldiers at demobilization over stature of recruits.", "9918573882206676X49", null, "1921", "1921", "Page 76 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "(C)                                   ARMY  ANTHROPOLOOY.  Taihe  I 1.  -Mean stature, by States,  of soldiers at demobilization (1919).  United States.......  Alaska..............  Mississippi..........  Tennessee...........  Texas...............  Alabama............  Georgia.............  Oklahoma...........  Nebraska...........  Kansas..............  Arkansas............  South Dakota.......  Oregon..............  Washington.........  Montana............  Arizona.............  South Carolina......  Minnesota...........  Iowa................  Idaho...............  Florida.............  North Carolina......  West Virginia.......  Utah................  Wyoming...........  Kentucky...........  Colorado............  Virginia.............  Missouri.............  North Dakota.......  Nevada.............  California...........  Louisiana...........  New Mexico.........  Wisconsin...........  Indiana.............  Illinois..............  District of Oolumbia  Ohio................  Michigan............  Delaware............  Maryland...........  Vermont............  Maine...............  Connecticut.........  Pennsylvania.......  New Jersey.........  New York..........  New Hampshire___  Massachusetts.......  Rhode Island.......   Number   of men  measured.         102,301         13      2,099      2,807      4,361      1,930      3,397      2,310        819      1,012      2,576        416      1,069      2,025        264        130        828      1,950      1,609        161      1,022      1,815      1,686        101         80      2,921        225      1,920      2,836        358         18        481      2,070        229      2,675      3,994      6,687        231      7,076      3,715        300      1,-138        446        693        996      10,874      3,180      9,207        413      4,782        403  Mean ;  tature.  Inches.      Centime-ters.  67.72  172. on  69.43        170. 3.-,  68.61     171.2s  68.61 171.26  68.60 174.21  68.57 174.16  68.51 174.01  68.44 173.8»  68.44 173.84  68.43 173.82  68.41 173.76  68.39 173.70  68.38 173.68  68.38 173.67  68.35 173.60  68.33 173.55  68.32 173.51  68.31 173.51  68.28 173.42  68.26 173.39  68.22 173.28  68.22 173.27  68.20 173.21  68.19 173.21  68.16 173 13  68.13 173.05  68.12 173.02  68.01 172.75  67.98 172.66  67.96 172.61  67.91 172.50  67.91 172.49  67.86 172.36  67.82 172.27  67.79 172.18  67.73 172.03  67.65 171.83  67.60 171.70  67.48 171.39  67.32 170 99  67.26 170.83  67.20 170.70  67.19 170.67  67.17 170.60  67.08 170.38  67.01 170.21  66.93 169.99  66.92 169.98  66. S0        169.67  66.77 169.60  66.54 169.00  Table 15.—Increase  in stature  of  soldiers  at demobilization  over  stature of  recruits,  1917-1919                                                   ( inches).  State.  Increase  (inches).  United States..  Alaska.........  South Carolina.  Florida........  Alabama......  Georgia........  Washington...  Connecticut___  Wyoming......  Nebraska......  Tennessee......  Utah...........  Mississippi.....  Montana.......  South Dakota..  West Virginia..  New Mexico...  Oregon........  Pennsylvania..  Minnesota.....  Louisiana......  Illinois.........  California......  Iowa...........  Kansas........  Arkansas......  State.  Increase  (inches).  Virginia.............  New York...........  Arizona..............  Texas................  Wisconsin...........  Idaho................  New Jersey..........  Oklahoma\"...........  Rhode Island........  Maryland............  Kentucky..........  Ohio.................  Michigan............  Nevada..............  North Carolina.......  Vermont.............  Delaware............  North Dakota.......  Missouri.............  Massachusetts.......  Indiana..............  Colorado.............  District of Columbia.  Maine................  New Hampshire.....  >.21  .20  .20  .20  .19  .16  .16  .16  .14  .12  .11  .10  .0!)  .OS  .07  .07  .07  .04  .03  .01  -.02  -.03  -.03  -.11  -.17", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-2978.ecm5_wk7i", "00000000-0000-0000-B9D1-B2CE5CC4DEEF", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Characteristics and composition of the population of the various sections of the United States", "9918573882206676X50", null, "1921", "1921", "The Army Medical Department's analysis of soldiers' physical status drew partly on characteristics of the troops' regions of origin, as set out in this table. Page 80-87 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "8", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "80      ARMY  ANTHROPOLOGY.      Table 17.—Characteristics and  composition of  thc popu-  Désignât ion of     section.  Characteristics.  Alabama 1..  Alabama 2.  Alabama 3.  Alabama 4.  Alabama 5.  Arizona !..  Arizona 2...  Arkansas  1.  Arkansas  2.    Arkansas 3.    California  1.  California 2.  California 3.  California 4...    California 5...  Colorado 1___  Colorado 2___  Colorado 3___  Colorado 4___    Colorado 5___  Colorado 6___  Connecticut 1.  Connecticut 2.......    Delaware............  District of Columbia  Floridal............  Florida2............  Florida3............  Florida4............  Georgia 1............  Georgia 2.  Idaho.  Illinois 1.    Illinois 2.    Illinois 3.    Illinois 4.    Illinois 5.  Illinois 6.  Illinois 7.  Illinois 8.  Indiana 1.  Indiana 2.  Indiana 3........    Iowa 1..........           'Indian.  Mining and manufacturing area.  Large Negro population........  Large native white poputation.  Large Negro population........  Urban and suburban area......  Large  Indian  population,    sparsely settled.  Chiefly white population.......  Negro, Mississippi bottoms.....,  Large native white  population,    hill country.  Large native white population..    Chiefly agricultural area.......  Mining area.  Sparsely populated.  Urban area.  .do.  Large native white population.  Russian population............  English population.............  Prevailingly agricultural.......   Urban population..............     Austrian and Italian population  /Prevailingly  agricultural and  \\\\  near metropolitan.     Manufacturing area.............   State un divided................   District un divided..............   More white and maritime.......   MoreNegroand rural population  fCuban, Spanish,  West Indian  \\\\  population.   Peninsular......................   Mixed population,  native white     predominating.   Large Negro population.........  State un divided.   Densely populated.............  fMixed native and foreign popu-  lo lation.   Agricultural area, native.......     Largely German population___   Urban area.....................   Negro population (Egypt)......   Agricultural area...............,   Agriculture and manufacturing     area.   Manufacturing..................   Agricultural, considérable Ger-     man.     Agricultural area, native stock..  fForeign  white,  German  and  \\\\  Scandinavian.             * Chinese.    760,740    563,441    577,627    122,817     95,308     57,953      146,371    641,940    212,005      720,504    1,433,895      93,226         114,318         319,198       416,912       108,622      89,813      78,716       139,574       213,381       159,918     \\\\ 400,100       714,656       202,322     331,069     248,836     220,302     \\\\  21,563       261,918   1,334,222       1,274,899         325,594         434,972     \\\\ 753,575       995,129       344,621     2,185,283        52,591     805,587     266,833         282,521       128,679       2,289,676     U.442.410      • Japanese.  SI  49.0  44.0  35.0  31.0  41.0   2.9  36.0  19.0    30.0    16.1  2.5   9,689. 0         3.4       8.0      10.0         5.0     3,679.0         8.0       114.7       536.5       103.0   5,518.0      18.7      21.0        19.0         8.7      43.0          45.0           3.9         192.5        68.2        51.0        80.0    11,812.0        80.0      49.0      45.3         117.0        37.0          76.0        38.0  Citiesof 25,000or       over.  Birmingham.  Montgomery.             54. Ô         12.2          38.4          17.6 3.4      11.3  fOakland (Sacramento.....       lit. 9 10.5   43.9  Los Angeles___    San Francisco..  Colorado Springs    Denver......    Pueblo.......  Norwich.....  fNew Haven.  \\\\Bridgeport..   Wilmington.   Washington.   Jackson ville.  Tampa.  Atlanta.  fSavannah.  \\\\Augusta..  rJoliet.........  lAurora.........   Peoria........   Rockford......  'Springfleld___   Decatur........  26.9  14.8   5.3  100.0   18.1   28.8   29.4     33.5    100.0     46.5     78.2   48.0  100.0   35.0   14.1     92.5     30.9   19.4      121.9       21.5  East St. Louis.  Chicago.......      Bloominçton..  South Bend.  flndianapolis..  lEvansville...  /SiouxCity...  I Davenport...  J-63.5    J43.9    V24.3     45.3    100.0     41.9   31.1   28.2       69.3     18.6      j-40.4    129.6  1 Russian.83.2 52.9 20.4 60.4 71.0 54.1 50.6 76.2 82.5 50.7                                                           Cn CO tO 4k CO Ol                       63.2 50.4 54.2 40.5 16.8 55.9 68.6 37.3 62.5                                                    CO o c» 50.1 52.4 44.3          OS Cncn-4 10 CO 4k 4k CO --4 Cn OlUIS kj                                co' to  47.2 57.6               42.8 41.7 96.9 83.9 49.6                        71.5 2«.5 67.6 26.9 46.3 34.2                                           Native p:ir-entage.     2 o o F  10.8 29.6 41.8 7.4 20.2 31.5 27.0 16.8 11.0 34.2                                                             00 to                   to eo i—k-i CO 1— ~4 1— t-\"COCOtO i-, i-, coi-, e» ©eoenoo                                                  CO Ol CO        to to to CO 10 00 en CO -4              H WHH W 00 k^coen Ci to h- oc -a co                             IO co 4k        -■ to \"0 -4 ji eo              tO IO Cn to i-* tO CO to co — z r                       CO -4 tO — en to ce en to                                               Foreign par-entage.       4.4 13.5 35.8 2.1 8.2 14.2 21.8 6.4 3.9 14.8                                                                »-» to Ol CO O CO                       i-1 to to » * ki-JÏ 4k Oi OS O* CO COO^O)                                                       CO 1-0 o        tO tO !-• 4k tO 00 CO 4k IO             ,_. H\" eo O -4*00 H kj enen en 4k                             CO o    M co y> co              o O i-> O CO Ci,               IO o, OCO      e, co i-, 4k to k- en co h-                                           Foreign-born white.       to to en .u ~. t                    CO HO H ÇO ►-> OOO, O 00 en                                    b b                     Cn to to to k- CO00 ^1 to o coen !-■                                    j, 4k toi-\" klOOOÇn » en Cn 4k                4k      H M JO to CO en         4k -110 4* 4k                           to 4k   H- tO           to 00 O -4              Cn Cn.-, «0>-> 4k-*3CO -4tO to to k- p en en oo oo o en cn                                             Negro.                                                                                ikCOH                                                                                                                                                           p en                                                    Indian, Chinosi', Japanese.                                                                                                                                                                                 ■ ffjj OOHOl                    !-• K. k-> ^ ÇO k- M IO JO COCn-^cncnoCncni—'                   Cn -4                   co en en                                                          4.3 17.4 19.5 3.5 10.2 15.2 17.2 8.1 5.6 15.9                                                               o               o       ÇO 00                                           4k eo ob        4k      Ol ÇO ^1 >-, en en             00 COO0 Cn —1                           p CO    ÇO 4k 4k eo             en ~4           IO to   Cn-l                                            German.   to Cn               -• tO k-i -o O in       «k MH -si tO - OiO Cn h-                                .o      CO ÇO en en                     4k                      kJ O                    4k en toto      en to   p JO 4k en CO 00                k- tOf-k-, ÇO co oo to en h-                           ;0 00   -a o            ÇO -4           IO      o o     -       -       -       -               Irish.    in                                  tO H* o o>                      to 4k           v3 30   ÇO 4k                   to en                   toeo    ÇO 4k   ÇO to ÇO O 00 cO                00 k- 00 CO Ol                          ÇO Cn   IO p Cn o               to 00   —:                                                              English.          es                                  JOOH to COCOCnOO                H- k-CO 4k , >«k004keO •                                                                               t-* tO CO CO 4k tO 00 4k k- k-> H- H- '/\"-' J-- k- J-» -*4Cn©cncnC004kCO-4tOCOCOOcno>                                                          t3 IO   c       #.k                                                                             Ausirian and Rus-sian.    00 o                to ■ 4k *. o            p CO                    ;-4 CO          en to   Çn -4                                           : !     CO to   [O tO ÇO Cn O -J                IO 4k ÇO J-» 4k tO 00 CO tO 4k                          r;              en      en                                      -                       -       Scandinavian.                 ! ; o                   CO CO                   to to           0 to                    to                      to ton-         ÇO en   çn oo to I-\" en CO             ►-> to k- O             tO 4k           co              en o    co 00           IO                                                      Italian.                                                                                                                                                      00      çn • ■          :                                               i               — ■;■           -       -                                       Canadian, French.                                            '• Oi                           '       CO      CO              -                               ; ;     i       '• CO o         r- co to eo 00                          to CO   0 ►-• O 4k              o o                                                             _       Canadian, other.                     ! '. en ! ! co                  ! -^                            o '•                                                                    to              i i                                                                                                                                     Ilungarian.                                                                                                           to o: to 4k00tO                 o • '•          i                       : \t\t\t\tcn o \t\t\tSj s.\t\t\t\t\t Mexican.   \t\t \t\t\t\t\t\to \ti\t\t\t\t\t \t\toeo o \t! -\t\t■ eo \t\t\t\t \t\t\t\t\t\t\t Scotch.   82  ARMY  ANTHROPOLOGY.  Table 17.—Characteristics and  composition of the  population  Désignation of     section.  Iowa 2...    Kansas 1.  Kansas 2___    Kentucky 1.    Kentucky 2.    Louisiana L.  Louisiana 2.  Louisiana 3.  Maine 1.....  Maine 2___    Maine 3....  Maryland 1......  Maryland 2......  Maryland 3......  Maryland 4......  Massachusetts 1.    Massachusetts 2.  Massachusetts 3.  Massachusetts 4.  Michigan 1.    Michigan 2.    Michigan 3.    Michigan 4.  Michigan 5.  Minnesota 1.  Minnesota 2.  Minnesota 3.    Minnesota 4.    Mississippi 1.    Mississippi 2.  Missouri 1.    Missouri 2.  Missouri 3.    Missouri 4.    Montana 1.  Montana 2.  Nebraska 1.    Nebraska 2.  Nevada 1..........    New Hampshire*l.    New Hampshire 2.    New Jersey 1......    New Jersey 2......    New Jersey 3......               i Austrian.  Characteristics.  Native White..................  Russian population............    Native and German population  Mountainous area, native white    Agricultural area...............  Mississippi bottoms and upland,    large Negro population.  Urban area......................  Rural, chiefly white population  English Canadian...............  Native white stock, maritime..  French Canadian population___    Urban area.....................  Peninsular area................  Large white population........  Large Negro population........    Mountainous area..............    Manufacturing center..........  Peninsular région..............    Urban area.....................   Finnish population.............    [Prevailingly native white popu-'  \\\\  lation.     Foreign population..............     Urban area....................   Dutch and other foreign popu-     lation.   Scandinavian population.......,   German   and   Scandinavian     population.     Scandinavians and Finns.......  Urban area, \"Twin Cities\"......    Rural area, large Negro popu-    lation.  Rural area, large native white    population.    Native white, agricultural.......  Mississippi bottoms,  considér-    able Negro population.  Native white, Ozark région__  Urban area.....................,   Mimng area, foreign population..   Sparsely settled,  mountainous     area.     German and Irish, foreign stocks.  f German, Austrian, and Russian  \\\\  stocks.    I State undivided, sparse popula-  I  tion.  Mountainous area..............    Manufacturing area............    Densely populated.............    Plains section, rural............      rountainous area plus Atlantic    County.                       * Russian.   782,361     198,998    ,491,951     569,797    ,720,108     599,548     339,075   717,765   222,741   124,729     394,901     680,834     176,412   400,354    43,741     148,850   465,766     259,078     558,953   752,212       207,388     557,155    ,029,399     714,715      , 936,845     510,181     159,280     687,029     225,098   150,955       776,717     413,497        81,875      88,721     341,851    , 514,588     733,624     288,955       44.0       12.0       23.0       44.0       63.0       36.8    1,695.0     24.8     13.0     37.0       37.0    1,001.0       65.0     77.0     41.0       89.0  306,884 454.0  179,345 144.0  731,388 14,341.0  206,943 21.0  158,767 34.0  613,048 65.9  /Kansas City.  \\\\Wichita.....      12.0      31.0          15.0       766.0        45.0        32.0          41.0        38.0        24.0    11,263.0        5.6      1.4          13.0        23.0     19.0       75.0    2,145.0      177.6      107.9  Cities of 25,000 or       over.  Des Moines.  /Louisville..  \\\\Covington..   Shreveport.  New Orléans.  fPortland..  \\\\Lewiston..     Baltimore.   Pittsfield.....  JWorcester___  \\\\Fall River...   Brockton.....  /Boston.........I  \\\\Chelsea......../  'Grand Rapids.  Kalamazoo—  ^aginaw.  Détroit...   Duluth......    /Minneapolis.  /Mmneapc  \\\\St. Paul..  /Kansas City..  \\\\St. Joseph___  St. Louis.  Butte....  /Omaha..  \\\\Lincoln.  f Manchester.  (Nashua___  /Newark___  VerseyCity.  f Trenton___  \\\\Camden___  Atlantic City....  'Japaneses \ten 4k -0 15.7 30.9 27.3 64.5 81.4 76.6 94.4 39.3 37.5 51.5 54.3 52.9 33.1 60.8 51.6 28.7 \t\t\t \t\t\t\t\t\t\t \t\t\t\t.oto oco ->■ to O -4 \t4k O\tp en en\tCD m en\teo eo eo Cn -4\tCn-4p OCO en 0 4-01 \t\t4k CO en -4 43.5 61.0 59.3 86.1 \t\t\tCO -4 00 4k \tCO en 4k es O M\t-4 eo Native par-entage. 3 < g  en \tto\tco to to to co to toeo eo >-■ i-, 4k co ■.4 4k k- en «o co eok-> en eo o o o oo en en en en en co co 4k coc0 4kco co co oo eo \t\t\t \t\t\t\t\t\t\t \t\t\t\t«. 4. CO 4k kjco k- o XO CO 4k \t\tW >4 en 4k 4k\tCO 00 to to\teo 4k -4 eo o -4 k-,-4 1-, COCO -4 \t\tg » to\tto to -4COtOk-00CO-4CO \t\t\tco en !_> co \t-4 M. en\ttO -4 -4\tForeign par-entage.   en -o \t00 k- CO -4 tO 4k Cn 0000 00 k- CO 4k cn -4 4k o co o k-en eo 4k k- 4k en \t\t\t\t\t\t\t \t\t\t\t\tIO -4 J, to \to\tto to oen — to — eo 35 eo o en \tO o 4k en\teo to 00 co Cn 0» 8 CO\tM to to ■«4 COI--00 4kW   \tto CO\t35 CO en en i—oo ooeo-4 to \t\t\tto to o to \teo\t-4 4k to -4 -4 en\tForeign-born white.   p to \tp en *° . : to ►\"• • \t\tp en k- co coen 4k  \t\t\t\t\tu\to to .0 to -o M 4k k4 10 o\t00\t•\ten to \ten 4k\tJ_, co\tto o 00\t00\t«kk->C0 kJ4.H oo oo en \t co\tto\teoto 4kp tO tO 4k CO \t\t\ten h p 4k O *■ \tto en p 4k eo\tit\tNegro.    : : : \t\t\t\tk- »_ ço _«. ». p *-■ k-k- 4k k- Cn k- co  \t\t\t\t\t\t\t \t\t\t\tto\t2 \t\t4k\teo \t\t\t\t\t\t \t\ti\t\t\t\t \tto\t\t\t\tIndian, Chinese, Japanese.   4k \tp en 4k b\t\to 4k w to Cnp p CO Cn tO tOCn O  \t\t\t\t\ten en 00\t4k -4 \t\to to Cn en tOkJ top coco 00 4-»4 en \t3i eo <l to\tp to to -4 o\ten\t4k to\t\tp co \t\t;\t p cn\t\tp 00 \t\tp 4k p\t4k CO German.   CO \tp en\tp o \t35 ■a _ p en 4k \t\tto ooo p 351— O \t \tto\t\t\t\t4k to oo tOk-i enco k- U *k h* \tto cn\tto 00 to to o CO en (k co\tP 4k U\t\t p 4k\tU Cn toco\t p\t\tto\t\ten \tto\t\tIrish.   to 4k \tto\tÇO en \tco\tMi 4k o b \tto\t— 4k Op t-oo co \t\t\t \t\t\ten\tcO i\tk-> tO en oo \tto en\to 00 en\tto CO to co 4k -4 to en\t \ti *\t\t\t; \t\t\t\t\t\tCO o\tH* en English.   t0k-4k J0^4 4k O 4k 4kk-» k^ 4k  \t\t\ten\t{-* tO p k. ÇO to tocn coco \t\t\t— kÇkeoto en en en en \t\t\t\t \ttoptop ^eo-kicn \t\ttOk-CC4k ►-Çn*. toeneo \tCn\ttop 4k Cn \tp to\tw eo k-ipco *k 00 COCO 4k \t\t\t\t\tenj-, 4k-4 eo\ti\t\t \t\t\t\t\"\"* e. to ;_ 00\t\tAustrian and Rus-sian.    \t\t\t\t \ttO 35\t35 .n 35 4k in-kj 45-4 \t \t\t\t\t\ttO to o M\tk-eo 35 ^J 00 4k to 4k\t\t to\tto fo Cn\tM\tto o \t\t\t: \t\to\t\t\t\t \t\tt-â en J3 J3\tto co Scandinavian.   p -JJ \t\t<k p K CO \t\ten\t\t MM H en 4k en \t \t\t\t\t\t\t\t \t-1\t\t\t\t4k 4k ^4 eo eo\tto 00 eo to \t\tU\t\t 1 f!*\" , eooo \t\t\tto to \t\t\t\t\tItalian.    \t\t\t ^4 co\tto CO \t\t\t \t\t\t\t\t\t\tto to co CO\t4k\t\too to\t05 en »\tto k4 o \"4 -4 en\t \t\t\to en p oo\t\t\t \t\t\t\t\t\tCanadian, French.   \t\t \t\t4k -0 ÇO en\tto Cn \teo 4k «*4 I-1 \t\t\t\t \t\t\tto 4k p 4k\tk-tO tO p en kJ \tp p CO\tM to -o\tp o p\t4k\t \t\ti\tp\ttop coco  \t\t\t\t\t\t\t Canadian, other.   to \t\teo to 4k tO  \t\t\t\t\t\t4k \t\t\t\t\t\t : :\t\ten \t\tr j\t\t \t\t\t\t\t\t\t \t\t\t\t\t\t\t \t\tHungarian.    \t\t\t\t\t \t\t\t\t\t\t\t \t\t\t\t\t\t\t \t\t:\t\t\t\t \t\t:\t\t\t\t \t\t\t\t\t\t\t Mexican.   \t\t \tr\t\t\t\t\t en\tto\t\t \t\t\t\t\t\t\t \t\t\t\tr\t\t4k M\t\t\t\t: \t\t\t\t\t\t \t\t\t\tScotch.   84  ARMY  ANTHROPOLOGY.  Tablk IT.—Characteristics and composition of.thc population  32  35  36  37  Désignation of     section.  New Mexico 1.  New Mexico 2.  New Mexico 3.  New York 1...  New York2...  New York 3...  New York 4...  New York 5...  New York 6...  New York7.....,  New York 8.....  North Carolina 1.  North Carolina 2.  North Carolina 3.  North Carolina 4.  North Carolina 5.  North Carolina 6.  North Dakota L.  North Dakota 2.  North Dakota 3.  Ohiol..........,  Ohio 2.......  Ohio 3.......  Ohio 4........  Oklahoma 1.  Oklahoma 2.  Oregon 1.....  Oregon 2.  Pennsylvania 1..  Pennsylvania 2..  Pennsylvania 3..  Pennsylvania 4..  Pennsylvania 5..  Pmnsylvania6..  Pennsylvania 7..  Rhode Island___  South Carolina 1  South Carolina 2.  South Carolina 3.  South Dakota L.  South Dakota 2..  South Dakota...  Tennessee 1.......  Tennessee 2......,  Tennessee 3.......  Characteristics.  Indian population..............  Native white population.......  Noteworthy Mexican élément...  Suburban territory.............  Urban area, densely populated...  Eastern manufacturing région...  Western manufacturing région...  Mountainous Catskill région___  Urban area......................  Agricultural and dairying.....  Mountainous Adirondack area...  Sparsely populated mountainous    area.  Intermediate...................  Native white of Scotch origin__  Large Negro population.........  Island and peninsular area.....  Remainder of State............  Scandinavian   and  Canadian    population.  Scandinavian population.......  Russian population.............  Dense foreign population........  Intermediate.  Agricultural area...............  Urban area......................  Marked  Indian  and  Negro    population.  Chiefly white population........  Fairlv densely populated........  (Columbia  River  Valley  and  <  coastal dry  plain,  sparsely  l  populated....................  Urban area.....................   Rural area, native stock..   Mining area...............   Coal mining...............   Manufacturing............   Rural area................  /Allegheny County plus a smalll  \\\\  rural area....................f   State undivided.................   Native white...................   Large Negro population___\" \"..   Peninsular and rural areas......   Dry farming area................   Large Russian population.......   Indian population.............   Negroes, Mississippi bottoms....   Agricultural région..............   Mountainous région.......      59,970     212,657      54,614     565,449   4,766,883     658,978   1,361,257     284,857     423,715     774,620     277,855     375,905     657,162     296,425     651,669      55,975     133,408     113,603     262,681     200,772     989,804     919,823   2,493,883     363,591     615,973   1,041,182     445,464     l 227,   1,549,008   1,877,385   1,067,487    357,356    750,892    892,495   1,363,333    542,610    300,348    638,941    576,111    480,230     87,826     15,832    352,510  1,148,013    683,266  5 o      2.0      3.0      1.7     210.0  16,667.0     85.0     141.0     101.0  301      62.0      25.0      38.0      62.0      40.0      51.0      19.0      29.0      12.0       8.0       6.0     478.0     114.0      81.0   7,279.0      24.0      23.0      29.5         2.8  11,647.0    132.0    245.0    118.5    182.0      74.0    181.0    508.0      77.0      50.0      41.0      9.0      8.0  Cities of 25,000 or       over.  /Yonkers.....  \\\\Mount Vernon..   New York City..  /Albany.........  \\\\Schenectady.   iRochester___   Syracuse.....   Newburgh...   Kingston.....   Buffalo......   18.5   13.1  1.57.9  100.0  fBinghamton.  \\\\Elmira........  Charlotte.  Wilmington.  /Cleveland___  \\\\Toledo........  /Youngstown.  \\\\Akron.........  /Columbus___  \\\\Dayton.......   Cincinnati.....   Muscogee......   1.0  57.5  51.5  51.5  Oklahoma City  Portland.......   Philadelphia...  /Reading......  \\\\Harrisburg.....  fScranton.....  \\\\Wilkcs-Barre...  /Johnstown...  \\\\Altoona......  /Erie..........  \\ ew Castle...  /Pittsburgh...  \\\\McKeesport..  /Providence...  (Pawtucket...  Columbia..  Charleston.  /Memphis.....  \\ ash ville.......   Chattanooga___  W6.3  W.7  \\\\ 39.9  100.0  J37.7   26.5    7.4   21.7    3.8   16.1    0.0   19.3   10.9   12.8  W.3  J51.3  J38.2  100.0   17.2  20.6  56.9  100.0  J42.5   66.7   33.7   37.7   40.5   •70.4  j-96.7   16.9   12.8   16.1   15.2    4.2   9.5  27.8  12.9                         COMPOSITION  OF  SECTIONS.        cf  the  various  sections  of  the  United  States—Continued.  85  Native white. a u O ,o , co bjci3 eo o d bo eu co 05 a 3 ® O w CP 3 ■ ta B g< ■O B B c3 S eu o d 3\tça 3 «•a .2 3 < d 2 '> c« a •S a co ai B .2 ■3 -d e> 3 eu h. a\" 2 -3 a 3 d O u eu s) 0 b\" 2 •3 (3 B C3 O ri .2 c3 60 B 3 W à a 0 '53   il, C3 o.„.-\"A a 6 e? ta c <y \t \t\t\t\t\t\t\t \t\t\tsi 0 u ai  61.1 86.9 61.5 44.7 19.3 59.4 47.8 60.4 28.2 70.8 62.5 90.8 74.7 60.9 51.9 69.6 57.1 21.2 31.8 27.3 33.1 64.7 78.7 42.6 72.6 82.7 55.5 74.5 37.7 79.5 42.5 61.3 56.8 64.1 45.6 29.4 67.8 39.5 35.7 44.7 33.5 8.1 54.5 74.1 89.5 3.8 6.2 19.8 27.6 38.1 24.2 30.9 20.0 43.2 17.9 24.7 .5 .4 .4 .3 .3 .9 47.7 43.3 41.4 37.1 20.0 13.7 36.4 2.9 7.2 23.1 14.3 32.1 9.8 32.5 18.1 19.7 20.5 29.3 35.9 .4 .5 1.2 37.2 43.8 1.6 .8 2.4 1.1 6.2 5.0 16.8 24.6 40.4 15.7 20.6 16.0 28.0 10.5 12.0 .2 .3 .2 .2 .1 .7 28.6 24.2 29.9 28.4 15.0 4.8 15.6 1.5 3.7 18.5 9.1 24.7 7.9 23.8 18.4 22.2 14.8 22.3 32.8 .3 .3 .6 16.8 22.3 2.5 .4 1.1 .6 .1 .6 .6 2.9 1.9 .7 .5 2.5 .4 .6 .2 8.3 24.7 38.1 47.3 29.8 41.2 1.3 1.1 2.8 5.4 13.7 5.0 .3 .1 5.4 2.6 .2 2.2 1.3 .6 2.7 1.8 31.4 59.9 62.2 '29.1 U.2 11.3' \t\t \t\t\t\t\t\t1.6 1.5 14.3    \t\t\t\t1.4 1.2 7.3 12.7 6.6 10.8 5.8 27.9 4.9 1.1  \t\t\t1.1\t \t\t\t   \t\t\t\t\t\t\t \t\t\t\t\t\t   \t\t\t\t 10.6 11.7 8.2 7.6 7.6 6.1 5.3 5.4 2.8 2.3 2.4 3.6 2.1 2.4 1.8 /2 2.8 IM.O /2 6.1 \\\\315.1 /2 1.6 \\\\3 2.7 /2 2.1 \\\\3 2.7 /2 1.8 V<2.6 /23.4 V<3.8 3 1.0 }..... }..... }..... }..... }..... }..... 2.4 8.3 11.1 3.7 4.7 5.4 4.4 2.4 1.0 \t\t1.5 2.3  1.1   \t\t\t\t\t\t\t \t\t\t\t\t\t   \t\t\t\t \t\t\t\t\t1.8 \t\t\t   \t\t\t\t\t\t \t\t\t\t3.4 \t\t  \t \t\t\t\t\t\t\t \t\t\t\t\t   \t\t\t\t\t \t\t\t\t\t4.7 \t\t   \t\t\t\t\t\t\t \t\t\t\t\t\t   \t\t\t\t \t\t\t\t\t6.7 5.1\t\t\t   \t\t\t \t\t\t\t\t\t\t \t\t  \t \t\t\t\t\t\t\t \t\t\t\t\t   \t\t\t\t\t \t\t\t\t\t\t\t   \t\t\t \t\t\t\t\t\t\t \t\t\t   \t\t\t\t\t\t\t \t\t\t\t........... \t  \t\t \t\t\t\t\t\t\t \t\t\t\t   \t\t\ti 2.4 i .6 i 1.2 \">*9.*2 U.7 M.4 \\ .5 l 5 .6 |il.4 r-3 ls -3 6.2 8.0 8.5 18.9 6.3 7.1 24.3 1.5 1.5 1.2 4.2 2.3 1.6 5.3 1.2 \"i.y 3.1 3.9 »2.2 «1.4 3 26.7 /28.5 V»4.8 /2 2.8 \\\\31.2 24.0 30.6 13.9 î..... }.....  \t16.0 3.8 2.9 1.8 \t\t   \t\t\t\t\t\t \t\t\t...... \t...... \t......   \t \t\t\t\t\t\t\t2.1 3.0 ...... \t6.2 3.6    \t\t\t \t\t\t\t\t\t\t \t\t\t   \t\t\t\t\t\t\t \t\t\t\t\t\t   \t\t\t\t \t\t'2.2\t 1.0\t\t\t1.9 \t   \t\t\t\t\t\t\t \t\t\t\t\t\t   \t\t\t\t2.4 l 3.2 9.7 3.2 5.6 1.6 4.5 5.4 10.7 1.7 \t\t» 1.2 IU.3 \\\\3l.8 /21.9 \\\\38.8 /21.6 \\\\3 1.0 210.0 311.0 /2 6.1 V2.8 *11.4 3 2.4 f23.8 VH.7 ( = 6.5 V»4.7 f'1.6 \\\\3 2.7 \t\t\t \t\t  \t \t\t\t\t1.9 1.4 12.8 2.5 6.7 1.7 1.5 2.7 5. S 13.5 2.2 1.4 3.7 1.0 6.4J 2.3 2. OJ LS 2.9 7.8 \t\\\\ 5.2 2.7 }..... i:: } 2.8 } 2.3 1.2 \t2.6 1.7 \t\t   \t\t\t\t\t\t\t \t4.8 1.9 3.9 5.3 5.9 4.1 3.3 7.8 \t\t1.1 1.2 2.3 3.5 5.5 1.7 \t   \t\t\t\t\t\t \t\t\t\t\t\t\t   \t\t\t \t\t\t\t\t\t\t \t\t  \t \t\t\t\t\t\t\t \t\t\t\t\t   \t\t\t\t\t \t\t\t\t...... \t\t\t.7   \t\t \t\t\t\t\t\t\t \t\t\t   \t\t\t\t\t\t\t \t\t11.4\t1.9 \t\t1.8   \t\t \t\t\t\t\t\t\t \t\t\t   \t\t\t\t\t\t\t \t\t\t\t\t\t   \t\t\ti 1.1 10.7 10.3 2.4 1.2 1.6 1.0 in.7 \\\\3 1.3 /23.3 V25.6 },.. \t\t1.3 \t\t   \t\t\t\t\t \t\t\t\t\t\t\t   \t\t\t44.2 22.0 9.3 ■ 87.2 \t\t\t \t\t\t\t\t\t   \t\t\t\t \t\t\t\t\t\t\t \t  \t\t \t\t\t\t\t\t\t \t\t\t\t   \t\t\t\t\t\t \t\t\t\t\t\t\t   \t\t\t \t\t\t\t\t\t\t \t\t  'Indian.  * Austrian.  3 Russian.  •Chinese.  * Japanese.86            ARMY ANTHROPOLOGY.      Table 17.—Characteristics  and composition of thc population  Désignation of     section.  Characteristics.  cû «  Cities of 25,000 or       over.  42  43  46  49  Texas 1.    Texas 2.    Texas 3.  Texas 4.  Texas 5.  Utah L.  Utah 2..  Utah.........  Vermont.....  Virginia 1.....  Virginia 2.....  Virginia 3.....  Virginia 4.....    Washington 1.  Washington 2.  Washington 3...    West Virginia 1.    West Virginia..    Wisconsin 1....    Wisconsin 2....    Wisconsin 3___    Wisconsin 4....    Wyoming......  Large Mexican population.  Sparsely settled, white.........  German and Negro population.  Coastal native population......  Large Negro population........  Sparsely populated.............  More densely populated........  Mining area....................  State undivided................   Peninsular région and east shore   Large Negro population........   Native rural région.............   Mountain, white...............  f Coastal région plus eastern coun-  \\\\  ties.  Puget Sound, foreign white.   Mountainous area..............    .....do..........................   Agricultural région.............  f Scandinavian and German popu-  lo lation.   German population............     Urban and foreign stock.......     Lake counties..................  /State undivided, sparsely popu-  lo  lated.    606,641    2,663,848      199,787    268,413    157,853     88,753      254,504       30,094    355,956      324,242      601,358    495,840    640,172    } 436,342        569,055    136,283      186,238    1,034,881    l 496,265    1,053,772      433,187      350,636    \\\\ 145,965    8.0     16.7     32.5   17.5   24.0    1.3     44.0      3.0   39.0    130.0     50.0   44.0   43.0     14.0     54.0          6.0       29.0       59.0       24.0       35.0    1,881.0       84.0        1.5   iSan Antonio..   El Paso.......   Dallas........   Houston......   Austin........   Galveston.....      /Sait Lake City  \\\\Ogden........      /Norfolk.\".'!!!!!  \\\\Portsmouth...   Richmond___   Lynchburg___   Roanoke......     Spokane......    /Seattle........  \\\\Takoma.......            /Whëeïingï.*!!!  \\\\Huntington...     La Crosse.....  /Oshkosk .....  \\\\Green Bay___     Milwaukee___  /Superior  ___  (Racine........  [33.8    [22.3    22.8  31.5   6.3  17.2    [60.6    11.4  47.5  27.9  16.6  15.6    43.4  17 7    13.3    19.6   26.4    J30.4  90.9    45.5    29.686.8 85.7 31.8 38.0 21.6 31.7 55.3 \t\t\t \t\t46.6 64.8 88.0 57.6 44.5 59.4 \t\t\t\t\t4k 43 je 3>4k t. 4k. <kcn  4k -O en _nco en eo -a O^JtOÇO ^1 eneococo en \t \t\t4k\tNative par-entage. 3 ta < |  3.7 4.9 44.2 43.2 48.3 45.9 22.3 \t\t \t\t\tS -o to i-to Oî to CO COCO 4k \t\t\t\t\tj0 35 •oco ^io \tj3 31 X) .O k-- to -, -kjeoen 4k frOHOl k- \t\t\t\tto o Foreign par-entage.   4.8 4.6 23.1 18.2 29.8 21.9 18.6 \t\t\t \t\t1-, to l-i çn en M # # J-4 en i-1 -kl oo-ki*. \t\t\t\t\tto 4k p s to \t» en — jtkMk- JO klO-J*- CO \t\t\t\to to Foreign-born white.   en k-, to k- i-, *.  \t\t\t\tj, en\t■kl\th- CO 4k oco co tk co tO Ci \t\t\tJk 4k O en- \t4k\tCn to tO k-. i-* en oo en k-, i-, oo i-, co \t\t \tCO O\tNegro.   k-O CO \ten 00\t \t\tkÇkt-*coootoe754keo -o \t\t\t\t\t\t o\t\t•kl\t.o \t\t\t\t\tIndian, Chinese, Japanese.   CO k-.  tk tO H* .o çn ço o co o \t\ten\t\tjk CD -4 35 to\t\t \t\t\tJO 4k \teo 04k<I kJ o -ai-1 o \t\t\t\tçn en German.   to to cn t- \tto to _. CD  kj\t\t\tk* o> IO\tto -5 \t\t\t MO\t\tr\t\tCO \t\t\t\tIrish.   ÇO 00 .o -■ -0 \t\t\t\tto 4k M to\tO \t\t\t\toto \tto\t00 eo \t\t\t\t English.   ÇO tô *. J. o en eo >- \t\t\t \t\"\"\tk-to HM JOu-i Hkk to t-,en oco \t \t\t\to\t\t\t çok-çn OCO 4k \t \t\t\tAustrian and Rus-sian.   \t kÇk h- k- k_. eo enoooeo to \t\t\t \t\t\t\t\t\t\t \t\t\t\t\t\t\t \t  \t4k en k-i-1 to \tto o \t\tÇO co O en en -4\t\t \t\tÇO\t\t3 Cn o 4k \t\t\t\t\tScandinavian.   O r\t\t\t\ten to 4k\t00 en\tO\t \t\t►-■eo 00-J \t\toenen \t\t \t\t\tItalian.   : \t\ten\t \ti\t\t\t\t\t \t;~i 00 \t\t\t\t\t\t\t \tCanadian, French.   to \t\tto en \t\t\tÇO co en o eo\t\t\t\trfk 4k \t \t\t\t\t\t\t\tCanadian, other.   \tCD \t\t\t\t:\t \t\t\t\t\t\t\t \t\t\t\t\t\t\tHungarian.   \t \t\t\t\t\t\t\t \t\t\t\t\t\t\t tO-J\t\t\t^4 \tMexican.   to O \t\t \t\t\to\tto \t\t\t\t\tr\tto O 00\tto \t\t\t\t\tScotch.   0>     £", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-re33~t38d_jya5", "00000000-0000-0000-BBDF-EBA0237F5779", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Average weight, by States, at mobilization,1917-1918, and demobilization,1919", "9918573882206676X51", null, "1921", "1921", "This table showed the variation in the average soldier's weight between entering military service and discharge, according to state of origin. Page 122 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "122  AR.MY  ANTHROPOLOGY.  Table 30.—Average  weight,  by  States, at  mobilization,  1917-18, and <h mobiliiahon,  UJ9 (m    pounds); States arranged in order of standing, with proportional weight for each 'inch ofjieight, and    chest circumference (expiration) for each pound of weight, for the first million draft recruits.  State.  Alaska...............  South Dakota........  North Dakota........  Minnesota............  Oregon...............  Montana.............  Washington..........  Nevada..............  Idaho................  Nebraska.............  Iowa.................  Wyoming............  Wisconsin............  California............  Kansas...............  Mississippi...........  Utah.................  Arizona.............  Oklahoma............  Texas................  Michigan.............  Illinois...............  Indiana.............  West Virginia.......  North Carolina......  Missouri.............  Ohio................  Alabama............  Arkansas............  Colorado............  Maine...............  Georgia.............  District of Columbia.  South Carolina......  Maryland...........  Virginia.............  Vermont............  New Hampshire.....  Tennessee...........  Kentucky...........  Connecticut.........  Pennsylvania.......  Louisiana...........  New York...........  Delaware............  Florida..............  New Jersey..........  New Mexico.........  Massachusetts.......  Rhode Island.......   Number   of men  measured.    106   3,892   6,444  27,341   2,748  11,648  13, 316   1,441   4,031  10, 774  19,537   1,927  18, 433  35, 461   9,571   8, 543   4,568   3,850  19, 429  34, 531  41, 872  69, 491  23,194  12, 367  14,668  24,964  52, 814  15,988  10,111   6,635   3,315  20, 305   4,486   9,343   9,192  17,616   2,077   2,240  14,426  15, 502  13,585  77,186  12, 356  87, 818   1,891   5,895  29, 958   2,690  29, 534   3,928    Mean  weight at  | Mean weight.    démo-   Mean height.  bilization.  I  Pounds.    150. 49    146.96    146.95    146.41    146. 38    146. 32    145. 44    145.35    145.31    144. 74    144.72    144. 61    144.50    143.98    143.72    143.23    143.13    143.04    142.35    142.22    141.99    141.77    141.64    141.53    141.49    141. 43    141.38    141. 28    141.28    141.06    141. 03    140. 82    140.53    140.49    140. 40    140. 34    140. 33    140. 33    140.10    140.00    139. 82    139. 72    139.62    139.53    139. 45    139. 32    138. 81    138. 47    138.40    136.44  Pounds.      2.208      2.159      2.163      2.150      2.150      2.151      2.140      2.143      2.133      2.126      2.126      2.130      2.137      2.127      2.107      2.100      2.109      2.099      2.084      2.079      2.110      2.103      2.090      2.085      2.076      2.081      2.098      2.077      2.071      2.069      2.100      2.071      2.077      2.077      2.090      2.070      2.091      2.095      2.052      2.058      2.095      2.094      2.065      2.091      2.075      2.061      2.079      2.051      2.070      2.060  Mean chest.  Mean weight  Inch.     0.223      .228      .229      .230      .228      .228      .230      .232      .232      .229      .230      .231      .232      .231      .231      .231      .231      .232      .232      .232      .235      .234      .233      .235      .235      .233      .234      .233      .234      .234      .237      .235      .232      .235      .236      .236      .238      .236      .235      .235      .239      .236      .236      .238      .237      .237      .239      .239      .239      .241  Mean weight  \"Mean chest.  Pounds.      4.472      4. 382      4. 353      4. 354      4. 368      4.372      4.347      4.307      4.307      4. 354      4.332      4.332      4.307      4.312      4.319      4.330      4.319      4.301      4.293      4.307      4.258      4.260      4.274      4.251      4. 255      4.275      4.268      4.277      4. 259      4.265      4.221      4.241      4.303      4.244      4.240      4.230      4.198      4.227      4.249      4.245      4.182      4.221      4.221      4.200      4.212      4.214      4.170      4.178      4.181      4.156  Demobili-       zation.     DifTer-  (average     ence.  weight).         Pounds.       162.00      11.51  152.19 5.23  150.89  3.94  151.37  4.96  148.32  1.94  151. 11 4.79  148.39  2.95  149. 50 4.15  150. 97 5.66  151. 23 6.49  150. 05 5.33  148. 44 3.83  147. 87 3.37  145.37  1.39  150.14  6.42  147. 54 4.31  149. 25 6.12  148. 34 5.30  14S. 47 6.12  147.36  5.14  145.07  3.08  145.42  3.65  144. 78 3.14  146.60  5.07  146.17  4.68  145. 70 4.27  144.45  3.07  144. 79 3.51  146. 83 5.55  147. 38 6.32  142. 97 1.94  143. 94 3.12  140. 80 .27  144. 89  4.40  141. si 1.41  146. 05 5.71  136. 95 -3. 38  142. 67       2.34  145. 54 5.44  144. 50 4.50  141. 05 1.23  142. 46 2.74  146. 41 6.79  140. 43 .90  142. 22  2.77  141.50  2.18  140. 29 1.48  144.00  5.53  139. 74 1.34  140.19  3.75     Hère, again, the numbers at demobilization from  certain of  the  States  and  Terri tories, like Alaska, Nevada,  and  Wyoming,  are  so small that no stress  must be laid upon the average that they  show.  7. INCREASE IN WEIGHT AT DEMOBILIZATION OVER MOBILIZATION (TABLE 31).       For the United States  as  a whole, the troops show an increase in weight  of  3.35 pounds.   The greatest increase was found in men from Alaska,  11.51  pounds, where the number weighed was  too small to furnish reliable averages.  In the upper half of  the list,  showing an increase of 4 pounds or more, we find  certain Southern States,  such as Louisiana,  with an  average  increase  of  6.8  pounds; Oklahoma,   6.1   pounds;  Virginia,   5.7; Arkansas,   5.6;  Tennessee,  5.4;  Texas.  5.1; West  Virginia, 5.1;  North Carolina,  4.7;   Kentucky,  4.5;  South Carolina, 4.4;  and Mississippi, 4.3.   On the other hand, the only Southern", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-d3d2.9ib9_q3w9", "00000000-0000-0000-CFCC-2FDEF7880544", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Mean chest circumference by U.S. section, with correlation to height and weight", "9918573882206676X52", null, "1921", "1921", "Page 145-147 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "3", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "CHEST CIRCUMFERENCE.  145  by the  Key  West  Section (Florida 3),  containing many Italians  and Cubans.  The  district  around Mobile  (Alabama 5) affords a  population with chest cir-  cumference of only 32.82, and indeed many southern sections, especially those  containing few Negroes,  are found in  the  lower part  of the table.   Rather  striking is the position, toward  the bottom,  of Denver (Colorado  5),  (associated  with a large  number of rejections for  tuberculosis)  and Philadelphia (Pennsyl-  vania 1), Cincinnati (Ohio 4), St. Louis  (Missouri 4), Baltimore (Maryland 1),  Los Angeles   (California 4),  Boston  (Massachusetts  4), and  even  New York  city (New York 2),  (mean  chest  girth, 33.14).   It is  clear  that the inhabitants  of cities tend to hâve reduced chest girth, possibly due to  a smaller amount  of  exercise of the upper appendages  and to the small races that congregate in them.  This is illustrated by comparing the twin cities of Minnesota with the rest of the  State.   The  men of the former hâve a chest circumference about 0.75 inch less  than the latter.      Table 43.—Mean  chest  circumference (expiration) by sections; sections arranged in order of standing      inlh proportional chest circumference (expiration) in inches for each inch of height and each pound      of weight; also  standard déviation for each chest circumference; first million draft recruits.  Average   for   United States  Minnesota.......     Do..........     Do..........  North Dakota...  California.......  Nevada.........  Idaho...........  South Dakota...  Wisconsin.......  New Hampshire.  North Dakota...     Do..........  Wisconsin.......  Alaska..........  Maine...........  North Carolina..  Michigan........  Washington.....  South Dakota...  Montana........  Utah............  Connecticut.....  Wisconsin.  Iowa......  Oregon........  New Jersey___  California.....  South Dakota.  Indiana.......  Michigan......  California.....  New York.....  Washington...  Maine.........  Indiana.......  Utah..........  Vermont......  Michigan......  Illinois........  Oregon........  Nebraska......  Washington...  Illinois........  Sec-  tion.  Characteristics of sections.    3    2    1    2    2    1    1    3    4    1    1    3    1  Ail.    2    1    1    3    2    2    1    1      2    1      1    3    1    1    2    3    3    7    1    1    1    3    1    2    8    2      2    2    4   Scandinavians and Finns............   German and Scandinavian population   Scandinavian population.............  .....do................................   Mining area..........................   State undivided, sparse population...   State undivided......................   Indian population....................   Lake counties........................   Mountainous area....................   Scandinavian and Canadian population   Russian population...................   Scandinavian and German population   Undivided............................   Native white stock, maritime.........   Sparsely populated mountainous area.   Firmish population.................   Mountainous area..................   Large Russian population..........   Sparsely settled, mountainous area___   Sparsely populated...................,   Prevailingly agricultural and near    metropolitan.   German population..................   Foreign white, German and Scandi-    navian.   Fairly densely populated.............   Mountainous area plus Atlantic County  Chiefly agricultural area..............  Dry f arming area.....................  Agricultural, considérable German...  Foreign population...................  Sparsely populated...................  Agricultural and dairying............  Coastal région plus eastern counties..  English Canadian....................  Manufacturing.......................  Mining area...........................  State undivided.......................  Prevailingly native white population..  Agricultural and manufacturing area..  Columbia River Valley and coastal dry    plain, sparsely populated.  German, Austrian, and Russian stocks.  Puget Sound, foreign white...........  Largely  German population...........  Number               Stand-ard dévia-tion.             of men      Mean chest.             Mean chest.     Mean chest.  ured.                    Mean height.    Mean weight.                  (chest).                      Inches. Inches. Inch.   Inch.  873,159       33.22   2.01    0.492   0. 234  3,515        33.95   1.98    .502    .232  7,585  33.86   1.93    .497    .229  6,448  33.86   1.86    .495    .228  3,305  33.82   1.88    .497    .230  942     33.81   1.87    .499    .231  1,438  33.75   2.08    .497    .232  4,031  33.74   2.04    .495    .232  247     33.74   1.74    .495    .228  2,883  33.73   2.01    .500    .234  667     33.72   2.09    .501    .238  1,131  33.72   1.91    .498    .230  2,005  33.70   1.86    .496    .228  3,290  33.68   1.89    .494    .232  106     33.65   1.94    .493    .223  828     33.64   1.95    .497    .237  2,738  33.64   1.82    .489    .238  2,340  33.63   1.96    .501    .232  1,539  33.62   1.83    .493    .230  594     33.61   1.87    .495    .228  6,521  33.60   1.85    .493    .229  1,224  33.59   1.82    .492    .233  4,877  33.58   2.10    .503    .240  7,678  33.56   1.97    .495    .232  12,139 33.54   1.93    .492    .230  2,747  33.54   2.10    .492    .228  3,196  33.52   2.02    .501    .240  11,712 33.52   2.06    .494    .231  3,050  33.51   1.95    .492    .228  835     33.49   2.01    .491    .231  6,290  33.49   2.09    .497    .235  2,106  33.48   1.97    .490    .231  6,465  33.48   2.06    .496    .236  5,174  33.47   2.02    .492    .230  1,238  33.46   1.86    .497    .235  3,609  33.45   2.12    .497    .235  562     33.44   1.77    .494    .232  2,079  33.43   1.90    .498    .238  12,560 33.42   1.98    .493    .235  2,451  33.42   1.98    .493    .233  1,076  33.42   1.91    .490    .229  3,138  33.41   1.95    .489    .229  6,599  33.41   1.96    .492    .230  4,238  33.40   2.03    .494    .233  146  AR.MY  ANTHROPOLOGY.  Table 43.—Mean chest circumference  (expiration) by sections:  sections arranged in order of standing       with proportional chest circumference (expiration) in inches for each inch of height and each pound       of weight; also standard déviation for each chest circumference; first million draft recruits-—( ontd.  State.  Illinois........  New Jersey__  Texas.........  Maryland.....  Illinois........  Wyoming.....  Texas.........  Virginia.......  New York.....  Connecticut...  New York.....  Virginia.......  Georgia.......  Illinois........  Wisconsin.....  New York.....  Pennsylvania.  California.....  Colorado......  Montana......  Colorado......  Missouri.......  Arkansas......  Characteristics of sections.  Louisiana.  Pennsylvania.  Kansas........  Arizona.......  Alabama.......  New Mexico___  Arizona........  Illinois.........  New Mexico___  Texas..........  Kansas.........  North Carolina.  Mississippi.....  New York......  Oklahoma......  Maine..........  Colorado.......  South Carolina.  Iowa...........  Ohio...........  West Virginia..  Kentucky......  New Jersey.....  Arkansas.......  Florida.........      Do.........  Michigan.......  Minnesota......  North Carolina.  Pennsylvania..  Nebraska.......  New York......  Louisiana......  Texas..........  Alabama.......  New York......  North Carolina.      Do.........  Massachusetts..  North Carolina.  Pennsvlvania..      Do.........  Michigan.......  New York......  Colorado.......  Ohio...........  Missouri........      Do........  Delaware.  Georgia..  MLxed native and foreign population.  Plains section, rural..................  German and Negro population.......  Large white population...............  Densely populated....................  State undivided, sparsely populated...  Large Negro population...............  Native rural région...................,  Urban area............................  Manufacturing area...................  Mountainous, Adirondack area.......,  Mountain, white......................  Large Negro population...............  Agricultural area......................  Urban and foreign stock...............  Eastern manufacturing région.........  Mining area...........................  Urban area...........................  Large native white population.......  Mining area, foreign population.......  Russian population..................  Native white, Ozark région............  Large native  white population, hill    country.  Mississippi bottoms and upland, large    Negro population.  Rural area............................  Native and German population.......  Large  Indian  population,   sparsely    settled.  Large Negro population...............  Native white population..............  Chiefly white population..............  Urban area...........................  Indian population.....................  Large Mexican population.............  Russian population...................  Island and peninsular area............  Rural area, large Negro population___  Western manufacturing région........  Chiefly white population..............  French Canadian population..........  English population....................  Large Negro population...............  Native white..........................  Dense foreign population..............  Mountainous area.....................  Mountainous area, native white.......  Densely populated....................  Negro, Mississippi bottoms............  Peninsular............................  Negro and rural population...........  Urban area............................  Urban area, \" Twin Cities \"............  Intermediate..........................  Rural area, native stock...............  German and Irish, foreign stocks......  Mountainous, Catskill région..........  Rural, chiefly white population.......  Coastal native population.............  Large Negro population...........Y. Y.  Suburban tenitory..................  Native white of Scotch origin....  Remainder of State....................  Manufacturing center...............\" \"  Large Negro population.........  Coal mining......................  Manufacturing..............YY.Y.YYYY  Dutch and other foreign population. ! '.  Urban area, densely populated......  Prevailing agricultural.............  Agricultural area..............  Native white, agricultural......!......  Mississippi   bottoms,   considérable    Negro population.  State undivided....................  MLxed population, native white pre-    dominating.  Stand-    ,ard    Mean chest.  devia-  Mcan height   Mean chest.  .\\\\Ie:in weight.  chest ).   7,803   8,985   1,415   2,675   7,803   1,927   1,346   3,866   6,544   8,708   2,986   5,499  10,070   5,442   4,513   5,131   7,293   7,189   1,053   5,117   1,099   1,138   1,559     4,072     8,616   8,505   1,027     3,327   1,851   2,821  33,905     290   6,676   1,066     254   5,149  14,222  10,958   1,247     380   3,976   7,404  17,208   1,506   4,029  17,772   4,933   2,339     995  17,751   9,757   4,309  14,218   7,621     795   5,227   2,722     665   4,919   2,050     744  18,352   4,558   4,813   8,892   2,889  46,651   1,222\"  17,548  13,571   3,448     1,891  10,235        Inches.     Inches.  33.40        2.03  33.40   2.12  33.40   2.09  33.39   1.99  33.38   2.02  33.38   1.89  33.36   2.05  33.36   1.94  33.35   2.08  33.34   2.20  33.34   2.00  33.33   1.87  33.33   1.91  33.33   1.98  33.33   2.11  33.32   2.07  33.32   2.10  33.32   2.09  33.32   1.77  33.31   1.93  33.30   1.75  33.30   1.76  33.29   1.80  33.29   1.97  33.29   1.98  33.28   1.99  33.28   1.91  33.27   1.90  33.26   1.84  33.25   1.99  33.25   2.12  33.25   1.84  33.24   1.98  33.24   2.68  33.24   1.84  33.24   1.88  33.23   2.13  33.22   1.95  33.22   1.93  33.21   1.86  33.20   1.85  33.20   1.92  33.20   2.08  33.20   1.87  33.19   1.80  33.19   2.12  33.18   1.95  33.18   1.95  33.18   2.02  33.18   2.08  33.18   2.01  33.18   1.90  33.18   2.02  33.17   1.93  33.17   2.01  33.17   1.87  33.16   1.99  33.16   1.84  33.16   2.08  33.16   1.82  33.16   1.85  33.15   2.04  33.15   1.91  33.15   2.00  33.15   1.98  33.14   1.96  33.14   2.15  33.14   1.88  33.13   2.00  33.11   1.90  33.11   1.89  33.11   1.97  33.10   1.88  Inch.     0.494      .499      .488      .496      . 495      . 192      .487      .489      . 198      .499      .497      .489      .490      .491      .497      .498      .500      .495      .489      .491      .490      .485      .484        .491        .494      .488      .489        .489      .493      .487      .495      .494      .487      .486      .491      .488      . 495      . 485      . 495      .487      .490      .488      .495      .488      .486      .497      .487      .491      .490      .496  .493  .488  .487  .486  .497  .485  .489  .497  .489  .4%  .497  .491  .498  .486  .489  .486  .486    .492  .486CHEST  CIRCUMFERENCE.  14  Table 43  ^ ™.—Mean chest circumference (expiration) by sections; sections arranged in order of standinq  with proport ional chest circumference (expiration) in inches for each inch of heighi and each pound  of weight; also standard déviation for each chest circumference; first million draft recruits__Contd  State.  Arkansas........  Oklahoma.......  Virginia.........  Alabama........  Illinois..........  Indiana.........  Ohio............  Massachusetts...  South Carolina..  California.......  Tennessee.......  Pennsylvania...    Marvland.......     Do..........  New Hampshire.  South Carolina..  Missouri.........  Ohio............  Tennessee.......  Alabama........  Florida..........  Pennsylvania...  Massachusetts...  Kentucky.......  Tennessee.......  Texas...........  Utah............  Massachusetts...  Colorado........  Illinois..........  Virginia.........  Mississippi......  Rhode Island..  Alabama......  Colorado......  Florida........  Dis. of Columbia.  New Mexico......  Louisiana........  Sec-  tion.  Characteristics of sections.   Large native white population.......   Marked Indian and Negro population   Large Negro population..............   Large native white population.......   Agricultural area, native.............   Agricultural area, native stock.......   Intermediate..........................   Urban area............................   Peninsular and rural areas............   Urban area...........................   Agricultural région....................   Allegheny County plus a small rural    area.   Peninsular area.......................   Urban area............................   Manufacturing area....................   Native white..........................   Urban area..........................  .....do.................................   Mountainous région...................   Mining and manufacturing area.......   Largely white and maritime..........   Urban area............................   Mountainous area....................   Agricultural area......................   Negroes, Mississippi bottoms..........   Sparsely settled, white................   More densely populated...............   Peninsular région.....................   Urban population.....................   Negro population (Egypt).............   Peninsular région and east shore.......   Rural area, large native white popula-    tion.   State undivided.......................   Urban and suburban area.............   Austrian and Italian population.......   Cuban, Spanish, West Indian popula-    tion.   District undivided....................   Noteworthy  Mexican élément.........   Urban area............................  Number   of men   meas-   ured.   3,589   8,471   5,339   2,666   8,900  18,725  14,443   8,553   3,795   7,428   6,305  17,238     1,066   5,420   1,581   1,563   6,7,84   3,554   5,898   8,833   2,477  16,053   1,373  11,419   2,217  22,372   2,781   1,123   1,640    409   2, SS(i   3,387     3,925    479   1,224     84     4,486    540   3,040  Mean  chest.  Inches.   33.10   33.09   33.07   33.07   33.07   33.06   33.06   33.06   33.05   33.04   33.02   33.01     33.00   32.99   32.98   32.97   32.96   32.96   32.93   32.93   32.92   32.91   32.90   32.90   32.90   32.90   32.89   32. 88   32. 88   32. S7   32. ,84   32.83     32.83   32. .82   32. 79   32.74     32.66   32.63   32.63  Stand-   ard  dévia-   tion,  j Mean height.  (chest).  Mean chest.  Inches.    1.78    1.87    1.89    1.80    1.94    2.00    1.98    2.14    1.85    2.02    1. S5    2.08      1.88    2.08    2.00    1.83    2.07    2.09    1.85    1.84    1.83    2.02    2.09    1.91    1.84    1.95    1. 8,8    2.12    1.83    1.95    2.05    1.86      2.11    1.96    1..89    1.99      2.00    1.85    2.09  Inch.     0.485      .485      .490      .484      .4,87      .487      .491      .494      .491      .487      .484      .495        .490      .493      .493      .484      .488      .489      .481      .484      .486      .494      .492      .484      . 483      .480      .4,85      .491      . 485      .482      .4,87      .480       .494     .485     .484     .487       .4,82     .480      .487  Mean chest.  Mean weight.  0.235   .233   .236   .235   .232   .233   .234   .237   .23,8   .234   .237   .236     .236   .235   .237   .235   .235   .232   .231   .233   .237   .239   .237   .234   .232   .231   .230   .237   .234   .236   .233   .231     .241   .234   .235   .240     .232   .234   .237       9.  STANDARD  DEVIATIONS OF  CHEST  CIRCUMFERENCE  BY SECTIONS.          Table 44 shows the variations in  the standard déviations of chest circum-  ference for the various sections.   For the United States as a whole the standard  déviation  is  close to  2  inches.   In  western Kansas it  is  2.68  inches, a high  variability associated  with the mixture of Germans and  large  Scandinavians,  on  the  one  hand, and of  smaller Russians  on the  other.   In  manufacturing  Connecticut, in New  York City, Boston, Chicago,  suburban New Jersey,  and  Rhode Island,  the standard déviation  is  also high.   In  gênerai, the  eastern  cities attract both extrêmes in body size.  Greater uniformity (smaller standard  déviation) is found in the Southern States.   Extremely low variability is  found  in South Dakota  3, with S7  per  cent Indians;  Colorado  2; and  Missouri  3,  the  Ozark Mountains, 94 per cent native whites and mostly  big men.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-nmfj.n6gf-fe8f", "00000000-0000-0000-F87E-0A46DD3C57F6", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Total and Proportionate Measurement by States--Height with Relative Weight and Chest Circumference", "9918573882206676X53", null, "1921", "1921", "This graph showed correlations of height, weight, and chest circumference of World War I soldiers with their States of origin. Page 178 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "ARMY A XI HllOPOLi Mi Y.  TOTAL AND PROPORTIONATE MEASUREMENTS BY STATES (P,)                  HEIGHT WITH RELATIVE WEIGHT AND CHEST (EXP.)  «8.00                                        \"nn            nnnnnrTn        I»                                                                              •1                                                    Ji      I       I               nnnnnr                                  ï                                                                                                                                                                                                                                                                                           [-1     -                                                                                                     f       -]-     -j                                                                                                                                                                                                                                  -                                                                                                                                                                                                                                                                                                                                                                                   A G  2.00 1.90 1.80 1.70                                                                      1                                                                       3                     \"                                                                                                                                                                                            L                                                                       r t  .49 ; •*8 .47 .46                                                                                                                                                                                                                                                              1                                   PLATE XV.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-sfzg-d4nj-zf8n", "00000000-0000-0000-F4A4-C8987FD8A223", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Distribution, Height, Weight, and Chest Measurements, and States of Nativity", "9918573882206676X54", null, "1921", "1921", "This map showed correlations of height, weight, chest circumference of World War I soldiers according to State of origin. Page 184 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes that item to be in the public domain.", "Public domain", null, null, "184  AR.MY ANTIIKdi'OUKlV.  DISTRIBUTION. HEIGHT. NEIGHT a CHEST MEAS.                 STATES OF  NATIVITY  MEAN HEIGHT  MEAN WT.-i-MEAN HT. = LBS.  MEAN WEIGHT  MEAN CHEST (EXP.)  LINES OF EQUAL INDEX OF CHEST CIR.         fA\"--» DEMOBILIZATION. 191» Hi.Y3^%-,     /y  f /\"'^t^—Jr=Hr~N4i-^»<   \\\\y^-50O  l w-__Lat] *87 :.*e^f-,/J^ -/;46S^ *9o  ♦90 ^^-fL—rAf W !          ■*9° v/ V ■-^■x       MEAN WT.-i-MEAN CH. (EXP.) = LBS.  MEAN CH. (EXP.) -i- MEAN HT. = IN.  LINES OF EQUAL HEIGHT (IN)  PLATE XX.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ykvw~y2vs.8nja", "00000000-0000-0000-7653-56521E0DA521", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlations, White and Negro Troops, demobilization--1919", "9918573882206676X55", null, "1921", "1921", "This bar graph showed correlation measurements between white and Black soldiers at the time of discharge in 1919. Page 270 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "270  ARMY ANTHROPOLOGY.  CORRELATIONS. WHITE AND  NEGRO  TROOPS                 DEMOBILIZATION-1919                  .0000   .1000   .2000    JOOO  COMPLETE CORRELATION   STATURE « STERNAL NOTCH     STATURE* SPAN     STATURE S PUBIC ARCH     STATURE S SITTING HEIGHT     CHEST CIR. S WEIGHT     ARM LENGTH S FOREARM   CHEST CIR. « NECK     LEG LENGTH «KNEE HEIGHT     WAIST CIR. «PELVIS. TRANS.     CHEST CIR. S PEI VIS. TRANS.     CHEST TRANS. «ANT. POST.     CHEST CIR. & SITTING HEIGHT       STATURE t STERNAL NOTCH     STATURE & SPAN     STATURE & PUBIC ARCH     CHEST CIR. « WEIGHT     STATURE « SITTING HEIGHT     ARM LENGTH & FOREARM     CHEST CIR. t NECK     WAIST CIR.» PELVIS.TRANS.     LEG LENGTH A KNEE HEIGHT     CHEST CIR. t PELVIS,TftANS.     CHESTCIR.SSITTMGHEI    CHEST TRANS.» ANT.POST.  .5000    .6000    .7000    .8000  9000    1.0000           1.0000  PLATE XXIX.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e7ex-un5m~vbqy", "00000000-0000-0000-EC93-13B4DA47D154", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Distribution, Hair and Eye Color, Demobilization--1919--States of Nativity", "9918573882206676X56", null, "1921", "1921", "This map showed the distribution by State of eye and hair color among demobilized U.S. Army soldiers in 1919. Page 295 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Cartographic materials", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "HAIR AXD EYE COLOR.  295  DISTRIBUTION. HAÏR. AND EYE COLOR DEM.-1919                  STATES OF NATIVITY            HAIR                         EYES  FLAXEN AND RED  CLEAR BLUE AND BLUE WITH BROWN SPOTS  LIGHT AND MEDIUM BROWN  DARK BROWN  fH% ~ -—~- ,                           mjT w       llbÉJ                 m  m 287.50- 375.00 Tjjggl ■ 376.00-475.00 W^ 5 476.00 - 525.00 WF\" ■ 526.00-665.01 *g&                LINES OF EQUAL PROP. OF FLAXEN HAIR                                         è=n \"b^aBBh 5                                                                      S 325.63-■ 501.00-S 601.00-■ 651.00-        mi h —.                               ■ 500.07W ■600.00 '4J ■650.00 ■745.26           Wl\"^-5=i                     LIGHT BROWN           S 93.63-150.00 ■ 151.00-175.00 = 176.00-200.00 ■ 201.00-386.89                  \"W \" 1  DARK BROWN  LINES OF EQUAL PROP.  OF CLEAR BLUE EYES        (Mf-^ 45    { ~~~Yf[yh      _—3^-30  s-1£j^               y*-zo p'o  30 \\\\ f    ^tr^ ,«> )     \\\\J  PLATE XXX.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-txih.gz4y~ypcq", "00000000-0000-0000-3C89-3D095044A5E2", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and weight in recruits with pulmonary tuberculosis in recruits", "9918573882206676X57", null, "1921", "1921", "This table showed correlations between height and weight, and the presence of pulmonary tuberculosis in U.S. Army recruits in World War I. Page 302 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Table  IM. -Corrélation between height and weight in recruits with tuberculosis (pulmonary), first (P,) and second (P2) million draft recruits.  Height, in   inches.  5S and under.  59............  60............  61............  62............  63............  so and over  W eight, in pounds.  Total.    89   and  under     ll    14    35    64   122   240   505   7S3  , 19S  ,444  , 5X4  , 496  , 212   xxs   50X   2S3   135  Total.....!   10,701  90-  94  95-  99  100-  104  105-   109  110-   114    1    4    9   20   43   74  123  128  121   84   66   24   18    2  115-  119    2    3   14   15   39   96  141  221  232  190  130   51   31   11    1    1    1  167 '  372   718 i  1,186  120-  124    4    3    4   11   15   38   84  134  215  255  263  1X7  133   80   21   11    3  1,462  125-  129    1    5    3    9   19   5.7  106  184  246  248  252  191  101   49   14    7    4    2  130-  134    2    2    3    8   20   38   68  123  209  270  235  205  146   77   27   16  1,498    1,460  135-   139    1    3    1    5    5    6   25   51  102  113  152  213  192  122   N5   40   17    6    2  140-  144    2    3    3    4   14   19   45   81  132  138  166  123   91   54   24    8    3    1    1  145-   149    1    3    3    2   11   34   42   83  116   88  102   92   44   13    4    3  150-   154  155-  159  160-  164  165-  169  170-   174  175-   179  180-   184  4        1  4  3  4  4  9  4  6  3  8 3 3      7 1  435  235   167   133  185-  1S9  190-   194  195-   199  200-   204  ',-                                                        P->-   Ntimher ofcases- 4 653                                      Number ofcases: 6,048.   Height: Mean, 0X.01  inches; standard déviation, 2.70±0.02    Height: Mean, 68.12 inches; standard déviation, 2.76±0.02     inches                                                      inches.   Weight: Mean, 131.77 pounds; standard déviation, 14.95±    Weight: Mean, 129.42  pounds; standard déviation, 14.36±     0.11 pounds.                                                0.09 pounds   Corrélation: o. 1551±0.0078.  Corrélation: 0.5533±0.0060.  Pi and P-2—    Number ofcases:  10,701.    Height: Mean, 68.07 inches: standard déviation, 2.74±0.01      inches.    Weight: Mean, 130.44 pounds; standard déviation,  14.74±      0.07 pounds.    Corrélation: 0.4754 ±0.0050.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-r2u4~d2ib-w36d", "00000000-0000-0000-6AEF-3FCDF6AB241B", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and chest circumference in recruits with pulmonary tuberculosis", "9918573882206676X58", null, "1921", "1921", "This table showed correlation between height and chest circumference measurements, and the presence of tuberculosis in U.S. Army recruits in World War I. Page 303 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Table  140.—-Corrélation between height  and chest circumference (expiration) in recruits                                                                                     recruits.  ith  tuberculosis (pulmonary), first (.P,)  and second (I'2) million draft  Chest, in inches.  co       Total. 9 12 34 64 121 236 502 782 1,192 1,435 1,579 1,489 1,237 884 566 2X2 135 53 23 5 X 1                                                                                37 1    3X                                        oo Height, in inches. Oj CO cr. 1          2x and under.   29      30      31      32      33 1 11 21 33 61 107 212 247 274 285 248 185 127 56 30 6 (j     34      35      30                      39      40      41      42      43 and over.  b: 58 and under.............                    1       3 4 11 29 41 89 158 210 211 206 176 117 70 32 15 12 3   3 4 7 17 25 50 102 179 250 318 330 275 219 122 68 30 16 8 2     2 3 5 11 18 41 104 151 223 317 328 351 290 200 135 62 35 11 3 2 1                       1                                                                 159............................................                                                                                                                                       60......            3 2 6 2 20 14 22 13 14 17 7 6   6 7 12 32 60 74 92 100 99 64 43 26 4 5 1 2                                      2 3 X 18 38 57 111 123 171 178 172 141 114 54 18 14 S   2 1 1 13 19 24 44 6.5 94 X9 90 xo 44 28 14 4 2 2                                        ..1................                       1 r>1 •                                                                             1                                       1                 IJ 62..............                                                                                                         1                         ° 63.                                                                               4 11 20 32 41 30 36 42 30 27 6 2 1 1 1  1 1 4 6 6 14 14 9 11 11 3 3 3 1 2 1 1 3 3 3 1 1 1 1 1                             64.                                                                                                                                   65....                                                                                                              1                       1  00.............                                                                                                                                      67.                                                                                                                                   6X.......                                                                                                   5 2 2 1                           69...............                                                                                                                                   1  70...                                                                                                                      1                 71......                                                                                                                                      72..............                                                                                                                                                                                                                                          1               .1                74......                                                                                                                    i                                                                                                                                     1                 76........                                                                                                                                    77                                                                                                                                    78                          1       1               1       3                       1                               |                 79.....                                                                                                                     \"T\"                                                                                                                                                   Total.............  10,649 126     62X     1,3xx  2,026  2, 293 1,918  1,233  617     290     X9      22      14      2       l               2                                                                                                                                       Number ofcases: 4,627.  Height: Mean, 68.02 inches; standard déviation, 2.69±0.02    inches.  Chest circuinference (expiration): Mean, 32.33inches; stand-    ard déviation, 1.X7±0.01 inches.  Corrélation: 0.2391 ±0.0093.  Number ofcases: 6,022.  Height: Mean, 6X.12 inches; standard déviation, 2.76±0.02    inches.  Chest circumference (expiration): Mean,31.90inches; stand-    ard déviation, l.S0±0.01 inches.  Corrélation: 0.2499±0.0081.  Pi and P-2—    Number ofcases: 10,649.    Height:  Mean, 6X.07 inches; standard déviation, 2.73±0.01     inches.    Chest circumference (expiration):  Mean,32.09inches; stand-     ard déviation, 1.85±0.01 inches.    Corrélation: 0.2412±0.0062.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-udrz.gf3i~cbbq", "00000000-0000-0000-4E71-810355BAF774", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and weight in recruits with myopia", "9918573882206676X59", null, "1921", "1921", "This table showed the correlation between height and weight measurements and the presence of myopia in U.S Army recruits in World War I. Page 312 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Table  145.—Corrélation between height and weight in recruits with myopia, first (J\\\\) and second (P2) million draft recruits.  Weight, in pounds.  Total......  2,420  Total.    3   10   11   32   58  108  185  304  299  348  350  235  209  132   86   24   17    1    6    2  89 and  under.  100-  104  105-  109  110-  114  115-  119  120-  124  210  125-  129  130-   135-  134   139  316  140-  144  145-  149  150-  154  172  155-  160- 165- 170- 17.5-  159   164   169  I 174   179  180-  184  185-  190-  189  194  134   97 I  57  195-  199  200- ; 20.X  204 ] 209  210-  21.X ! 220-  214  219  224  P,—                                                         P*~    N umber of cases 77X                                         Number of cases, 1,642.    Height- Mean 67 23 inches; standard  déviation, 2.83±0.05     Height: Mean, 67.01 inches; standard déviation, 2.77±0.03      inches      '                                                 inches.    WeiL'hf'Mean  140.23 pounds: standard  déviation, 18.07±     Weight: Mean, 138.75  pounds;  standard déviation, 18.61±      <U1 pounds. '                                               _ 0.22 pounds.   Corrélation: 0.5121 ±0.0178.  Corrélation: 0.4806±0.0128.  Pi and P^    Number of cases, 2,420.    Height: Mean, 67.08 inches; standard déviation, 2.79±0.03     inches.    Weight: Mean, 139.23 pounds; standard déviation, lx.45±     0.1X pounds.   Corrélation: 0.4912±0.0104.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-523n.casr~9un9", "00000000-0000-0000-C7E7-D4BEA4740B56", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and weight in recruits with mitral insufficiency", "9918573882206676X60", null, "1921", "1921", "This table showed the correlation between height and weight and the presence of mitral-valve insufficiency in U.S. Army recruits in World War I. Page 336 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Table  157.—Corrélation between height and weight in recruits with mitral insufliciency, first (PJ and second (P.J million draft recruits.  Weight, in pounds.  Height, in inehes.   58 and under.   59...........   60...........   61............   62...........   63............   64............   65............   66............   67............   68............   69............   70............   71............   72............   73............   74............   75............  76............  77............  78............  Total...................   8,860  Total.      H     30     52    115    276    45g    732  1,034  1,244  1,380  1, 140    984    620    399    205    101    44    19     4     5  89 and  under.  95-100-; 105-  99 ' 104  109  110-  114  115- | 120-  119  124  125-  12g    3    4   11   32   86  110  164  157  138  113   53   34   11    6  130-  135-  134     13g  2        1  1  2  5  2  3  1  7  11  2g        20  61        42  96        85  167       142  206      181  191      228  139      154  107      123  35       71  22        38  8 18  3 3  1  4 1  140-  144  145-.150-  14g  154   1     3  2    1   3  g  2  12 g  27 13  56        46  ge        54  148       g2  îgs       131  164      142  128      134  85       99  45        60  25        21  7 13  2 5  1  1  8  44  112 j 273  523  778 j 923 | 1,083  1,127 | 1,008 I  830   686   484  Number ofcases:  4,257.  Height: Mean, 67.86 inches; standard  déviation, 2.73±0.02   i nches.  Weight: Mean,  139.11  pounds; standard déviation, 16.62±   0.12 pounds.  Corrélation: 0.4860 10.0071'  155-  160-   159  164  165-  16g  356  230  170-  175-  174  179  180-j îax  184; 189  190A95-  194 : 199  2OO-I20.X 210J215- 220- 225-  204 , 209 214  219 | 224  229   2   4  .   2 '    Al  52  36  22 ! 20  22  P2-    Number ofcases: 4,603.    Height: Mean, 67.82 inches; standard déviation, 2.73±0 0->     i nches.    Weight: Mean, 138.87 pounds; standard déviation, 16.94±     0.12 pounds.    Corrélation: 0.5029±0.0074.  Pi and Pr-    Number of cases: 8,860.    Height: Mean, 67.84 inches; standard déviation, 2.7.i±0 01     inches.                                     '    Weight: Mean, 138.99 pounds; standard déviation, 16.79±     0.09 pounds.    Corrélation: 0.494g±0.0054.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-i5wm-4nr9_8mmv", "00000000-0000-0000-B49B-90E481F2F634", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and chest circumference in recruits with mitral insufficiency", "9918573882206676X61", null, "1921", "1921", "This table showed the correlation between height and chest circumference measurements and the presence of mitral-valve insufficiency in U.S. Army recruits in World War I. Page 337 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Taule 158.    Correleitwn betireen height and chest circumference (expiration) in recruits with mitral insufficiency, first (PJ and second (P.J million elreift recruits.  Height, in inches.  58 and under.  59.........  60.........  61............  62.........  63............  tH............  Total.  Chest, in inches  Total. ! )28 and ! under.                29      30      31      32      33      34 1 5 3 10 23 46 89 118 169 214 194 164 127 87 39 26 9 S 1 3   35 3 2 2 4 11 42 37 80 106 147 98 110 76 55 25 15 6 3 1 36 1    37      3 S     39 40 11                        12      43 and over.  s 9 2g 51 115 275 45g 730 1,032 1,238 1,376  4 2 4 6 4 5 5 13 3 1 6 3        2 1 3 2 11 25 31 42 51 45 35 24 21 10 3 2 2     1 6 11 16 37 54 95 109 113 110 87 47 26 12 6 2 1        2 1 4 12 14 55 105 101 188 200 184 156 115 65 41 17 8 3 2 1 2 7 25 57 g2 177 22g 272 2g3 222 184 120 64 36 10 6 1       2 3 10 24 50 71 143 îgg 233 272 231 209 118 79 40 IS 2 ï 1                                                                                                                                                                                                                                                                                  4                                                                                                                                                                                                                                                           3 6 7 25 32 57 71 66 73 42 35 20 13 2 2 3 4 3 11 12 24 22 36 37 15 9 12 4 4 2 1 1 2 6 6 9 7 16 13 9 5 4 2 3     2 1 2   .......                                                                                                                                                                                                                                                                                                                                                                                           ........1\"'\"                                                                                                        2 7 4   1 2                                                                                                                                   1       1       1 1  1,137                                                                                                                             g78                                                                                         4                                 6ig 3g8                                                                                             2 5 3   1       1               1  205                                                                                                        1                         100                                                                                                                           43                                                                                          1                               1  19 1                                                                                                                         4                                                                                                                                                                 1                                                                                                                                                                                                                       8, 830     57      310     733     1,271  1,801  1,713  1, 336 823     459     199     83      33      5       2       1       4  Pl-    Number ofcases: 4,240.    Height: Mean, 67.86 inches; standard déviation, 2.73±0.02      inches.    Chest circumference (expiration): Mean, 32.86 inches; stand-      ard déviation, 1.94±0.01 inches.    Corrélation: 0.1972±0.0100.  Number of cases: 4,590.  Height: Mean, 67.82 inches; standard déviation, 2.73±0.02    inches.  Chest circumference (expiration): Mean, 32.65inches; stand-    ard déviation, 2.05±0.01 inches.  Corrélation: 0.2886±0.0091.  P, and P2—    Number ofcases: 8,830.    Height: Mean, 67.84 inches; standard déviation, 2.73±0-01     inches.    Chestcircumference(expiration): Mean, 32.75inches; stand-     ard déviation, 2.00±0.01 inches.   Corrélation: 0.2338±0.0068.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qgy9-95fk~esnt", "00000000-0000-0000-46DC-281FD205D257", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and weight in recruits with varicose veins", "9918573882206676X62", null, "1921", "1921", "This table showed the correlation between height and weight measurements and the presence of varicose veins in U.S. Army recruits in World War I. Page 349 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Table  103.—Corrélation  between height and ueight in recruits with varicose veins. first (PJ and second (PJ million elreift recruits.  Weight, in pounds.  Height, in inches. Total. 1 3 6 20 25 51 152 219 329 425 536 475 421 312 233 107 60 30 13 1 3 1    89 and 90- 9X under.1 94 ! 99                   100-104 10.X 109        110-114 115-119 120-124 12.X 129        130-134 1       135-139 140-144 145-149 150-154 15.X 159        160-164 16.X 169        170-174 17.X 179        180-1S4 185-189 190-194 19.X 199        200- 20.X 204 209               210-214 21.X 219        220-224 22.X 229        230-234  58 and under.........                                                                                                                                                                                                                                                          59...................               l                                               2 1 1 6 8 15 14 18 10 14 7 1 3  3 4 3 5 20 25 22 22 22 15 13 2  1 3 6 12 24 34 45 43 41 31 19 4 4 3                                                                                                                                                                       60.................         . |                             1 1     \"4\" 2 3 7 H 10 5 5                            1 1 2 7 18 32 50 43 56 52 28 14 10 2 3                                                                                                                                                            61................          1                                                                               2 2 4 16 29 46 55 77 46 44 26 14 2 2    1 1 2 17 21 36 61 76 69 55 41 24 10 6 3 2       1 2 4 16 11 38 66 63 70 57 49 23 8 7 3  1       ___                                                                                                                       62...................                                                                                                                                       1                                                                                                                             I                                                                                                       3 6 17 30 35 49 46 54 28 28 6 6 3                       1 1 3 6 15 28 23 32 23 13 10 2 1 1      1               1                                                                                 lit         1 ....          1       2 1 1   5 3 2 2                                                                         3 5 7 30 31 44 34 38 26 15 8 1  1 5 5 18 30 32 30 23 32 24 5 2                                  1                                                                         65..                                                                                                                                                        3 8 6 14 12 17 19 14 9 3 7 2    3       2                               1                               1                 66....................                              1                                                                                                                                               3 2 4 3 4 11 3 3 1                      1                                                 67............                                                                                                                                                              5 8 8 14 19 13 4 6 3    6 6 6 8 8 6 2 4 3 1                                                                               68___                                                                                                                                                                                       4 5 1 2 2 2     1 1 2 2 2 2     5 3 1 5 6 2 4   2 1 1 3 1                                         69 .                                                1                                                                                                                                                                                       1                 70...............                           l                                                                                                                                                                                       .... 1  1 ....  1                 71............                                      1               1                                                                                                                                                                               1                 72....................                                                                                                                                                                                                                                                        73...................                               1               i... - -                                                                                                                                                                                                        1  74...................                              1               1                                                                                                                                                                                                         75 .                                1                                       1                                                                                                                                       1 1                                       76                                                                                  ■>                                                                                              2 1 1 1                                                           77...............                                                                                                                                                                                                                                                     78...............                                                                                   ::::*'i::::::                           1               1       1                                                                                                         79                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Total...........    3, 423 2               2       4       15      45      100     157     270     322     365     425     418     312     242     209     160     115     83      53      42      26      10      28 ; K)         3       2       2               1                                                                                                                                                                                                                                                       Number ofcases: 1,409.  Height: Mean, 68.34 inches;  standard déviation, 2.70±0.03    inch.  Weight: Mean,  146.43  pounds; standard déviation, 18.39±    0.23 pound.  Corrélation:  0.4833±0.0138.  Number ofcases: 2,014.  Height: Mean, 68.49 inches;  standard  déviation, 2.77±0.03    inch.  Weight: Mean,  146.45  pounds; standard déviation, 18.62±    0.20 pound.  Corrélation: 0.4608±0.0118.  l'i and P2—   Number ofcases: 3,423.   Height :  Mean, 68.43 inches; standard déviation, 2.74 ±0.02     inch.   Weight:  Mean, 146.44 pounds; standard déviation,  18.53±     0.15 pound.   Corrélation: 0.4696±0.0090.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-aksq.92ed-kiin", "00000000-0000-0000-2AAD-57BB385CA8C6", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and weight in recruits with asthma", "9918573882206676X63", null, "1921", "1921", "This table showed the correlatation between height and weight measurements and the presence of asthma in U.S. Army recruits in World War I. Page 358 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Table  Jti'J.—< 'eu-relation between height and weight in recruits with asthma, first (PJ and second \\\\PJ million draft recruits.               i                                                                                               Weight, in pou                         Kl S.                                                                                                             Height, in inches. Total. 1 1.89 and under         90- I 95-91 99          100- 105-110-104 1109i114                       11X 119 120-124 12X 129 130-134 13X 139 140-144 145-149 150-154 155-159 160-164 16X 169 1 1     170-174 17X 1.80-179 184                18X 190- 19X 189 194 199                        200-204 20X 209 210-214 21X 219 220- 22.X 224 ! 229 1           230- 235 and 234 i over.          58 and under.................       .,     1 1 .......1________                    |               1                                                                                                                                                                                               j             6 7 15 21 72 112 178 196 242 231 186 138 93 40 27 7 3 4                                                                 1 1 1 4 11 25 21 26 28 23 17 9 1 2 1    1 1 1 10 9 23 29 31 25 21 13 6 3        1       3                                                                                                                               1                         60............................                                      1       \"ï 1 5 6 5 4 2 2 4 3 5 5 11 4 10 2 2 1 1 2 2 15 12 13 17 13 11 8 3     1 5 9 24 26 17 25 18 13 6                                                                                                                                               1                                         61............................              i                                                                                       2       1 2 1 5 11 16 25 31 18 13 15 7 4        1       1                                                                                                                       1         62..................                .......|....            1       1 2 1 1                                                                                                 1 2 1 2 2 5 4 7 5 4 1 2                                                                                                 t         63............................                                                                                              6 9 28 27 39 33 21 20 13 7 2 1  2 4 17 26 27 28 29 23 8 4 1             2 8 5 14 17 19 10 22 4 3 1      2 2 3 12 5 12 12 9 4 3 2 ....   1 3 3 6 5 11 5 13 7 5 1 1                                       i 1                                             i                       J         64............................                                                                                                                                                      1 3 2 4 5 7 3 2 1 \"Y 4 2 2 3 2 î       \"i'                            1               j                                         65............................                              1                                                                                                                                                                               1               1                                 66............................                                                                                                                                                                                              1                                                                 67............................                      ......          1                                                                                                                               \"4 1 \"2       1 1 3 1 1       3 \"2\"                                                         1         68............................                                                                                                                                                                                              2                                                                 69............................                                      1 2                                                                                                                                                                                                                       70............................                                                                                                                                                                                                                                      t                         71............................                                                                                                                                                                                                      i                                                         72............................                                                                                                                                                                                                                      1                                       1  73............................                                                                     1                                                                                               1       2               1                               1                                 74............................                                                                                                                                                              1 1                                     1                                                       1  75............................                                                                                                                                                                     1                                                                                         76............................                                                                                                      1       1                       1               1                                                                                                         77............................                                                                                                                                                              1                                                                                                 78 ...........................                                                                                                                                                                                                                                                                79............................      1                                                                                                                       1                                                                               i                                                                                                                                                                                                                                                             1                                                 Total...................    1, 581                 2       10      27      50      97      145     171     174     207     175     150     106     69 62           38      40      20 10           11 5            4       3 ! il il                                                       0  1                                                                                                                                                                                                                                                            Number ofcases: 614.  Height: Mean, 67.22 inches; standard déviation, 2.77±0.05    inch.  Weight: Mean, 139.38 pounds; standard déviation, 17.28±    0.33 pound.  Corrélation : 0.3833±0.0232.  Number of cases: 967.  Height: Mean, 67.26 inches; standard déviation, 2.67±0.04    inch.  Weight: Mean, 138.78 pounds; standard déviation, 18.35±    0.28 pound.  Corrélation:  0.4226±0.0178.  Pi and P2—    Number of cases: 1, 581.    Height: Mean, 67.24 inches: standard déviation. 2.71±0.03     inch.    Weight: Mean, 139.01 pounds; standard déviation, 17.94±     0.22 pound.    Corrélation: 0.4069± 0.0142.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8hw2.fd58_vhcw", "00000000-0000-0000-FCA7-26B2C07D1FF5", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and chest circumference in recruits with asthma", "9918573882206676X64", null, "1921", "1921", "This table showed the correlation between height and chest circumference measurements and the presence of asthma in U.S. Army recruits in World War I. Page 359 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Table  170.—Corrélation betueen height and chest circumference (expiration) in recruits with asthma, Jirst (PJ and second (P.J million elraft recruits.  Chest, in inches  Heighl in inches. Total. 28 and under.            29      30      31      32 1 2 2 2 4 20 31 36 40 56 44 27 31 10 4 2 1   33      34      35 1 2 1 3 1 6 12 19 29 22 35 29 23 15 S 5 1    36      37      38      39      40      41      42      43 and over.  X and under................     6 7                                                                                                                                                           j                       2 4 15 16 42 39 44 41 27 27 20 9 8 1 1  1 1 2 5 8 19 22 32 44 45 28 23 16 5 1 1 1               1 1 1 2 1 3 11 9 16 15 18 10 12 3 6     1                                       j         60......................                            1 . 2 1 1 6 6 10 11 17 12 2 2 2 1 3 3 16 17 22 22 28 22 21 7 7 3 2                                                                                                61.......................   15 21 72 111 178 195 242 231 186 138                                                                                                    .               l         62......................                                                                                    8 S 9 3 s 4 6 2 ï 1     1 1 1 3 2 2 4 4 4 4 1                           1                             3                                                                               4 1     1 2 ï                     64......................                                                                                                                                      65          1 2 1 1 1       6 2 4 7 5                                                                                                                 66..................                                                                                                                                          67........................                                                                                                  5 1 3 2 1 1                               68........                                                                                                          ........ 1      i       1       1  69......................                                                                                                                                     70...                                                                                                                                         71...........................       93 40 27 7 3                                                                                                                              72...............................                                                                                                           1       2                                                                                                                                                       74...............................                           1                                                                                                       1  75                                                                                                                                   76...............................   4                               1                               1                       .......|........1........                                         77.................................                                                                                         .i ... .. .                                       7s                                                                                                                                    79...........................       1                       ,                                              1               j                               1                                     j                                                               j                                         Total.....................................  1, 579 1 6 1           -       74      175     313     298     254     213     110     52      27      18      6       3       1 •?.                                                                                                                                           I'l-    Number ofcases: 612.    Height: Mean, 67.23 inches; standard déviation, 2.77±0.05      inch.    Chest circumference   (expiration): Mean,  33.57  inches;      standard déviation. 2.11±0.()4 inch.    l'orrelal ion : 0.1274± 0.0268.  Number ofcases: 967.  Height: Mean, 67.26 inches; standard déviation, 2.67±0.04    inch.  Chest circumference (expiration):  Mean, 33.19 inches; stand-   ard déviation, 2.11±0.03 inch.  Corrélation: 0.1628±0.0211.  P,and P2—    Number ofcases: 1,579.    Height: Mean, 67.25 inches; standard déviation, 2.71±0.O3     inch.    Chest circumference (expiration): Mean, 33.34 inches; stand-     ard déviation, 2.12±0.03 inch.    Corrélation: ().1477±0.0166.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7ns7~8y8r_izc3", "00000000-0000-0000-2CB0-608F40AE4FDD", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Correlation between height and weight in recruits with hernia", "9918573882206676X65", null, "1921", "1921", "This table showed the correlation between height and weight measurements and the presence of hernia in U.S. Army recruits in World War I. Page 366 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Table  174.—Corrélation between height and weight in recruits with hernia, first (PJ and second (PJ million draft recruits.  Weight, in pounds.  Height in inches.     Total. 47 71 120 341 642 1,288 2 249 4, 433 4,871 5 017 4,242 3,249        h«» and ' 90-under. 94 1 3      9.X 99 2 3 -- 4 0 3 i 2 100-104 3 1 2 18 23 24 21 18 8 12       10X.110-109 114         115 119 2 5 14 59 95 203 249 281 261 227 116 62 18 5 4 1 1      120-124 3 9 19 53 98 198 336 433 124 372 283 145 73 27 6        125-129 3 8 s 46 104 214 340 515 635 573 476 251 135 62 16 3 1  130-134 135-139 140-144 2 G 20 24 87 176 330 536 708 736 651 422 231 96 49 16 2 3       145-149 4 3 2 12 19 45 107 240 384 546 606 577 462 301 134 56 19 8 4    150-154 3 1 4 11 25 75 123 267 308 478 476 438 307 142 71 28 6  155-159 4 2 3 3 9 14 35 83 161 239 321 409 340 250 155 80 33 6 2 ï 2    160-164 1 2 1 1 5 11 27 55 87 161 238 227 257 210 144 83 35 17 3 3      165 170- 175-169 174 179                        181V 18X 190- 195-,200- 20.X 210-184 189 194 1991 204 209 214 1                                                21X 219 22(1- 225-221 229  58 and under. . .. 59                                      .... 3 6 4 11 , 25 26 38 31 1 86 62 118 61 152 56 159 32 133                                   5 3 10 27 68 155 353 493 641 690 589 117 255 120 46 15 1        1 11 24 61 116 278 450 707 714 730 560 374 186 87 13 8 2 2 1 2 3                                                l 1 2                                           ,                                                                                                                                                                    7 1 1 ....... 2 2 , 2                                          ___: i |...                               60          1 i....                                                                                                                                                                 i               i                 61                                                                                                                                                                                  l                                             ........... .......!\"i ........... ..... i                                                                                                                     8 14 40 52 102 150 171 174 154 120 65 37 10     1                                               1                       i  63                                                                                                                                         2 8 18 41 51 87 109 119 111 71 48 26 14 1 7 10 18 32 62 59 64 55 67 31 27 12 2  i 4 8 23 25 40 37 43 47 41 30 13 10                             ::::l::::::::i::::                        64                                                                                                                                                                  1 4 11 ,8 22 25 21 24  \"ï 4 10 26 23 17       3 1 6 9 14 9 14 8       i                       ....|....                                                                                                                                                                                                                     ....L...  66......                                                                                                                                                                                            3 ... 7 A... 12 4 '.... 12 1 1 14 t 1 11 ... 2 24 1 1 1         .... ....         67. .. ...                                  21 11   61 32 12 3 1                                                                                                                                                                      68          i i                                                                                                                                                                                                       >9          !                                                                                                                                                                                                         70                                                                                                                                                                                                                    71.         .....,........r .                                                                                                                                                                                                        72. 1,209 607 109 49 17 11 6                                                                                                                                                               26 ; 17 25 14 8 11 6 9 2 3 1 1                                  2 1     1 ....  73...............                                     1                                                                                                                                               7 11 .... 2 7 0 2 1                             2  74                                                                                                                                                                                                                 2 ....  75                                    '\"' I                                                                                                                                          2 1 1   4 ........                      ___l___  76           i                                                               1                                                       5 ■■ 8                                                  3 . ... 2 4 ........                                  i                                                                                                                                       2 2 1 i 2                                                                             !                                                       1       1               1       1 1     1                                                                               1 ........                                    i                                                                                                                               1                               1       1       1 i........                       ko and over......           | 1                                                                                             1                                                       1                                                                                                                                                                                     i               1                                                                 Total...... 34, 324        2 ! 5   19      135     318     827     1,603  2,479  3,391  3, 890 4,337  4,101  3,531  2,780  2,152  1,568  1,122 1721             452 328         1,85 'l45              88      114 j 13        10      3       3 | 2  Pi  Number ofcases: 13,870.  Ueight: Mean,67.40 inches; standard déviation, 2.74±0.0t    inch.  Weight: Mean, 141.69 pounds; standani déviation, 17.22±    0.07 pound.  Corrélation: 0.5285±0.0041.  P2—    Number ofcases: 20,454.    Height:  Mean, 67.47 inches; standani déviation, 2.77±0.01     inch.    Weight:  Mean, 140.91 pounds: standard déviation, 17.12±     0.01 pound.    Corrélation: 0.5130±0.0035.  Pi and P>—    Number of cases: 34,324.    Height:  Mean, 67.44 inches; standard déviation, 2.76±0.01     inch.    Weight:  Mean, 141.23 pounds; standard déviation, 17.17±      0.04 pound.    Correlation: 0.518s±o.0O27.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wkct~86na-9hsv", "00000000-0000-0000-1D39-96FDFBAEC980", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Height measurement, special diseases in relation to the average inducted man", "9918573882206676X66", null, "1921", "1921", "This bar graph showed the relative occurrence of various diseases in inducted U.S. soldiers according to height measurements, in World War I. Page 394 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "HEIGHT  MEASUREMENT. SPECIAL DISEASES (RsR)                     IN RELATION TO THE AVERAGE.INDUCTED MEN (P,)                                            RATIOS PER 1000                        ABOVE AVERAGE                              BELOW AVERAGE   ________________100   .80    .60   .40    .20    0    .20   .40    .60   .80   1.00   1.20   1.40   1.60   1.80   2.00   VARICOSE VEINS   VARICOCELE   TUBERCULOSIS. PUL.   GOITER. EXOPH.   GOITER. SIMPLE   MITRAL INSUF.   HEMORRHOIDS   TACHYCARDIA. SIMPLE   CARDIAC HYPER.   MITRAL STENOSIS   HEART. VALV. DIS. OF   TONSILLITIS. HYPER   ING. RINGS. ENL.   HERNIA   CONG. GEN. DEFECTS   FLAT FEET   TEETH. DEF. DEFIC.  ASTHMA  HYPEROPIA  MYOPIA  ASTIGMATISM  DEF. PHYS. DEVEL.  UNDERWEIGHT                                     AVERAGE 67.49 IN.                                           PT.ATE XXXIX.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-tk3p_rad8-atav", "00000000-0000-0000-9CAA-B297367183E6", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Weight measurement, special diseases in relation to the average inducted man", "9918573882206676X67", null, "1921", "1921", "This bar graph showed the relative occurrence of various diseases in inducted U.S. soldiers according to weight measurements, in World War I. Page 395 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", null, "Copyright may apply", null, null, "NEIGHT MEASUREMENT. SPECIAL DISEASES (R&       IN RELATION TO  THE AVERAGE.INDUCTED MEH (P,)  R)  VARICOSE VEINS  FLAT FEET  GOITER. SIMPLE  TONSILLITIS. HYPER  VARICOCELE  HERNIA  CARDIAC HYPER.  HEMORRHOIDS  CONG. GEN. DEFECTS  ING. RINGS ENL.  MYOPIA  ASTIGMATISM  ASTHMA  MITRAL  INSUF.  HYPEROPIA  GOITER. EXOPH.  TEETH. DEF. S DEFIC.  TACHYCARDIA. SIMPLE  HEART. VALV. DIS. OF  MITRAL  STENOSIS  TUBERCULOSIS. PUL.  DEF. PHYS. DEVEL.  UNDERWEIGHT  ABOVE AVERAGE    3    2  RATIOS PER 1000    1     2     3  BELOW AVERAGE  5     10    15  AVERAGE 141.54 LB  PLATE XL", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-zrga_whu9.3apj", "00000000-0000-0000-E8D1-998D3448DB93", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Chest measurement, special diseases in relations to the average inducted man", "9918573882206676X68", null, "1921", "1921", "This bar graph showed the relative occurrence of various diseases in inducted U.S. soldiers according to chest circumference measurements, in World War I. Page 396 of The Medical Department of the United States Army in the World War. Vol. 15 (Statistics), part 1 (Army Anthropology). Washington, DC: Government Printing Office, 1921-1929.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "CHEST MEASUREMENT(e™,».SPECIAL DISEASES  (P,*.          IN  RELATIOH TO THE AVERAGE. INDUCTED MEN (P,) '  R)  1.00  VARICOSE VEINS  ASTHMA  TONSILLITIS. HYPER  GOITER. SIMPLE  HERNIA  HEMORRHOIDS  VARICOCELE  INGUINAL RINGS. ENL.  HYPEROPIA  CONG. GEN.DEFECTS  ASTIGMATISM  TEETH. DEF.V^DEFIC.  CARDIAC HYPER.  MYOPIA  GOITER. EXOPH.  TACHYCARDIA. SIMPLE  MITRAL INSUF.  MITRAL STENOSIS  HEART. VALV. DIS. OF  TUBERCULOSIS. PUL.  DEF. PHYS. DEVEL.  UNDERWEIGHT  ABOVE AVERAGE    .60    .40  RATIOS PER 1000   0    .20    .40    BELOW AVERAGE  60    .80    1.00  1.50  2.00  2 50   3.  AVERAGE 33.22 IN.         PLATE XI.I.", "United States. Army. Medical Department ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-vqhb_wug3.dibh", "00000000-0000-0000-68E9-A3C7169BC713", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Numbers of men rejected by draft boards, and numbers sent to mobilization camps in 1st and 2nd million draftees, by State", "9918573882206676X69", null, "1919", "1919", "This table showed the numbers of American World War I draftees rejected by local draft boards, and numbers sent along to mobilization camps, by State. Page 28 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jbnm~xkib-2uix", "00000000-0000-0000-E5DC-0B25426FBFFF", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total of draftees rejected for pulmonary tuberculosis and suspected tuberculosis, by State, with ratio per 1000", "9918573882206676X70", null, "1919", "1919", "This table showed the number of American World War I draftees rejected by draft boards or by mobilization camp physicians for having pulmonary tuberculosis or suspected tuberculosis. Page 82 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-pjka.r87t~j4fi", "00000000-0000-0000-77F2-1C1A9F9C7DE4", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total of cases of syphilis, chancroid, and gonorrhea infection found in the second million draftees, by State", "9918573882206676X71", null, "1919", "1919", "The tables on these pages showed the total number of American World War I draftees found to have syphilis, chancroid, or gonorrhea infections, in the 2nd million drafted, by State. Page 84-85 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kj8s.qiwx-pihu", "00000000-0000-0000-6BC4-80E14EA30554", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total of cases of curvature of spine, and grand total of cases of diabetes mellitus, with ratios per 1000", "9918573882206676X72", null, "1919", "1919", "These two tables showed the total number of American World War I draftees found to have curvature of the spine or diabetes. Page 89 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4v34-5zcp_f6ry", "00000000-0000-0000-9AB6-F5EA385479A7", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand totals of cases of deafness and defective hearing, by State", "9918573882206676X73", null, "1919", "1919", "These two tables showed the total number of American World War I draftees found to be completely deaf or with defective hearing. Page 98 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rqud-39ti_7f4m", "00000000-0000-0000-4463-FE2A3F9B8483", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total of rejections for mental deficiency, by State, with ratio per 1000", "9918573882206676X74", null, "1919", "1919", "This table showed the total number of American World War I draftees rejected for service due to mental deficiencies, by State. Page 101 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-eumf_ckar-bswx", "00000000-0000-0000-78F5-4761E7E57F4C", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total of cases of myopia, and grand total of cases of other types of defective vision, by State", "9918573882206676X75", null, "1919", "1919", "These two tables showed the total number of American World War I draftees found to have myopia or other defects of vision, by State. Page 105 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dqyp~anp9~a43w", "00000000-0000-0000-E6ED-0125B7B1BD14", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total of cases of trachoma", "9918573882206676X76", null, "1919", "1919", "This table showed the total number of American World War I draftees found to have trachoma infection. Page 107 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-b7vx.2788.4f4s", "00000000-0000-0000-628B-9667BED3AB16", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total rejected for endocarditis, valvular diseases of heart, etc., by State", "9918573882206676X77", null, "1919", "1919", "This table showed the total number of American World War I draftees rejected for service due to endocarditis, valvular disease, and other cardiac conditions, by State. Page 113 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-u6mk~8kfz.x6gy", "00000000-0000-0000-E646-584A240EDED9", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total for defective and deficient teeth, and Grand total for hernia", "9918573882206676X78", null, "1919", "1919", "These two tables showed the total number of American World War I draftees found to have defective or deficient teeth (Table 63) and hernia (Table 64). Page 122-123 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6ug9.5xeg~mg82", "00000000-0000-0000-241F-455F802D0252", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total for malunion of fracture in the upper or lower extremities; and Grand total for loss of all or part of upper extremity.", "9918573882206676X79", null, "1919", "1919", "These two tables showed the total number of American World War I draftees found to have badly healed bone fractures of the upper or lower extremities (Table 69) and the grand total of those who had lost all or part of an upper extremity (Table 70). Page 126-127 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-69zm~a7p7-uust", "00000000-0000-0000-EA7C-D6C26507555C", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total for hammer toe & hallux valgus, and Grand total for pes planus (flat foot)", "9918573882206676X80", null, "1919", "1919", "These two tables showed the total number of Amercan World War I draftees found to have hammer toes or hallux valgas (bunion) (Table 75), and flat feet (Table 76). Page 131 to 132 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xqst.8ek2.k9hy", "00000000-0000-0000-2E38-C6C01BD84A23", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Grand total for deformities, injury, infection of hand or loss of fingers", "9918573882206676X81", null, "1919", "1919", "This table showed the total number of American World War I draftees found to have deformities, injuries, or infection of the hands, or loss of one or more fingers. Page 137 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-24vm~5bxs~fukc", "00000000-0000-0000-C0CA-51F0FF203588", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Proportion per 1,000 of cases of each disease or defect listed to the total number of defects or disease in the particular group under which the proportion is shown in 1,961,692 inducted men examined at camp and in 549,099 men rejected by local boards", "9918573882206676X82", null, "1919", "1919", "The medical examiners at U.S. Army mobilization camps during World War I classified draftees into four groups: Group A included men in whom no disqualifying defect or disease was found; Group B included men with remediable defects/diseases, who would qualify for general military service once those problems were addressed; Group C included men with diseases or defects that precluded general military service, but who might serve in limited or special capacities; and Group D included draftees rejected on physical grounds for any military service. Group Vg in this study indicated men who had been rejected by local draft boards and were not sent on to mobilization camps. This table, part of a discussion of the defects and diseases found in Group C (special or limited service), showed how the various defects and diseases were distributed among the groups. Page 154 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kpqc~fy7f_tg6r", "00000000-0000-0000-1423-33C4FB4065EF", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Proportion of the various defects listed per 1,000 of the total for that particular defect as classified in the various groups by the medical boards at camps, and by the local boards", "9918573882206676X83", null, "1919", "1919", "The medical examiners at U.S. Army mobilization camps during World War I classified draftees into four groups: Group A included men in whom no disqualifying defect or disease was found; Group B included men with remediable defects/diseases, who would qualify for general military service once those problems were addressed; Group C included men with diseases or defects that precluded general military service, but who might serve in limited or special capacities; and Group D included draftees rejected on physical grounds for any military service. Group Vg in this study indicated men who had been rejected by local draft boards and were not sent on to mobilization camps. This table, part of a discussion of the defects and diseases found in Group C (special or limited service), showed the proportion of each disease/defect found in each group. Page 155 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rpgf~nnz7~arj3", "00000000-0000-0000-99AD-8189700745B0", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Distribution of defects and diseases in draftees, by regional, occupational, and racial groups", "9918573882206676X84", null, "1919", "1919", "This series of tables showed how diseases and defects in American World War I draftees was distributed among various regions of the United States, with their associated populations and predominant types of occupations (e.g., agricultural, manufacturing, mining, commuting, etc). Page 267-295 of Albert G. Love and Charles B. Davenport. Defects Found in Drafted Men. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "29", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, null, "Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-rzuy~6afy.dc46", "00000000-0000-0000-FD16-C7A986DF4EC8", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Statistical card used to tabulate diseases and defects found in medical examinations of American World War I draftees, and sample interpretation of coded statistical card", "9918573882206676X85", null, "1919", "1919", "These figures illustrated 1) the blank punch card used for recording data from medical exam forms, and 2) a sample statistical card with coded data punched in to it. Page 44 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "44  PHYSICAL EXAMINATION OF FIRST MILLION DRAFT RECRUITS.  tion, \" has been placed at the top of the list. Next in order of importance have  been placed syphilis, gonorrhea, valvular heart disease, tuberculosis, all degrees of  flatfoot, defective vision, defective hearing, defective teeth, overweight, under-  weight, malunion of fracture, loss of one or more fingers, etc. The foregoing list,  however, is used not as an absolute standard in all cases, but only as a general working  outline.  Fig. 2a.-Statistical card: Code numbers corresponding to diseases and physical defects are written in the  blank spaces at the top and left border. Each space has a corresponding column or group of columns of  figures from 0 to 9, which are punched out according to the code numbers, so that the card may be used  in the sorting and tabulating machine.  When determining the major defect, among defects stated, their severity and the  effect military service would have on them are taken into consideration. In the case  of a man with first degree flatfoot with no symptoms and marked defective vision,  the preference would be given to the defect of vision; but if he had third degree  flatfoot, with or without symptoms, and vision of 20/40 or 20/70, then the flatfoot  would be considered the major defect.  Fig. 2b.-Interpretation of the statistical card: The man is from Boston, board 3. His name is Clyde M.  Stetson, his serial number 2794, and his identification number 1685529. Boston is in (21) Massachusetts  and is (1) a city. His weight is 138 pounds, height 68 inches, chest inspiration 35 inches, and chest expira-  tion 32. The local board (1) \"accepted him for military service with no defect;\" he was (0) \"not exam-  ined\" by the advisory board, while the mobilization board (8) \"rejected him with two defects,\" the  major one being defective teeth. He -was examined in March, 1918. His physical examination report  was coded by clerk 11. The numbers given above are punched in the columns which form the body of  the card.  In the general examination of the body, scars of the head and face are coded as a  defect only when \"extensive,\" \"painful, \" or \"with marked cicatricial contraction.\"  A man giving a \"history of a fractured skull\" would not be considered as having a  defect unless it was stated on the examination report to be a \"depressed fracture\"  or one with symptoms of \"convulsions,\" \"mental deteriorations,\" etc.", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-i969.yhbd-6dm2", "00000000-0000-0000-CECD-0C5B18D1D118", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Key punch machine for coding diseases and defects onto statistical cards", "9918573882206676X86", null, "1919", "1919", "Photograph of the sorting machine used by the U.S. Army Medical Department to tabulate medical exam data from local draft boards and Army mobilization camps. Page 45 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Photographic prints", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "45  PHYSICAL EXAMINATION OF FIRST MILLION DRAFT RECRUITS.  Goiter, except exophathalmic goiter, is considered a minor defect.  The various deformities of the chest, as \"funnel,\" \"asymmetrical,\" \"pigeon,\" \"flat,\"  or similar terms, are not coded as defects unless the degree of deformity is stated to be  \"marked,\" \"severe, \" or of such degree as to interfere with standing erect, drilling, etc.  Relaxed inguinal rings are considered a defect; but when associated with such  defects as syphilis, flatfoot, tuberculosis, gonorrhea, or valvular heart disease, the  preference is given to the latter defects; when associated with hypertrophied tonsils,  varicose veins, various deformities of the hand or foot, etc., then the preference is  given to the relaxed rings.  Scars of the abdomen and extremities from operations or burns are not coded unless  \"extensive,\" \"painful,\" or \"with marked cicatricial contraction.\" If an abdominal  scar is associated with a \"weak abdominal wall,\" then it is considered a defect.  Curvature of the spine is considered only when \"marked\" or \"severe.\" Ankylosis  of the joints, either fibrous or bony, is always coded as a defect. Old fractures of the  Fig. 3.-Key punch machine: The card is inserted into the carrying arm of the machine (indicated by  arrow at left). The punch operator uses the keyboard (indicated by arrows at top), reading the code  numbers from the left border and top of the card while she punches. As each figure is punched, the  machine automatically carries the card to the left and in position for punching the succeeding figures.  extremities are not coded unless a \"deformity\" is present from malunion, or \"anky-  losis,\" or \"loss of motion\" is said to exist. A finger amputated at the distal phalanx  is not considered a defect. If amputated at the second phalanx or proximally, it is  as coded the loss of the whole finger. Varicose veins are considered only when  \"marked\" or \"painful, \" or \"with symptoms.\"  The common deformities of the nose, as a deviated septum, nasal spurs, or hyper-  trophied turbinates, being common defects and ones that can in all probability be  corrected, if necessary, are not considered for the statistics. Hypertrophied tonsils,  because they play such an important part as a source of various infections of the body,  are considered defects; but when associated with other defects as varicocele, venereal  disease, or flatfoot, the latter are given the preference.  In coding defects of the heart, \"functional,\" \"systolic,\" \"accidental,\" or \"cardio-  respiratory murmurs\" are not considered as matters of great importance. When a  definite disease of the heart is said to exist,- as mitral stenosis or regurgitation, aortic", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-38ix~cyv5.skka", "00000000-0000-0000-6F71-1D5339CACD26", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "A sorting machine for coded statistical cards", "9918573882206676X87", null, "1919", "1919", "Photograph of the sorting machine used by the U.S. Army Medical Department to tabulate medical exam data from local draft boards and Army mobilization camps. Page 46 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Photographic prints", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "46  PHYSICAL EXAMINATION OF FIRST MILLION DRAFT RECRUITS.  stenosis, or regurgitation, or stated as \"organic murmurs,\" it is coded as a defect and  ranks next to hernia and venereal disease in order of importance. Cardiac hypertrophy  or dilatation, whether slight or marked, is always considered a defect. Tachycardia is  considered only when characterized as \"constant,\" \"persistent,\" or by any other  term that shows it to be of a permanent character and not from nervousness as a result  of the physical examination.  In considering defects of the lungs, \"rales,\" \"dullness over lungs,\" \"bronchial  breathing,\" \"rough breathing,\" etc., are not taken as defects. If the man is rejected  - or discharged from the Army for any of these physical signs, it is considered a defect.  If any of the physical signs are found to fit in with  any previous illness with other parts of the examina-  tion, such as underweight, poor physical develop-  ment, persistent cough, hemotysis, etc., then the  symptoms for which the man was rejected or dis-  charged are coded as near as possible as a definite  disease. Asthma, tuberculosis, and similar diseases  are always considered major defects.  Diseases of the genito-urinary organs, like syphilis  and gonorrhea, are always considered major defects  and rank next to hernia in order of importance.  Varicocele is not coded a defect unless it is said to be  \"marked,\" \"large,\" or \"with symptoms.\" Hydro-  cele is always coded as a defect. Atrophy of the  testicle is not considered, but defects such as monor-  chism, cryptorchidism, or hypospadias are always  coded.  Hemorrhoids are considered only when \"ex-  ternal,\" \"large,\" \"bleeding,\" or \"painful.\" Small  epithelial or skin tabs are not coded. Fistula in ano  is always a defect.  All degrees of flatfoot are coded as defects. Hallux  valgus is considered a defect, but overriding toes are  not unless they are of such severity as to be a cause for  rejection, and pronated feet only when \"marked\" or  \"severe.\" Weak, flaccid and spastic feet or similar  conditions of the feet whose function can be restored  by treatment are not considered.  VI. GROUNDS OF REFERENCE TO MEDICAL  ADVISORY BOARDS.  The medical advisory boards were de-  vised as a result of the experience of the  first selective draft and became effective  with the second draft after the induction of about 527,100 men.  The organization of the medical advisory boards is indicated by  the following extracts from the Selective Service Regulations of  November 8, 1917. In these Regulations section 29 reads:  Each State shall be carefully districted with due regard to communication and  hospital facilities for the erection of a number of medical advisory boards computed  with a view to the equitable and practical distribution of the work of reexamina-  tion as provided herein and to the convenience of registrants and economy to the  Government in sending registrants before such boards.  Fig. 4.-Sorting machine: Cards are  set on the metal platform (A); the  motor (B) causes the cards to he  automatically fed into the machine  by a \"feeder\" (C). As the punch  holes of a single column of figures  come in contact with an electric  needle, the cards are sorted and  carried by a card-conveying belt  (D) into the collecting boxes (E).  Each box is numbered to corre-  spond with the series of figures that  appear on the punch card.", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-u9v4_hpzz-xk8b", "00000000-0000-0000-29F0-5F7DC65EEBED", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Maps showing incidence of pulmonary tuberculosis, asthma, and simple goiter, as found in U.S. draftees", "9918573882206676X92", null, "1919", "1919", "Page 160 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Cartographic materials", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Plate XIII.  TUBERCULOSIS, PULMONARY  FIRST 1.000,000 MEN  ' 213-9.1  9.0-56  55-28  27 - .40  ASTHMA  FIRST 1,000,000 MEN  2.9 _ LI I  LO _75 I  74 39  38 15  GOITRE, SIMPLE  FIRST 1,000,000 MEN  14.7 -5 8  5 7 -1.2 I  l.l -22l  31 -.051  Plate XIII.-Upper: Map of the United States showing by States the varying incidence of pulmonary  tuberculosis as found at mobilization camps in the first million men; in four grades. Middle: Map of  the United States showing by States the varying incidence of asthma as found at mobilization camps  in the first million men; in four grades. Lower: Map of the United States showing by States the vary-  ing incidence of simple goitre as found at mobilization camps in the first million men; in four grades.  160", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bf9i.wenk-vpmm", "00000000-0000-0000-6B6D-88A5E8C095DD", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Numbers of men accepted as qualified for general military service by local boards only (and not sent to medical advisory boards) from June, 1917 to May, 1918, inclusive, and number and percentage of the same men rejected at mobilization camps", "9918573882206676X89", null, "1919", "1919", "Page 66 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "66  PHYSICAL EXAMINATION OF FIRST MILLION DRAFT RECRUITS.  were found without defects; of December men less than 20 per cent  (but the number examined was very small). Thereafter the proportion  remained at about 75 per cent. In the case of men from Massachu-  setts the proportion found without defect started at 71 per cent,  fell to 40 per cent (November to December men), then rose to nearly  67 per cent in March, 1918, after which it fell slightly (fig. 6, p. 68).  Table 16.-Number of men accepted as qualified for general military service by local  boards only (and not sent to medical advisory boards') from June, 1917, to May, 1918,  inclusive, and number and percentage of same men rejected at mobilization camp.  State.  Number  of men  accepted as  qualified  by local  boards.  Number  of men  rejected at  mobiliza-  tion camps  who were  accepted as  qualified  by local  boards.  Percentage  of men  rejected at  mobiliza-  tion camps  to number  accepted as  qualified  by local  boards.  Maine  3,041  298  9.80  Rhode Island  3,550  324  9.13  New Hampshire  2,056  183  8.90  Connecticut  12,552  1,113  8.87  Massachusetts  27,275  2,331  8.55  California  33,495  2,858  8.53  Michigan  38,314  2,982  7.78  Nevada  1,359  102  7.51  Montana  10,974  797  7.26  Vermont  1,805  7.26  Mississippi  7,941  552  6.95  Utah  4,382  303  6.91  Idaho  3,841  264  6.87  Washington  12,144  831  6.84  Florida  5,459  364  6.67  South Carolina  8,406  558  6.64  Wyoming  1,700  108  6.35  Louisiana  11,738  684  5.83  Kentucky  15,012  801  5.34  Alabama  15,035  793  5.27  Georgia  18,135  943  5.20  New York  80,869  4,183  5.17  Delaware  1,640  82  5.00  New Jersey  27,605  1,336  4.84  Arkansas  9,241  435  4.71  Wisconsin  16,751  777  4.64  Oregon  Indiana  3, &amp;22  130  739  3.69  3.38  Illinois  61,588  1,981  3.22  Missouri  22,578  681  3.02  North Dakota  5,918  177  2.99  North Carolina  13,296  382  2.87  New Mexico  2,621  74  2.82  Ohio  47,402  1,253  2.64  Kansas  8,660  32,307  228  2.63  Texas  829  2.57  Arizona  3,567  90  2.52  Minnesota  25,412  636  2.50  Nebraska  10,222  256  2- 50  Oklahoma  18,067  415  2.30  Colorado  6,140  136  2.22  Iowa  18,306  380  2.08  Vest Virginia  11,354  223  1.96  Virginia  16,254  310  1.91  South Dakota  3,636  61  1.68  Tennessee  13,264  196  1.48  Pennsylvania  70,598  980  1.39  District of Columbia  2,440  31  1.27  Maryland  8,981  55  .61  Alaska  88  3,583  5.47  State not specified  65,478  Total  867,877  37,959  4.37  Seen by medical advisory board, also not from local boards  126,329  Grand total  994,206", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-mdtb-8cbv_37dd", "00000000-0000-0000-2CDA-8637712C67DD", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Graph showing the comparative percentages of rejection for cardiovascular disease at various Army camps, October, 1918", "9918573882206676X90", null, "1919", "1919", "This graph showed rejection rates for cardiovascular diseases at the 16 Army mobilization camps during World War I. Page 76 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "76  PHYSICAL EXAMINATION OF FIRST MILLION DRAFT RECRUITS.  To arrive at a conclusion concerning the adequacy of the physical  examination service at the camps we have to consider the admissions  to sick report, discharges for disability, and total days lost in the  Army on account of hernia. From the report of the Surgeon General  it appears that during the year 1917 there were 5,779 admissions to  sick report on account of hernia, or 8.51 per thousand of mean  strength during the year. There were 521 discharges for disability  on this account, or 0.77 per thousand of mean strength. There were  117,610 days lost, or 0.47 per thousand of the days spent by the total  number of men in the Army during the year. Also, there were 2,620  herniotomies performed, of which 2,592 were inguinal. These her-  CARDI0-VA5CULAR DEFECTS AT MOBILIZATION CAMP Octobers  CAMP % 1 2 3 4 5 6 ~ 8 9 SfSLZ.  I PIKE  2- LEWIS  ;  4 KEARNEY  5 BOWIE  6 UPTON <  7 DIX  8 DODGE  9 DEVENS ■  10 MCLELLAN -  II GORDON  12 HANCOCK .  13 FREMONT >  K-ShermanTF  15 FUNSTON <  16 GRANT  17 SEVIER  18 CODY  19 CUSTER  20 EUSTIS  21 GREENE  22 SHELBY  23 SHERIDAN  24 WHEELER  25 JOHNSTON  26 MEADE  8.76  6.40  3.88  3.54  3.27  2.87  2.73  2.34  2.31  2.00  I .96  I .64  I .59  1.22  I .18  I .10  I .07  I .04  .74  .62  .42  ' .39  .2d  ■ I6|  Fig. 21.-Graph showing the comparative percentages of rejections for cardio-vascular disease at various  Army camps, October, 1918.  niotomies constituted over 9 per cent of operations for the correction  of defects.  The distribution of cases of hernia, by States, is shown in Table 19.  Tn this table the States are arranged in order of the ratio of cases  found per thousand men examined from each State. Similarly, in  Table 20, is given the distribution of cases of enlarged inguinal rings.  Plates VIII and XX give in graphic form the geographical dis-  tribution of cases found at .mobilization camps with hernia and the  geographical distribution for enlarged inguinal rings.  The striking fact in both charts on plate XX is the large amount of  hernia, incipient and developed, in the territory tributary to Camp  Lewis, and this is probably due to the idiosyncrasies of the exami-  ners at that camp, although the possibility that it is associated  with the mining occupation of these mountain States must be kept", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p2mz_v3t3-7ata", "00000000-0000-0000-43C6-118023C102BF", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Tributary areas to the 16 National Army Cantonments, 1917, and a map of varying incidences of all venereal diseases, as found at mobilization camps", "9918573882206676X91", null, "1919", "1919", "Page 158 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Cartographic materials", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Plate XI.  STATES TRIBUTARY TO THE CANTONMENTS  NATIONAL ARMY 1917 \\\\  VENEREAL DISEASES, ALL  FIRST 1,000.000 MEN  905-3751  374  23.8-12 oE  11.9 -590C  Plate XI.-Upper: Map'of the United States showing the area tributary to each of the 16 National Army  Cantonments, 1917. Lower: Map of the United States showing by States the varying incidence of  venereal diseases combined as found at mobilization camps in the first million men; in four grades.  158", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-3fgp-zawn~9eut", "00000000-0000-0000-9293-2903E51E5516", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Numbers of men rejected for pulmonary tuberculosis by local draft boards, by medical advisory boards, and by mobilization camp examiners, by state", "9918573882206676X88", null, "1919", "1919", "This table showed the numbers of American men (of the first million examined) who were rejected for pulmonary tuberculosis in local draft board exams, or by medical advisory committees, or by mobilization camp medical examiners during World War I. Page 48 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "48  PHYSICAL EXAMINATION OF FIRST MILLION DRAFT RECRUITS.  From the foregoing extracts it will be seen that the function of the  medical advisory board was, as its name indicated, purely advisory.  Also it will be specifically noted that the medical advisory board  functioned only during the induction of the last 400,000 men con-  sidered in this report, or about two-fifth of all. Nevertheless, it will  be interesting to consider the principal grounds of reference to  medical advisory boards and the varying extent to which the same  diseases were referred by local boards to the medical advisory boards  in the different States. In the following sections will be considered  primarily those diseases for which the local board during a part or  the whole of the period under consideration was required to reject.  Table 3.- Tuberculosis, pulmonary  State.  Rejected  men seen  by local  board  only.  Per 1,000  of men  examined.  Rejected  men seen  by medical  advisory  board  also.  Per 1,000  of men  examined  at medical  advisory  board.  Per cent  rejected at  1 mobiliza-  tion camp  ! for tuber-  | culosis that  had been  referred  to medical  1 advisory  board.  Tennessee  8  0.58  10  11.49  55.56  Wisconsin  28  1.64  11  7.12  28.20  Kansas  47  5.09  17  23.29  26.56  District of Columbia  3  .69  1  6.41  25.00  Colorado  47  7.44  13  28.20  21.67  Missouri  180  7.68  43  24.86  19.28  New Hampshire  9  4.32  2  9.66  18.18  Oregon  9  2.52  2  6.21  18.18  New York  189  2.27  40  6.47  17.47  New Jersey  52  1.84  11  4.98  17.46  South Dakota  16  4.26  3  11.63  15.79  Vermont..  11  5.98  2  8.30  15.38  Illinois  149  2.26  25  5.50  14.37  Rhode Island  24  6.66  4  11.36  14.29  Michigan  111  2.81  17  6.13  13.28  Georgia  107  5.45  16  12.40  13.01  Oklahoma  53  2.80  7  7.24  11.67  Arizona  31  8.34  4  22. 73  11.43  Indiana  64  2.90  8  6.13  11.11  Nebraska  40  3.85  5  9.40  11.11  Utah  17  3.78  2  12.66  10.53  Connecticut  62  4.82  7  8.89  10.15  Minnesota  31  1.20  3  1.82  8.82  Kentucky  110  7.21  10  16.16  8.33  Maine  24  7.75  2  8.09  7.69  Pennsylvania  88  1.16  7  2.27  7.37  Virginia  51  2.82  4  7.42  7.27  Massachusetts  156  5.47  12  7.31  7.14  Ohio  84  1.64  6  2.36  6.67  West Virginia  29  2.40  2  3.84  6.45  Washington  59  4.73  4  3.83  6.35  New Mexico  30  10.92  2  21.98  6.25  Louisiana  106  8.57  7  18.23  6.19  Mississippi  97  11.46  6  16. 48  5.83  Iowa  86  4.47  5  3.68  5 49  Texas  158  4.52  7  7.82  4.24  Alabama  94  6.02  4  5.87  4.08  South Carolina  86  8.72  3  18.63  3.37  Montana  33  2.96  1  1.61  2.94  Arkansas  218  22.00  4  10.53  1. 80  California  243  6.97  4  4.01  1.62  North Carolina  161  10. 93  2  4.57  1.23  Florida  44  7.58  Wyoming  11  6.25  Idaho  18  4.61  Delaware  6  3.40  Nevada  2  1.43  North Dakota  7  1.16  Maryland  3  .32  State not specified  874  8.72 |  8  4.82  .91  Total  4,166  4.41  353  7.15  7.81", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-z434.id6q_g3dm", "00000000-0000-0000-59D6-0A482E1513E7", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Maps showing incidence of constitutional psychopathy, psychosis, and mental deficiency, as found in U.S. draftees", "9918573882206676X93", null, "1919", "1919", "Page 164 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Cartographic materials", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Plate XVII.  PSYCHOPATHS, CONSTITUTIONAL  FIRST 1,000,000 MEN t  2.62 - .46  .45 - .30  .29 - .17  .16 - .05  PSYCHOSES  FIRST 1,000,000 MEN  1.5 - . 67 I  .bb - .411  .41 -.251  .24 - .oo{  MENTAL DEFICIENCY  FIRST 1.000.000 MEN  tl.O -5.0  4.9 _ 2.0  1.9 -.89  ,«a _.4s I  Plate XVII.-Upper: Map of the United States showing by States the varying incidence of constitu-  tional psychopathic states as found at mobilization camps in the first million men; in four grades.  Middle: Map of the United States showing by States the varying incidence of psychoses as found at  mobilization camps in the first million men; in four grades. Lower: Map of the United States showing  by States the varying incidence of mental deficiency as found at mobilization camps in the first million  men; in four grades.  164", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-c8pw_7bbq_s8mm", "00000000-0000-0000-0156-0280746A32FA", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Maps showing incidence of flat feet, congenital foot defects, and defective physical development, as found in U.S. draftees", "9918573882206676X94", null, "1919", "1919", "Page 169 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Cartographic materials", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "rLAIh  PES PLANUS  FIRST 1.000,000 MEN  284.9- 1  2O9.9~180.0E  179.9-  \\\\ 149.9\" 73.2[  FEET, CONGENITAL DEFECTS  FIRST 1,000,000 MEN \\\\  27.27-8.83  8.82-5.00  499-2.64  243-1.26  DEVELOPMENT, DEFECTIVE  first i ,000,000 men  3.29 - 1.93  1.32- .69  .68- .31  •30- .16  Plate XXII.-Upper: Map of the United States showing by States the varying incidence of pes planus  as found at mobilization camps in the first million men; in four grades. Middle: Map of the United  States showing by States the varying incidence of congenital defects of the feet as found at mobilization  camps in the first million men; in four grades. Lower: Map of the United States showing by States  the varying incidence of defective physical development as found at mobilization camps in the first mil.  lion men; in four grades.  169", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-j7gn-6asc.9h8m", "00000000-0000-0000-6DA0-2A62BC7A6242", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Graphs showing the relative incidence of diseases and defects in Alabama, Arizona, and Arkansas", "9918573882206676X95", null, "1919", "1919", "Page 174 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "[DISEASES AND DEFECTS -ALABAMA-RATIO P ER 1,000.  Plate XXVI.  FES PLANUS ~  GONORRHEA '2 '  SYPHILIS (ALL)  CHANGRQ10  [MENTAL DEF  JTUBERCULQllS_RUh.  UNDERWEIGHT  ANKYLOSIS  I FEET CONGEN. P.  [ TEETH D.I3R UDIS  SKIN  PTlTtt MEDIA  FRACTURE  ARTHRITIS  [JiQftE PIS.  .ASTHMA  [PSYCHOPATHS CQNfl  , TESTES .CDNGEN. D.  [scoliosis' '  .HEMORRHOIDS . ....  [mydgarpitis J  VARICOSE VEINS  , HEARING D. |  ■'Oil  2204  'Hl  773  663  656  3.19  288  &amp;  114  1.481  1.40  129  'i?  .80  .80  .80  74  41  .61  .55  .31  TRACHOMA  GOITRE SIMPLE.04  DRUG AbDlCTS-  GOITRE, CXO PH J  iDISFASrS AND DEFECTS -ARIZONA- RATIO PER 1.000  GONORRHEA|  HERNIA}  T UBERCULD sis puu  HEART VALV. DIS. I  SYPHILIS lALLJJ  VARICOCELEI  FELT, CONGEN. D. 1  VISION D. I  OTITIS FkEP LA J  PSYCHOSES J  TRACHOMAI  TEETH P. OR UDSSJ  GOlTRfl. JEXQPH. J  AmRlTLS ' '1  CKANCRWD  SCOLIOSIS  HEARINfiD.  NOS£ PIS. J  SKIN DIE -J  MENTAL J?EF. J  JMttlQ. APPfcTS * J  147.66  33.37  27X7  na  *1$  82  105  2.05  1.29  r.8i  1.03  1.03  .77  .77  f'[  51  51  51  51  51  .26  26  26  heart. 9 ».rvNtf,  HEART, HYP - CHU.  HY OCA RD rm  NERVOUS PPR FUNCT  DISEASES AND DETECTS -ARKANSAS- RATIO PER 1.000°  THREAT PIS.  VENEREAL  jGONORRHEA  VISION D.  HERNIA  MENTAL PEF.  INGUINAL RINGS.FNL  VARICOCELE  OTITIS MEDIA  ANKYLOSIS  UNDERWEIGHT  ASTHMA  FEET CQNGEN. P.  uHEMdRRHOlDS  FRACTUREMAL'lfc  HEART D1K FUNCT.  ARTHRITIS  SKIN PIS, _.  .PSYCHOSES  HEART, HYK~ OIL.  EYE, REF. ERROR  VARICOSE VEINS  CHANCROID  SeRVOUA.ODJLEUNCJ  GOITRE .SIMPLE  MYOCARDITIS  HEARING P.___  fHYSiQAb.PEYEU.P  nDRuG ADDICTS .101  I PSVCMOfATHS CONST  I GOITRE. EXOPH. -  Plate XXVI.-Graphs of comparative frequency of various diseases and defects found at mobilization  camps in the first million men. Ratios per 1,000 recruits from the States of Alabama, Arizona, and  Arkansas.  174", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fnxz.2kk8-uiwu", "00000000-0000-0000-E970-5E586250BEE3", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Graphs showing the relative incidence of diseases and defects in New Jersey, New Mexico, and New York", "9918573882206676X96", null, "1919", "1919", "Page 183 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "Plate XXXV.  DISEASES AND DEFECTS -NEW JERSEY- RATIO PER 1.000.  THROAT PIS.  INGUINAL RlNGS.EMu  TEETH, P. OHLOSS  VENEREAL ALL •  -GONORRHEA  TUBERCULOSIS PLkl  .UNDERWEIGHT . 1  VARICOCELE J  -FAACTURE,nAL:UM|  MENTAL bEFJzj  ANKYLOSIS  -SKIN  HeV?ORRHO1DSU*Z  ARTHRITIS.__J  GO IT RE_ EXQ P rtZ)  PSYX HO  16.51  15.26  13.17!  7.97  7.66  5.031  3.74!  3.17J  2.78  2-721  2.23!  1.60!  1.47  137  1.31  1.31!  •*«  .«%  .591  •42  •54  _1  TRACHOMA - - - _.I3  DRUG APPICTS . __.13  MYOCARDITIS13  PSYCHOPATHS CONST. 10  NERVOUS DOR FJNCT.lOi  DISEASES AND DEFECTS -NEV/ MEXICO- RATIO PER 1.000  -GONORRHEA  -THROAT PLS. I  ' H EART. VALV. DIS71  FEET.CCNGEN.'p ■  MENTAL DEEI  'VISION tL I  mracAL-DEYEUpj  TESTESCONGEN.P. I  VARlCOtELE ~ .2- I  ANKYLOSISJ  TRACHOMA I  SKIN DIS.  I  -VARICOSE--VEINS ■  . TEETH.. EL OR LD S51  .QT1T12 MEP1A I  ARTHRITIS ■  .CHANCROID■  CMi5M  GQ1TRE. EXDPH. fl  PSYCHOSES___ZJ  w  na  11.63|  7.751  1:31  457  3:1?  £11  1.76  1.41  1.41  1.41  .70  .70  .33  J?  .35  .35  ASTHMA - ....  HCART HYP. PIU.  HEARING Cl . . .  NOSE DIS. ....  MYOCARDITIS  NERVOUS DPR. FUNCT  GOITRE . SIMPLE -  'DISEASES AND DETECTS -NEW YORK- RATIO PER W  PES-PLANUS  THROAT PIS.  VENEREAL (ALL  GONORRHEA  HERNIA  TEETH. Pl OR L.0  FEET GONGEkL C  EYE REEERROf  1TESTE1CONGEN.  .FRACTUREHAL'Uf  .SKIN PIS.  VARICOSE VEIN  .ANKYLOSIS  HEMORRHOIDS  DRUG ADDICTS  HEART. HYP. DIL  HEART P1S. FLNC  SCOLIOSIS  JMQSE PIS.  PSYCHOSES  HEARING R  -CHANCROID  ARTHRITIS  TRACHOMA  GOITRE, EXOPH  \"zrrso  18.02  lill  8.3 2  5.03  2.84  18  1.98  1:11  149  1.41  'if  .74  .71  .66  •.:?  :U  .37  *30  NERVOUS DPR. FUNCT-M  MYOCARDITIS33  PSYCHO PATHS CONST ,20|  Plate XXXV.-Graphs of comparative frequency of various diseases and defects found at mobilization  camps in first million men. Ratios per 1,000 recruits from New Jersey, New Mexico, and New York.  183", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xzjx~2uni~f486", "00000000-0000-0000-9EDB-F07DA952EED4", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Total number of men examined from each state at mobilization camps, and disposition of the same men--first million sent to camps, June 1917 to May 1918", "9918573882206676X97", null, "1919", "1919", "Page 191 of Albert G. Love and Charles B. Davenport. Physical Examination of the First Million Draft Recruits: Methods and Results. Washington, DC: Government Printing Office, 1919.", "Monographs, Excerpts, Charts (graphic documents)", null, "World War I: Collecting Medical and Anthropological Data in the U.S. Army, 1917-1919", "1", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "APPENDIX.  Table I.-Total number of men examined from each State at mobilization camps and  disposition of same men; covering approximately first million men sent to camp in  period from June, 1917, to May, 1918.  State.  Num-  ber of  men  exam-  ined  from  each  State at  mobil-  ization  camps.  Men accepted  as qualified  without  defects.1  Men accepted  as qualified  with defects.  Num-  ber of  men  ac-  cepted  with  defect  for  opera-  tion.  Men accepted  for limited  service.  Men rejected.  Num-  ber.  Per  cent.  Num-  ber.  Per  cent.  Num-  ber.  Per  cent.  Num-  ber.  Per  cent.  Alabama  16,291  10,185  62.52  5,031  30.88  1  9  0.06  1,065  6.54  Alaska  106  44  41.51  59  55.66  1  2  1.89  Arizona  3,894  2,733  70.18  981  25.19  1  21  .54  158  4.06  Arkansas  10,287  7,569  73.58  1,946  18.92  29  .28  743  7.22  California  35,839  19,998  55.80  12,117  33. 81  37  351  .98  3,336  9.31  Colorado  6,774  4,129  60.95  2,407  35.53  14  .21  224  3.31  Connecticut  13,653  8,644  63.31  3,776  27.66  2  12  .09  1,219  8.93  Delaware  1,909  1,149  60.19  661  34.63  1  4  .21  94  4.92  District of Columbia....  4,504  3,230  71.71  1,218  27.04  9  .20  47  1.04  Florida  5,982  3,488  58.31  1,954  32.66  6  .10  534  8.93  Georgia  20,936  13,533  64. 64  5,970  28.52  4  36  .17  1,393  6.65  Idaho  4,078  2,614  64.10  1,146  28.10  1  34  .83  283  6.94  Illinois  70,459  44,662  63.39  23,271  33.03  10  97  .14  2,419  3.43  Indiana  23,444  16,205  69.12  6,358  27.12  3  40  .17  838  3.57  Iowa  20,584  13,846  67.27  5,881  28.57  1  23  .11  833  4.05  Kansas  9,970  6,317  63.36  3,224  32.34  2  39  .39  388  3.89  Kentucky  15,875  11,773  74.16  3,191  20.10  23  .14  888  5.59  Louisiana  12,752  8,993  70.52  2,875  22.55  3  39  .31  842  6.60  Maine  3,344  2,131  63.73  886  26.50  1  3  .09  323  9.66  Maryland....  9,897  5,914  59.76  3,886  39.26  1  24  .24  72  .73  Massachusetts  30,181  18,396  60.95  9,204  30.50  8  31  .10  2,542  8.42  Michigan  42,212  29,664  70.27  8,948  21.20  6  184  .44  3,410  8.08  Minnesota  27,560  19,123  69.39  7,650  27.76  3  27  .10  757  2.75  Mississippi  8,827  6,317  71.56  1,773  20.09  2  42  .48  693  7.85  Missouri  25,172  14,826  58.90  9,135  36.29  10  119  .47  1,082  4.30  Montana  11,780  7,522  63.85  3,289  27.92  2  80  .68  887  7.53  Nebraska  10,930  7,170  65.60  3,391  31.02  1  25  .23  343  3.14  Nevada  1,443  844  58.49  475  32.92  11  .76  113  7.83  New Hampshire  2,288  1,532  66.96  551  24.08  6  .26  199  8.70  New Jersey  30,531  20,433  66.93  8,520  27.91  7  159  .52  1,412  4.62  New Mexico  2,838  1,928  67.94  777  27.38  3  4  .14  126  4.44  New York  89,339  55,540  62.17  28,896  32.34  11  166  .19  4,726  5.29  North Carolina  15,170  8,851  58.35  4,970  32.76  3  7  .05  1,339  8.83  North Dakota  6,621  4,465  67.44  1,944  29.36  6  .09  206  3.11  Ohio  53,817  35,230  65.46  16,663  30.96  1  19  .04  1,904  3.54  Oklahoma  19,864  13,574  68.33  5,586  28.13  6  22  .11  676  3.40  Oregon  3,900  2,271  58.23  1,417  36.33  2  60  1.54  150  3.85  Pennsylvania  78,706  52,434  66. 62*  24,774  31.48  10  152  .19  1,336  1.70  Rhode Island  3,958  2,395  60.51  1,200  30.32  13  .33  350  8.84  South Carolina  10,025  6,578  65.62  2,545  25.39  5  .05  897  8.95  South Dakota  4,013  2,703  67.36  1,211  30.18  2  7  .17  90  2.24  Tennessss  14,691  10,386  70.70  3,881  26.42  7  .05  417  2.84  Texas  35,852  24,659  68.78  10,089  28.14  9  27  .08  1,068  2.98  Utah  4,652  2,842  61.09  1,419  30.50  7  31  .67  353  7.59  Vermont  2,079  1,314  63.20  592  28. 48  4  .19  169  8.13  Virginia  18,600  9,351  50.27  8,844  47r55  27  .15  378  2. 03  Washington  13,523  8,287  61. 28  4,137  30.59  4  155  1.15  940  6.95  West Virginia  12,613  7,696  61.02  4,631  36. 72  1  25  .20  260  2.06  Wisconsin  18,610  12,840  69.00  4,794  25.76  4  47  .25  925  4.97  Wyoming  1,932  1,157  59.89  632  32.71  25  1.29  118  6.11  State not specified  101,901  66,302  65.07  26,099  25.61  11  126  .12  9,363  9.19  Total  994,206  643,797  64.76  294,875  29.66  182  2,434  .24  52,918  5.32  i Obtained by difference.  191", "United States. Department of the Army. Office of the Surgeon General ; Love, Albert G. (Albert Gallatin), 1877-1964 ; Davenport, Charles Benedict, 1866-1944", null, null, "United States. Government Printing Office", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-nina.7hdg-5m9j", "00000000-0000-0000-4443-00ED3AD94142", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Medical Descriptive List, Camden Street Hospital, Baltimore", "9918573882206676X148", null, "1863", "1863", "This medical form from a U.S. Army General Hospital in Baltimore provides an example of the case history information that army surgeons reported to the Surgeon General's Office after Surgeon General Hammond issued Circular No. 2 in 1862.", "Forms (documents)", null, "Background Information", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "MEDICAL DESCRIPTIVE LIST. — - ooce  General Hospital at /&WP@M/\\\\/ ______ \\\\wV Ea/%z. ___________ e , Age 3.1 ., Rank /Ofl/w) ...... b 9{’ _______ , Regiment /7[&%(.%@ ...........  ( Admassion,           Return to duty, cured,     : Hurlonshy .. SRR e e N Bl e s e e O o &     NoTE.—When a patient is first received into a General Hospital, the entries on this Descriptive List will be commenced. All important changes in his - - condition will be noted on it, (in ink,) from time to time, by the Surgeon in charge of the Ward. When the patient has been wounded, the date and character  of the wound will be stated, the nature of the operation, (if any,) and, above all, the result. - In case of transfer, this list will be sent, through the Officer in charge of the transportation, or failing one, by mail, to the Surgeon in charge of the Hospital receiving the patient. When this Medical History shall have  been completed, by the cure, discharge, furlough, or death of the patient, it will, with the treatment and result carefully noted, be transmitted directly to the Surgeon General.           DATE. TREATMENT. DIET. REMARKS AS TO CONDITION OF PATIENT, &c.", "United States. Department of the Army. Office of the Surgeon General", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hj8d~uwjw_99ct", "00000000-0000-0000-8072-7C5A0D916344", 0, 1750962675, null, 1750962675, null, "{ }", "Risk Factors: Measuring and Rating Health, Illness, and Mortality", "9918573882206676", "Report of Sloan U.S. General Hospital for the week ending October 15, 1864", "9918573882206676X149", null, "1864", "1864", "This hospital report from the Sloan Hospital in Vermont is an example of the weekly updates that Union Army hospital physicians were required to submit to the Surgeon General's Office during the Civil War.", "Official reports", null, "Background Information", "2", "pages", "Text", "English", "The National Library of Medicine believes this item to be in the public domain.", "Public domain", null, null, "e £ ettt e S g  e et ARSI 2 S        e     REPORT  OF     FOR THE  Week ending @/@/,// é/?é 186 %4                                         flec'd & G4, i s o F G    M the week ending ! TR e e e     186 24     ) 3                                                                                                                              The beds remaining “vacant,” and the number of ‘* Patients remaining,” should together equal the ‘ Total num nber of beds for  e — ———————— . T . o e | e Sk 5 5 3 22 Sh i 2 P | 2 o4 3 Ff a1 By ledlEg B B 20 | | : 8 28 ST LR R e SH s B 1S 28 | .2 |Rario OF DEATHS | NO. NAMES OF MEDICAL OFFICERS. RANK AND TITLE OF MEDICAL OFTFICERS. NAMES OF HOSPITAL STEWARDS. | « ?é 0 g © | 4 8 gé‘é m.é e e P—,é =% SR &,g | PER 1,000 OF | o e e B P R s sg | Sk, | ; 8 S a0 sl T R s E i = ' R Zi 2 Z 1 e 0B e = ol E 2 Z ; - | el g Ve E A e \" | mees | //7&; /% /4/4@%{ Wg% et ,%i Agiie 02/32/ 44,@55 57 /5 e S 4 / 49& 2;,&174 % fi//' a2l fl, e 7 %/ fi,fifW/ Y | | | | i | Al / | ‘ | %/ 04 G el gl e 7 7 | | | | o | § 'i/é/ /z%f,/ %é W\\\\ & i r 7 | * 1 [ *\\\\ ! _ | \\\\ ’ \\\\ \\\\ | oA a T | | | e | L 7 Lty fl% LA 7 7 7 ; | | | | | | | ' “ | : | | | | | | | i i E | | | | | | 1 | | | | | | \\\\ \\\\ 5 i | | | i | f ‘ | i | i t | | 1 i | ‘ | i | , | | ' ? | | OBSERVE CAREBFULLY THE FOLLOWING INSTRUCTIONS: x \\\\ \\\\ \\\\ ‘ The official title of each Medical Officer must be given. | | | j ' | ” ; The beds occupied by Guards and Hospital Attendants will not be included in the column ‘¢ Total number of beds for Patients.” 4) | | : | 4 | |  Puatients.”     All changes of Officers, Stewards, and Cadets must be noted on the first report subsequent to such change. Report also the name of the Chaplain, if there is one attached to the hospital, and when assigned.  These reports to be forwarded weekly, on Saturdays, to the Medical Director, who will forward them on the following Monday to the Surgeon General.", "Sloan U.S. Army General Hospital", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-pua7_cjd9.48jq", "00000000-0000-0000-C86B-A7CEE18D6416", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery Memorial Gateway", "101584575X116", "101584575X83,101584575X84,101584575X85,101584575X87,101584575X89,101584575X90,101584575X18,101584575X10", "1965", "[After 1965]", "The Avery Memorial Gateway was dedicated in 1965 on the grounds of Rockefeller University.", "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cs6m-9tai-zsns", "00000000-0000-0000-81D7-A237C43F5FBE", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery", "101584575X134", "101584575X520,101584575X30,101584575X157,101584575X41,101584575X158,101584575X212,101584575X159,101584575X160,101584575X213", "1943", "[March 1943]", null, "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Joshua Lederberg.", "Copyright may apply", null, null, null, "Coburn, Alvin F.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4tps~vbxu~c6ni", "00000000-0000-0000-69FE-051F7CB1DDED", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Rockefeller Institute for Medical Research hospital staff", "101584575X15", null, "1935", "1935", "Front Row, Left to Right: Donald D. Van Slyke, Alfred E. Cohn, Rufus Cole, Homer F. Swift, Thomas M. Rivers, and Oswald T. Avery.. Second Row, Left to Right: Julius Sendroy, Theodore J. Abernathy, Cornelius P. Rhoades, Irvine Page, Walther F. Goebel, Unknown, David Miller, and Thomas Francis, Jr.. Third Row, Left to Right: Mark P. Schultz, John Murray Steele, Jr., Alfred Mirsky, Rene Dubos, Lee E. Farr, Delavan V. Holman, Bacon F. Chow, and Robert T. Dillon.. Fourth Row, Left to Right: Rebecca C. Lancefield, Frank Babers, Colin MacLeod, Johannes K. Moen, Alma Hiller, Joseph E. Smadel, and R. Parker. Fifth Row, Left to Right: Ernest Stillman, Kenneth Goodner, T. F. McNair Scott, Thomas P. Magill, Raymond F. Holden, Jr., and Frank L. Horsfall, Jr.", "Photographic prints", null, "Shifting Focus: Early Work on Bacterial Transformation, 1928-1940", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-eety.cyze.a6ai", "00000000-0000-0000-EC37-7940E8E21DBA", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Rockefeller Institute for Medical Research hospital staff", "101584575X16", null, "1948", "1948", "Front Row, Left to Right: Maclyn McCarty, Henry D. Lauson, Oswald T. Avery, Thomas M. Rivers, Donald D. Van Slyke, Frank L. Horsfall, Jr., Rebecca C. Lancefield, and Sidney Rothbard.. Second Row, Left to Right: Fred M. Davenport, Harold C. Anderson, Duard Lee Walker, George C. Cotzias, William J. Eisenmenger, Henry G. Kunkel, and Harold S. Ginsberg.. Third Row, Left to Right: Paul H. Hardy (between Cotzias and Eisenmenger), and Howard A. Eder (between Kunkel and Ginsberg).. Fourth Row, Left to Right: Lewis K. Dahl, Oskar H. Lahelle, Sven E. Bjorkman, George E. Murphy, Edward H. Ahrens, Jr., Francis P. Chinard, and James R. Weisiger.", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-nczu_ws23-tbhq", "00000000-0000-0000-AD38-B1DCD90DAC3D", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery, Christmas", "101584575X17", null, "1940", "December 1940", null, "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-c8bp-c2kp_g3yg", "00000000-0000-0000-D2AC-2E9452DF020E", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Colin MacLeod, Maclyn McCarty, Detlev Wulf Bronk, Theodosius Dobzhansky, and Wendell M. Stanley at the Avery Memorial Gateway dedication ceremony", "101584575X18", "101584575X83,101584575X84,101584575X85,101584575X87,101584575X89,101584575X90,101584575X116,101584575X10", "1965", "29 September 1965", null, "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7qma~669i~txsc", "00000000-0000-0000-759E-5A6237E960ED", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Fred Griffith", "101584575X27", "101584575X346", null, "undated", "Caption in Joshua Lederberg's hand, \"From Stuart Elliott.\"  A copy of the photograph is printed at the end of Griffith's 1928 article.", "Negatives (photographic)", null, "Shifting Focus: Early Work on Bacterial Transformation, 1928-1940", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of Maclyn McCarty.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-r529~xz39~jwd7", "00000000-0000-0000-49FA-AE22E1FE1652", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery", "101584575X280", "101584575X37", "1937", "1937", "Portrait of Avery, cropped from a Rockefeller Institute for Medical Research staff photograph.", "Photographic prints", null, "Shifting Focus: Early Work on Bacterial Transformation, 1928-1940", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4pq2-67i2~thwa", "00000000-0000-0000-4E14-5AED3DAD3339", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Fred Griffith and \"Bobby\"", "101584575X30", "101584575X520,101584575X157,101584575X134,101584575X41,101584575X158,101584575X212,101584575X159,101584575X160,101584575X213", "1936", "[1936]", "This photograph is one of the few remaining of the English microbiologist who was killed in the Blitz in London in 1941. Although Griffith and Avery never met, the two scientists greatly respected each other's work on pneumococci transformation. Inscription on reverse reads \"Snap-shot of Dr. Fred. Griffith and \"Bobby\" on the Downs near Brighton from Al Coburn.\"", "Photographic prints", null, "Shifting Focus: Early Work on Bacterial Transformation, 1928-1940", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Joshua Lederberg.", "Copyright may apply", null, null, null, "Coburn, Alvin F.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xakc_2s5r.t7xc", "00000000-0000-0000-7E65-27DD81FC7A8A", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Rockefeller Institute for Medical Research hospital staff", "101584575X32", null, "1923", "1923", "Front Row, Left to Right: Arnold Branch, Hugh J. Morgan, A. Baird Hastings, Thomas M. Rivers, George R. Brow, and Harry W. Dahl.. Second Row, Left to Right: Harald A. Salvasen, Alfred E. Cohn, Ralph Boots, Donald Van Slyke, Herald J. Stewart, James M. Neill, Oswald T. Avery, and Michael Heidelberger.. Third Row, Left to Right: Christen Lundsgaard, Henry A. Murray, Carl A. L. Binger, Alma E. Hiller, Homer F. Swift, C. Philip Miller, Geoffry C. Linder, and William S. Tillett.", "Photographic prints", null, "The \"Sugar-Coated Microbe\" and the Search for a Cure for Pneumonia, 1919-1929", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jnh5.un7g_e8xz", "00000000-0000-0000-BD6F-483E72F3AB4C", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Colin M. MacLeod", "101584575X339", null, "1935", "1935", "Image is a close-up of MacLeod, cropped from a Rockefeller Institute for Medical Research staff photo.", "Photographic prints", null, "Shifting Focus: Early Work on Bacterial Transformation, 1928-1940", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-e6kg~uhww.2d2v", "00000000-0000-0000-FA19-21D673A49158", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Rockefeller Institute for Medical Research hospital staff", "101584575X34", null, "1932", "9 June 1932", "Front Row, Left to Right: Homer F. Swift, S.F. Seelig, Alfred E. Cohn, Rufus Cole, Oswald T. Avery, Thomas M. Rivers, and Robert T. Dillon.. Second Row, Left to Right: Ernest Vaubel, Harold J. Stewart, William H. Lewis, Jr., and Donald D. Van Slyke.. Third Row, Left to Right: David K. Miller, Osceola Currier McEwen, George P. Berry, and Thomas Francis, Jr.. Fourth Row, Left to Right: Edward E. Terrell, Francis F. Schwentker, John Esben Kirk, Walther F. Goebel, Bernard Benjamin, Julius Sendroy, Alfred E. Mirsky, and Irvine H. Page.. Fifth Row, Left to Right: Frank H. Babers, James Lionel Alloway, Alma E. Hiller, Douglas H. Sprunt, and Rebecca J. Lancefield.. Sixth Row, Left to Right: Cornelius P. Rhoads, Rene Dubos, Kenneth Goodner, John Murray Steele, Jr., Benjamin E. Hodge, and Cuthbert L. Cope.", "Photographic prints", null, "Shifting Focus: Early Work on Bacterial Transformation, 1928-1940", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-dmas~p7ak~57uw", "00000000-0000-0000-F00C-47C19305F71F", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Maclyn McCarty", "101584575X340", null, "1948", "1948", "Image is a close-up of McCarty, cropped from a Rockefeller Institute for Medical Research staff photo.", "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-x78b-t2bu~ki2e", "00000000-0000-0000-A1C1-126310BD4808", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Pneumococcus", "101584575X341", null, "1994", "1994", "Image is cropped from a Rockefeller University flyer on the 50th anniversary of Avery's article.", "Photographic prints", "Streptococcus pneumoniae", "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of Rockefeller University.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-uus6_x7hc-7kp2", "00000000-0000-0000-A22F-5AC4356DC1A2", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Pneumococcus colony", "101584575X342", null, "1994", "1994", "Image is cropped from a Rockefeller University flyer on the 50th anniversary of Avery's article.", "Photographic prints", "Streptococcus pneumoniae", "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of Rockefeller University.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-44zu.5rbr~dhj4", "00000000-0000-0000-2EF8-4DCE34D2B4CB", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Rockefeller Institute for Medical Research hospital staff", "101584575X37", "101584575X280", "1937", "1937", "O.T. Avery is in first row, third from the left. Rufus Cole is in first row, fourth from the left. Colin M. MacLeod is in second row (behind and to the right of Cole). Alfred E. Mirsky is in third row (behind and to the right of MacLeod). Rene J. Dubos is in fourth row, (behind and to the left of Mirsky). Rollin Hotchkiss is in back row, third from the right.", "Photographic prints", null, "Shifting Focus: Early Work on Bacterial Transformation, 1928-1940", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5eeu-u23t~t7u3", "00000000-0000-0000-B6B2-FFA41F7D35F2", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Rockefeller Institute for Medical Research hospital staff", "101584575X39", "101584575X515", "1951", "1951", "First Row, Left to Right: Regina M. Archibald, Maclyn Mccarty, Frank L. Horfall, Jr., Thomas M. Rivers, Alfred E. Cohn, Henry G. Kunkel, and Vincent P. Dole.. Second Row, Left to Right: George E. Murphy, Rebecca C. Lancefield, Dominic D. Dziewiatkowski, Harold S. Ginsberg, Robert J. Slater, and Jacques Genest.. Third Row, Left to Right: Solomon H. Blondheim, Irving L. Schwartz, Lewis K. Dahl, George C. Cotzias, and Chandler A. Stetson.. Fourth Row, Left to Right: Stewart D. Elliott, Igor tamm, William J. Eisenmenger, Herbert Jaffe, and Edwin D. Kilbourne.", "Photographic prints", null, null, "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-siib~scac.ba8h", "00000000-0000-0000-1670-2AD148DB2957", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery", "101584575X4", "101584575X497,101584575X26", "1958", "[January 1958]", "The photograph used in this 1958 publication was taken in the 1940s.", "Photographic prints, Portraits", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Joshua Lederberg.", "Copyright may apply", null, null, null, null, null, "National Academy of Sciences, Biographical Memoirs", "Columbia University Press", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-848c.7z7c.n8iw", "00000000-0000-0000-C151-3DA36A6D4BB4", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery with Alvin Coburn's son, Tim", "101584575X41", "101584575X520,101584575X30,101584575X157,101584575X134,101584575X158,101584575X212,101584575X159,101584575X160,101584575X213", "1943", "[March 1943]", "For this picture, Avery took a moment at Al Coburn's home in Bedford, New York, to pose with Coburn's son, Tim. The picture was taken one Sunday afternoon shortly after Avery reported his findings to the Rockefeller Institute Hospital Board on the discovery of the \"transforming principle.\"", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of Joshua Lederberg.", "Copyright may apply", null, null, null, "Coburn, Alvin F.", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-q2n9_jfm3_nudd", "00000000-0000-0000-CC1E-C4C9D0DFD2C4", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Colin M. MacLeod", "101584575X42", null, "1960", "[ca. 1960]", "MacLeod spent seven years in Avery's laboratory at the Rockefeller Institute before leaving for New York University in 1941.", "Photographic prints, Portraits", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-eng5.5yp3-t8mv", "00000000-0000-0000-86D4-895AD4C4EC61", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Maclyn McCarty with James Watson and Francis Crick at the Waterford Prize Ceremony", "101584575X43", null, "1979", "1979", "McCarty warmly greets Watson and Crick at the 1979 Waterford Biomedical Science Awards ceremony at the Scripps Institute.", "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of Maclyn McCarty.", "Copyright may apply", null, null, null, "Lensmen Photographic Agency ; Smull, Robert", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-eaa7.fjpt~he4h", "00000000-0000-0000-81C3-01F13A4D5DCC", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Maclyn McCarty with a portrait of Oswald T. Avery at the Rockefeller University Hospital", "101584575X44", "101584575X45,101584575X595", "1985", "[ca. 1985]", "The portrait of Avery on the wall behind McCarty was commissioned in 1928 by Avery's colleague at the Rockefeller Hospital, and close friend, Ernest Stillman. It was painted by Durr Reedley. In 1985, the portrait was donated to Rockefeller University by Avery's sister-in-law, Catherine Avery.", "Photographic prints", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-aq57_54uu-cndb", "00000000-0000-0000-E6BC-35E5D8F261E7", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Titration of Activity of Extracts 1/40, 2/40, 3/40", "101584575X46", null, "1940", "5 November 1940", "Caption by Maclyn McCarty reads: \"Tests of trial extracts made in presence of fluoride illustrating the variability of results and the lack of reliability of fluoride as an inhibitor of inactivation. Extracts 1/40 and 2/40 were prepared with small batches of organism and showed some (but quite limited) activity. Extract 3/40, using organisms from 30 liters of culture, lacked transforming activity.\"", "Laboratory notes", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "MacLeod, Colin M. (Colin Munro), 1909-1972", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-bpwn~4spu.s2xz", "00000000-0000-0000-281B-F5E1599A9724", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Effect of Ribonuclease on Deproteinized Extract 5-40 (image 1)", "101584575X47", "101584575X48,101584575X49,101584575X50", "1941", "28 January 1941", "Caption by Maclyn McCarty reads: \"An experiment in January 1941, carried out to reconfirm earlier work indicating that ribonuclease has no effect on the transforming substance. The diphenylamine test for desoxyribose was used for the first time in testing various fractions.  MacLeod's comment reads: 'Thus it would appear as though these transforming extracts may contain a little desoxyribosenucleic acid in addition to the large amount of ribosenucleic acid present.'\"", "Laboratory notes", "Ribonucleases", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Avery, Oswald Theodore, 1877-1955 ; MacLeod, Colin M. (Colin Munro), 1909-1972", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-y6z8-hjxe.ygee", "00000000-0000-0000-E146-976DB720201F", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Effect of Ribonuclease on Deproteinized Extract 5-40 (image 2)", "101584575X48", "101584575X47,101584575X49,101584575X50", "1941", "28 January 1941", "Some of Avery and MacLeod's lab notes on the experiments that led to the 1944 article with McCarty.", "Laboratory notes", "Ribonucleases", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Avery, Oswald Theodore, 1877-1955 ; MacLeod, Colin M. (Colin Munro), 1909-1972", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wvvm~84et_5c5g", "00000000-0000-0000-7F63-31087E2116DF", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Effect of Ribonuclease on Deproteinized Extract 5-40 (image 3)", "101584575X49", "101584575X50,101584575X48,101584575X47", "1941", "28 January 1941", "Some of Avery and MacLeod's lab notes on the experiments that led to the 1944 article with McCarty.", "Laboratory notes", "Ribonucleases", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Avery, Oswald Theodore, 1877-1955 ; MacLeod, Colin M. (Colin Munro), 1909-1972", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-utsk~u3qd_wbav", "00000000-0000-0000-01B0-D6A5971EDF45", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery's medals (back view)", "101584575X496", "101584575X600", "1950", "[ca. 1950]", "A photograph of Avery's medals.. Clockwise from upper right: Copley Medal, Royal Society of London (1945); John Phillips Memorial Award, American College of Physicians (1932); Paul Ehrlich Foundation Gold Medal (1933); Pasteur Gold Medal, Swedish Medical Society (1950); New York Academy of Medicine Medal (1944); Koeber Award, Association of American Physicians (1946). Center: Phi Beta Kappa Key, Columbia University.", "Photographic prints, Awards", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p9jn-vyg5~6gu7", "00000000-0000-0000-47B2-9F622B624417", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery", "101584575X497", "101584575X4,101584575X26", "1948", "1948", "This photo was likely one of the last official portraits of Avery at the Rockefeller Institute. In 1949, he retired to Nashville, Tennessee.", "Photographic prints, Portraits", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-jw7z~8p24-iemd", "00000000-0000-0000-3CCD-CA923E587185", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery postcard", "101584575X498", null, "1940", "[ca. 1940]", null, "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-njpx_82pk.9bbx", "00000000-0000-0000-5CA7-493D858B75D3", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Effect of Ribonuclease on Deproteinized Extract 5-40 (image 4)", "101584575X50", "101584575X47,101584575X48,101584575X49", "1941", "30 January 1941", "Some of Avery and MacLeod's lab notes on the experiments that led to the 1944 article with McCarty.", "Laboratory notes", "Ribonucleases ; Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Avery, Oswald Theodore, 1877-1955 ; MacLeod, Colin M. (Colin Munro), 1909-1972", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ytiy-ynhf~5qs7", "00000000-0000-0000-1361-ABC2F254BBA8", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery", "101584575X503", null, "1920", "[1920s]", "In the 1920s, Avery turned his focus on biochemical studies of the pneumococcus, collaborating with various members of his research team, including Glenn Cullen, Hugh Morgan, James Neill, and Theodor Thjotta.", "Photographic prints", null, "The \"Sugar-Coated Microbe\" and the Search for a Cure for Pneumonia, 1919-1929", "1", "pages", "Still Image", "No linguistic content", "Courtesy of The History of Medicine Division. Prints and Photographs Collection.", "Copyright may apply", null, null, null, null, "The History of Medicine Division. Prints and Photographs Collection", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9znq-hwez.9ze2", "00000000-0000-0000-D82E-80F582B4BEFC", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery", "101584575X504", null, "1930", "[1930s]", "Following Fred Griffith's demonstration in 1928 that R and S strains of pneumococci could be converted from one to the other, the focus of Avery's laboratory began to shift to the problem of bacterial transformation.", "Photographic prints, Portraits", null, "Shifting Focus: Early Work on Bacterial Transformation, 1928-1940", "1", "pages", "Still Image", "No linguistic content", "Courtesy of The History of Medicine Division. Prints and Photographs Collection.", "Copyright may apply", null, null, null, "Schmidt, Louis E.", "The History of Medicine Division. Prints and Photographs Collection", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-pemx.rjzn~qvuq", "00000000-0000-0000-0D8F-622F39AD3EB6", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Roy C. Avery", "101584575X505", null, "1940", "[1940s]", "Roy Avery was a bacteriologist at the Vanderbilt School of Medicine. In 1943, in a letter from his brother Oswald, he became one of the first individuals outside the Rockefeller Institute to learn that DNA was genetic material.", "Photographic prints, Portraits", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of The History of Medicine Division. Prints and Photographs Collection.", "Copyright may apply", null, null, null, null, "The History of Medicine Division. Prints and Photographs Collection", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-b64q-2c4e.r9qr", "00000000-0000-0000-F1C1-FDE8CE029BEF", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Alphonse R. Dochez", "101584575X506", null, "1935", "[Late 1930s]", "Dochez collaborated with Avery on some of his early research at the Rockefeller Hospital. The two scientists were close friends and roommates for over thirty years.", "Photographic prints, Portraits", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Courtesy of The History of Medicine Division. Prints and Photographs Collection.", "Copyright may apply", null, null, null, null, "The History of Medicine Division. Prints and Photographs Collection", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7h9k.s2wf~qks4", "00000000-0000-0000-7E5A-A7CDA78CB074", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Alphonse R. Dochez", "101584575X507", null, "1920", "[1920s]", "Dochez collaborated with Avery on some of his early research at the Rockefeller Hospital. The two scientists were close friends and roommates for over thirty years. This portrait of Dochez appears to have been taken shortly after he joined the faculty at Columbia University following World War I.", "Photographic prints, Portraits", null, "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "1", "pages", "Still Image", "No linguistic content", "Courtesy of The History of Medicine Division. Prints and Photographs Collection.", "Copyright may apply", null, null, null, "Schmidt, Louis E.", "The History of Medicine Division. Prints and Photographs Collection", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-z7u7~9e79_ya2y", "00000000-0000-0000-8DF9-C53A03FB697D", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Colin M. MacLeod", "101584575X508", null, "1940", "[ca. 1940]", "MacLeod and Avery returned their focus to the problem of bacterial transformation in 1940. After MacLeod left to join the faculty at New York University, Maclyn McCarty joined with Avery to complete the research. This photo appears to be signed by MacLeod.", "Photographic prints, Portraits", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of The History of Medicine Division. Prints and Photographs Collection.", "Copyright may apply", null, null, null, null, "The History of Medicine Division. Prints and Photographs Collection", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-97ig.f2pm~xxmr", "00000000-0000-0000-B125-0ED5DBB67025", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Maclyn McCarty", "101584575X509", null, "1940", "[Early 1940s]", "McCarty joined Avery's lab at the Rockefeller Institute in 1941. He and Avery continued to perfect purification techniques developed by Colin M. MacLeod that were essential to unlocking bacterial transformation.", "Photographic prints, Portraits", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of The History of Medicine Division. Prints and Photographs Collection.", "Copyright may apply", null, null, null, null, "The History of Medicine Division. Prints and Photographs Collection", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-q4ga_zz8e~qujr", "00000000-0000-0000-36B3-3427965AEDE3", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Desoxycholate - Saline Extract of Heated Intact Type III Cells", "101584575X51", null, "1941", "11 March 1941", "These lab notes appear to be in Avery's handwriting. A negative of this photograph is also in the collection.", "Laboratory notes", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Avery, Oswald Theodore, 1877-1955", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-eiwy_4pdy~y5cf", "00000000-0000-0000-04E6-24EC8761B59B", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Rockefeller Institute for Medical Research hospital staff", "101584575X515", null, "1951", "1951", "First Row, Left to Right: Regina M. Archibald, Maclyn McCarty, Frank L. Horfall, Jr., Thomas M. Rivers, Alfred E. Cohn, Henry G. Kunkel, and Vincent P. Dole.. Second Row, Left to Right: George E. Murphy, Rebecca C. Lancefield, Dominic D. Dziewiatkowski, Harold S. Ginsburg, Robert J. Slater, and Jacques Genest.. Third Row, Left to Right: Solomon H. Blondheim, Irving I. Schwartz, Lewis K. Dahl, George C. Cotzias, and Chandler A. Stetson.. Fourth Row, Left to Right: Stewart D. Elliott, Igor Tamm, William J. Eisenmenger, Herbert Jaffe, and Edwin D. Kilbourne.", "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, null, null, "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-9as6-2w3u.2xus", "00000000-0000-0000-1E92-94038A38E289", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Desoxy--Saline Extract of Heated Type III Cells", "101584575X54", null, "1941", "18 March 1941", "A page from a lab notebook with first line reading: \"Repeat of exp. of Mar. 11 -- using desoxy -- saline extract of heat-killed cells.\" Caption by Maclyn McCarty:  \"Experiment carried out by Avery in March 1941 in search for a procedure that would avoid the losses of activity of the transforming substance that were being experienced on extracting live organisms by lysis with bile salts [sodium desoxycholate]. His modification was to heat kill the organisms at 65 degrees at the outset. The organisms were then no longer susceptible to lysis with bile salt, but he reasoned that active material could be extracted by shaking the organisms in saline containing a higher concentration of desoxycholate. After some further trials, which MacLeod also participated in, this proved successful. The old method, which was essentially that devised by [Lionel] Alloway in 1934, was abandoned from then on, with the result that extracts were consistently obtained with higher activity than had ever been encountered in the past.\"", "Laboratory notes", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Avery, Oswald Theodore, 1877-1955", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "MacLeod, Colin M. (Colin Munro), 1909-1972" ]
, [ "row-kuce_wijq-6ffd", "00000000-0000-0000-1523-76810FC1F6D8", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Action of S III B Enzyme on Dried T[ransforming]. P[rinciple]. Extract (image 1)", "101584575X55", "101584575X57", "1941", "28 November 1941", "Caption by Maclyn McCarty reads: \"An example of experiments carried out in November 1941 by McCarty to reconfirm that elimination of the type III polysaccharide [the capsule material] from transforming extracts by the use of the Dubos enzyme that hydrolyzes the capsular antigen has no effect on transforming activity.\"", "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Reproduced with permission of Maclyn McCarty.", "Copyright may apply", null, null, null, "McCarty, Maclyn", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-xuw7-prrt_uhu3", "00000000-0000-0000-D166-5525DF050700", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Action of S III B Enzyme on Dried T[ransforming]. P[rinciple]. Extract (image 2)", "101584575X57", "101584575X55", "1941", "28 November 1941", "Some of McCarty's notes on the experiments that led to the 1944 article with Avery and MacLeod.", "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Reproduced with permission of Maclyn McCarty.", "Copyright may apply", null, null, null, "McCarty, Maclyn", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-wsyv-3csm_dqrs", "00000000-0000-0000-2939-FB55120B3F1A", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "O.T. Avery, Hoagland Laboratory, 1907-13", "101584575X577", null, "1907", "[1907-1913]", "Prior to going to work at the Rockefeller Hospital in 1913, Avery cut his teeth as a medical researcher at the Hoagland Institute in Brooklyn, one of the first privately-funded medical research laboratories in the United States. As part of his duties, Avery often lectured nursing students. It is here that he acquired the nickname \"Fess,\" short for \"The Professor,\" that would remain with him for the remainder of his career.", "Photographic prints", null, "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-d5cg-reg3-pnda", "00000000-0000-0000-3BD8-3D8CD8B37985", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery", "101584575X578", null, "1944", "[ca. 1944]", null, "Photographic prints, Portraits", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-4fm7~6it4-a3ma", "00000000-0000-0000-3C2D-4E339569DF45", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Dr. O. T. Avery--Deer Isle, Maine", "101584575X579", null, "1940", "[1940s]", "Avery vacationed each summer on Deer Isle, Maine, with his brother Roy, sister-in-law Catherine, and their daughter Margaret. On many occasions, several of Avery's Rockefeller associates would join his family on vacation.", "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cvzx_azuk-jvyk", "00000000-0000-0000-40CF-20F048968ED4", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Dr. O. T. Avery (in lounge chair)", "101584575X580", null, "1940", "[1940s]", "This photo of Avery was likely taken on Deer Isle, Maine, where he vacationed every summer with his brother Roy, sister-in-law Catherine, and their daughter Margaret. On many occasions, several of Avery's Rockefeller associates would join his family on vacation.", "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bmjg.yxqr~hug7", "00000000-0000-0000-981A-96C88FC142B1", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "O. T. Avery, Rockefeller Institute", "101584575X581", null, "1920", "[1920s]", "Oswald Avery looks through a microscope in his lab.", "Photographic prints", null, "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-cnvi_gsyq~wd9f", "00000000-0000-0000-15CB-07B44888ACC6", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery during the First World War", "101584575X582", null, "1918", "[1918]", "During World War I, many of the scientists at the Rockefeller Hospital took part in the war effort. Avery lectured military officers as a member of the Army Medical Corps. In this photo, from left to right: Colonel A. R. Dochez, unknown, Captain Oswald T. Avery.", "Photographic prints", null, "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ymr8-8ifz~rm77", "00000000-0000-0000-9D08-284A08C06C9D", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald Avery at the College of Physicians and Surgeons in New York", "101584575X583", null, "1900", "[1900-1904]", "This undated photograph of Avery appears to have been taken while he attended medical school in the early 1900s.", "Photographic prints", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-6n4u.s6hc.9jr8", "00000000-0000-0000-1A54-FF70E8015F24", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Board for the Investigation and Control of Influenza and Other Epidemic Diseases in the Army", "101584575X584", null, "1942", "[12-13 May 1942]", "This photo was taken at the fifth meeting of the board, at Washington, D.C.. On the reverse, members of the board are identified as: Front row, left to right: Dr. Andrew J. Warren, Dr. Ernest W. Goodpasture, Dr. Francis C. Blake, Dr. Oswald T. Avery, Dr. Kenneth F. Maxcy, Dr. A.R. Dochez. Second row, left to right: Dr. O. H. Perry Pepper, Colonel James S. Simmons, M.C., Lt. Colonel S. Bayne-Jones, M.C.", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hkxb~gbd2-puww", "00000000-0000-0000-823C-04B7F4E55135", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Army Epidemiological Board", "101584575X585", null, "1946", "[15-16 April 1946]", "This photo was taken at the annual meeting of the A.E.B. in Washington, D.C., in April of 1946. A card attached to the photo indicates that it was provided to Avery \"with the compliments of James Stevens Simmons, Brigadier General, United States Army.\". On the reverse, the members are identified as: Left to right: Brig. General J.S. Simmons, AUS; Dr. A. R. Dochez; Dr. A. J. Warren; Dr. O. T. Avery; Dr. F. G. Blake, President; Dr. K. F. Maxcy; Dr. O. H. Perry Pepper; Brig. General S. Bayne-Jones, MC.", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, "United States. Army", "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-xhq8-csrf_7dp2", "00000000-0000-0000-63AA-376E56344910", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Army Epidemiological Board", "101584575X586", null, "1945", "[26-27 April 1945]", "This photo was taken at the annual meeting of the A.E.B. in Washington, D.C., in April of 1945.. On the reverse, individuals are identified as: Back row, left to right: Dr. J. J. Phair, Dr. J. Stokes, Jr., Dr. J. R. Paul, Dr. K. F. Maxcy, Maj. J. H. Dingle, MC, AUS, Dr. A. R. Dochez, Dr. C. M. MacLeod, Dr. A. J. Warren, Dr. C. S. Keefer, Dr. O. H. Robertson, Dr. H. E. Meleney, Lt. Col. A. C. McGuinness, MC, AUS. Front row, left to right: Dr. T. Francis, Jr., Dr. O. T. Avery, Brig. Gen. James S. Simmons, USA, Dr. Francis G. Blake, Brig. Gen. S. Bayne-Jones, USA, Dr. E. W. Goodpasture, Dr. O. H. Perry Pepper.", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, "United States. Army", "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-p33w-cyui~thjj", "00000000-0000-0000-6AE7-6E1D9FC97BD6", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Board for the Investigation and Control of Influenza and Other Epidemic Diseases in the Army", "101584575X587", null, "1943", "[6-7 May 1943]", "Present in this photo are: Front row, seated, left to right: Ernest W. Goodpasture, Alphonse R. Dochez, Brig. Gen. James S. Simmons, Francis, G. Blake, Brig. Gen. S. Bayne-Jones, Oswald T. Avery, O. H. Perry Pepper, A. J. Warren, K. F. Maxcy. Back row, standing, left to right: Captain D. W. Walker, MC, J. J. Phair, Morton J. Hamburger, John Holmes Dingle, Thomas Francis, Jr., O. H. Robertson, Colin MacLeod, Chester S. Keefer, Joseph Stokes, Jr., Robert Ward.", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, "United States. Army", "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-f4u7_i9y5-76y9", "00000000-0000-0000-0437-2AED8D0BCD0F", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Army Epidemiological Board", "101584575X588", null, "1946", "[15-16 April 1946]", "On the reverse, individuals are identified as: Front row, left to right: Dr. A. R. Dochez; Dr. A. J. Warren; Brig. General J. S. Simmons; Dr. F. G. Blake, President; Brig. General S. Bayne-Jones, MC; Dr. O. T. Avery; Dr. K. F. Maxcy; Dr. O. H. Perry Pepper. Back row, left to right: Dr. A. B. Sabin; Dr. O. H. Robertson; Dr. H. E. Meleney; Dr. C. S. Keefer; Dr. T. Francis, Jr.; Dr. C. MacLeod; Dr. J. J. Phair; Major J. H. Dingle, MC; Dr. J. Stokes, Jr.; Major E. B. Schoenbach, MC.", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, "United States. Army", "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-88qt_waxc.ma6v", "00000000-0000-0000-016C-21ECDF1AB4E8", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery, Rockefeller Institute", "101584575X589", null, "1940", "[1940s]", "In this photograph, Avery is in his office at the Rockefeller Institute Hospital.", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-7nsp-34wa~u3nd", "00000000-0000-0000-8255-C63E59C9841B", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery, Deer Isle, Maine", "101584575X590", null, "1949", "1949", "Avery vacationed each summer on Deer Isle, Maine, with his brother Roy, sister-in-law Catherine, and their daughter Margaret. On many occasions, several of Avery's Rockefeller associates would join his family on vacation.", "Photographic prints", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-u58i.bq87~hrhw", "00000000-0000-0000-F5BC-63621B15B4D8", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery and the Colgate University band", "101584575X591", null, "1900", "1900", "On reverse: \"Colgate University Band--1900. Oswald Theodore Avery third from left seated, holding cornet. He was a senior at this time. Picture reproduced in a special issue of their Year Book about 1968/9.\"", "Photographic prints", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Public Domain", "Public domain", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hc7c.j8kx~9snq", "00000000-0000-0000-40D0-6A73A8A463D7", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery at his desk", "101584575X592", null, "1920", "[ca. 1920]", "In this undated photograph, Avery is taking notes at his desk at the Rockefeller Institute.", "Photographic prints", null, "The \"Sugar-Coated Microbe\" and the Search for a Cure for Pneumonia, 1919-1929", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-wfqp-br79~h7m4", "00000000-0000-0000-4002-413785E07B5A", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery at his desk", "101584575X594", null, "1950", "[ca. 1950]", "In this photo, Avery is at his desk making notes in a lab notebook.", "Photographic prints", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-f8bp~4n7s_4et7", "00000000-0000-0000-6729-2E7FFFAB6523", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Oswald T. Avery", "101584575X595", "101584575X44,101584575X45", "1928", "1928", "This portrait of Avery by Durr Reedley was commissioned in 1928 by Avery's colleague at the Rockefeller Institute, and close friend, Ernest Stillman. In 1985, the portrait was donated to Rockefeller University by Avery's sister-in-law, Catherine Avery.", "Photographic prints, Portraits", null, "The \"Sugar-Coated Microbe\" and the Search for a Cure for Pneumonia, 1919-1929", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, "Reedley, Durr", "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yia2_vttj~6i9x", "00000000-0000-0000-5093-2CBD7F3D320F", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery's medals", "101584575X600", "101584575X496", "1950", "[ca. 1950]", "A photograph of Avery's medals.. Clockwise from upper right: Copley Medal, Royal Society of London (1945); John Phillips Memorial Award, American College of Physicians (1932); Paul Ehrlich Foundation Gold Medal (1933); Pasteur Gold Medal, Swedish Medical Society (1950); New York Academy of Medicine Medal (1944); Koeber Award, Association of American Physicians (1946). Center: Phi Beta Kappa Key, Columbia University.", "Photographic prints, Awards", null, "Biographical Information", "1", "pages", "Still Image", "No linguistic content", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, null, null, "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-min7_y4we~hjsp", "00000000-0000-0000-ECDA-E7B937722888", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Extract #24 Overnight Incubation [without] Added Glucose (image 1)", "101584575X61", "101584575X62", "1941", "2 December 1941", "Caption by Maclyn McCarty reads: \"An extract prepared with pneumococci from 51 liters of culture omitting the addition of extra glucose for the final two hours of growth to reduce the amount of polysaccharide formed. The activity (transformation with .03 ml of a 1:10,000 dilution) proved to be the highest yet encountered.\"", "Laboratory notes", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Reproduced with permission of Maclyn McCarty.", "Copyright may apply", null, null, null, "McCarty, Maclyn", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-bcyy_89ia~vrcu", "00000000-0000-0000-C541-C05844F7DEF5", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Extract #24 Overnight Incubation [without] Added Glucose (image 2)", "101584575X62", "101584575X61", "1941", "2 December 1941", "This page of a laboratory notebook also contains notes for December 15, 17, and 19, 1941.", "Laboratory notes", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Reproduced with permission of Maclyn McCarty.", "Copyright may apply", null, null, null, "McCarty, Maclyn", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-6xqa-8ecd.3b47", "00000000-0000-0000-D9C9-05016F48B264", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Repetition of Dog and Rabbit Sera Experiments (image 1)", "101584575X65", "101584575X66", "1942", "1 October 1942", "Caption by Maclyn McCarty reads: \"Experiment showing the effect of dog and rabbit sera in destroying transforming activity. The effect of dog serum was inactivated at a lower temperature than rabbit serum, and the same temperature differential was shown to obtain on testing the activity of these sera in depolymerizing purified mammalian DNA.\"", "Laboratory notes", null, "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-rfsm_nv7j_33dw", "00000000-0000-0000-1F8D-FA9020D3D6A5", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Repetition of Dog and Rabbit Sera Experiments (image 2)", "101584575X66", "101584575X65", "1942", "2 October 1942", "Some of McCarty's lab notes on the experiments that led to the 1944 article with Avery and MacLeod.", "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-ew4q.mh84_xty4", "00000000-0000-0000-182E-20EDA92F71B0", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Time of Entrance of T[ransforming]. P[rinciple]. into Susceptible Cell - 2nd Attempt", "101584575X67", null, "1945", "24 August 1945", "Caption by Maclyn McCarty reads: \"An example of the experiments making use of purified DNase to show that transforming DNA was not 'taken up' by the growing rough pneumococci until 3 to 4 hours after inoculation. This was the first indication that the state of 'competence' took time to develop. Competence developed only during growth in the complete culture medium used for transformation.\"", "Laboratory notes", "Transformation, Genetic", "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-hbe3~686t_pcf9", "00000000-0000-0000-4F71-FB038CF10B76", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Test of 'Fixation' Time of T[ransforming]. P[rinciple]. by Sensitized Cells", "101584575X68", "101584575X64", "1946", "13 August 1946", "Caption by Maclyn McCarty reads: \"One of the experiments carried out by Harriet Taylor (and written in her hand) showing that pneumococci that reach the competent state in the absence of transforming DNA will take up added DNA in as short a period as 5 minutes. She later found that the competent state would persist for some days at refrigerator temperature (2 to 4 degrees C).\"", "Laboratory notes", "Transformation, Genetic", "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Ephrussi-Taylor, Harriett", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "McCarty, Maclyn ; Avery, Oswald Theodore, 1877-1955" ]
, [ "row-29n8-piin-4hev", "00000000-0000-0000-F222-EB334DEE72A4", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Exp. 1 (T[ransforming]. P[rinciple].)  Effect of Fluoride on Autolysis of Pneumococcus Type III and on Preservation of the Transforming Principle (image 1)", "101584575X69", "101584575X70", "1940", "22 October 1940", "Page one of two from a lab notebook with caption by Maclyn McCarty: \"Note-book page marking the reinitiation of research on the pneumococcal transforming substance by MacLeod and Avery in the fall of 1940 after a three year hiatus.\"  The notes are in MacLeod's hand, but the title at the top, 'Exp. 1 (TP),' is by Avery. These experiments were carried out to test the possible effect of fluoride as an inhibitor of inactivation of the transforming substance by autolytic enzymes of the pneumococcus.", "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Avery, Oswald Theodore, 1877-1955 ; MacLeod, Colin M. (Colin Munro), 1909-1972", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8u8a~xnkh-b9xb", "00000000-0000-0000-7424-D42637CF3EF4", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Exp. 1 (T[ransforming]. P[rinciple].)  Effect of Fluoride on Autolysis of Pneumococcus Type III and on Preservation of the Transforming Principle (image 2)", "101584575X70", "101584575X69", "1940", "22 October 1940", "Page two of two from a lab notebook with caption by Maclyn McCarty: \"Note-book page marking the reinitiation of research on the pneumococcal transforming substance by MacLeod and Avery in the fall of 1940 after a three year hiatus.\" The notes are in MacLeod's hand, but the title at the top, 'Exp. 1 (TP),\" is by Avery. These experiments were carried out to test the possible effect of fluoride as an inhibitor of inactivation of the transforming substance by autolytic enzymes of the penumococcus.", "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, "Avery, Oswald Theodore, 1877-1955 ; MacLeod, Colin M. (Colin Munro), 1909-1972", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ufev.7ykx_hf2c", "00000000-0000-0000-9AA7-B3708772CBA7", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Transforming Activity - T.P. #43 - 44 (image 1)", "101584575X72", "101584575X73,101584575X74,101584575X75", "1943", "7 June 1943", "Caption by Maclyn McCarty reads: \"Titration of the last preparation of purified DNA from pneumococcus type III before beginning the process of preparing the paper for publication. All procedures in the purification process had been carried out in the cold, except for treatment with ribonuclease and the Dubos enzyme hydrolyzing the capsular polysaccharide. This preparation showed transforming activity in 50&#037; of the tubes containing 0.003 micrograms of the final product.\"", "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", null, "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-n6cg_djgm-ey4m", "00000000-0000-0000-9172-2951A5E250BB", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Transforming Activity - T.P. #43 - 44 (image 2)", "101584575X73", "101584575X72,101584575X74,101584575X75", "1943", "8 June 1943", "The first line reads, \"Dilutions from same material as that used in activity test of 6/7 - saline.\"", "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-nd5t.f6iu~xace", "00000000-0000-0000-32CD-14962A800160", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Transforming Activity - T.P. #43 - 44 (image 3)", "101584575X74", "101584575X72,101584575X73,101584575X75", "1943", "8 June 1943", null, "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-87uf.zsnn-aa5h", "00000000-0000-0000-F2B0-14228024153C", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Transforming Activity - T.P. #43 - 44 (image 4)", "101584575X75", "101584575X72,101584575X73,101584575X74", "1943", "8 June 1943", null, "Laboratory notes", "Transformation, Genetic", "DNA as the \"Stuff of Genes\": The Discovery of the Transforming Principle, 1940-1944", "1", "pages", "Still Image", "English", "Courtesy of Maclyn McCarty.", "Copyright may apply", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, "Avery, Oswald Theodore, 1877-1955" ]
, [ "row-bm9m~968c-rnrd", "00000000-0000-0000-A3A3-8F5DB8E31B92", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Letter from Oswald T. Avery to Roy Avery", "101584575X1", "101584575X519", "1943", "[13, 26 May 1943]", "The second part of this letter describes experiments conducted by Avery, MacLeod, and McCarty and the role of DNA as carrier of genetic information.", "Letters (correspondence)", "Transformation, Genetic ; DNA", null, "14", "pages", "Text", "English", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, "suet.  EE  sisi.  ee 7        tees vaoereeapemenses ope SSM SET eT I Oe oS RR eae ress ts mse srt gen Se     >  (oem) areas  Ae fe      “Wt Ly  ples. 7 Mottin,”  aan son Hira | |  he lrg ‘eps 4 /E- faut “ha, hat a.     { :              Mop A Kedar, - asap th Setrale.- Ma | Mirra, Thue hh. ba rae tak heh Yl fe OT Haines x Cone tor tyre  lat Mh Cr. bac. Aa. Be tig ie! tes ftir om Yue, ee  Me  espe y        . a hn  MM me wa . * \", ‘ 5 : . : woe : nop gt a . ; . . , . . 7 X : : ~ . : . we ee eS a ee cee meee came ee nice meme mene eva tat ee en ine ene iia telah en OC ROE A ts 2, inert ore ee rate 9 Dot ae Le af . : : oe : me. . . . an « « . so ee . S       2 PR        & ptials) tak be Spa hd, he Wack In, Ua fn fea,  Ue tiring —~ Ak bog hp Pert og az . A - rite a roa «fla a jet     “4 /                    ‘ aie ne . fi . - Fon, . . Le R . a . ae ee . Co ae fen Nae wy , . . ff. Se 2 ty ore aot ! . ‘ 7 ‘ - 1 “4. a oe cet e ren . mo : * ‘ \" c a - yi Wh lpmnee lay us» bem. Yes te ee . fe so roy coe i x eo EE, : a PR Bg 4 bey te tf tien wees . . me ER A AE foe, B is een 44              Yermuy lag joke ent gente Sod oo ht 4 Anaifey ja es Hac todi nate 7 os bahay fess $4 |     oo A Gets daa pl. aye  Ce hee Spee Sra 2 | yo mH (tT hn gui A idioms Gy hi, |}                                       bee bye  St hy     I Mibs M Hb               ;  7 7 . Me herd baad he V5 thread” Lives) ba Cafticlagy     ee oe a a ena          re ne oe eee ae a        | Pani ins pr pa        Pay 4 fa fed Tah te vt 4e K hbk Mbbinte 4 & bachapn.y txTnch het Ee Sy oo en TE apt e Mage K na nied 1) | Bh seat Pier, sat ge Ay, bpd a ue ¢ tur as:  _ beh ple sie Te: [Om igte Md, a, ttt he td  4 parte + han Ahn His                   ad alti eae A te ee       >  >  x é a S \\\\\\\\ 8 oe)                                  . ' . | . Nt eco. , a ‘ se : ne . thy, ON be , a wo Esa yee Dt age - 2 . Robt . . . * . . ‘* cease oh or of Si EERE W he gL RN ak Da RLS os IRS GES Se achat om me  2a ER er aad tf  ICN SA TIE Seite ye Nye        — Xfm  ’     oy  hic otneahber Dia tae nck spanked Jee .  Ply iun  OR LEI 083 9 0 thle gm ow Hee hy Tt Sigs he A hace she     Pipe tte gta in faa. a mee “Maat Ib jab tea gh for     Pe hn dentin hy pang ban     t                   Pky fy Wyrrte Hin fe tle 7 7 cle winnie Asie Mato                            “. : A tag Ab Sia Ge he Reerinl - te Lx  a, Lana fa) Fda Es, Nida — Meuse Lie 2 5                             25 an i  ae  TOT Bee te ene > FR cee ip reminaxtiy,  See Mes  ROE wy ne poe  ~~ eine tet NN AO HPPA RE Vi  pr : ee ' 7  J                   ete i, ‘  Een te en A MeCN Seapine cone a             tev, Ay KY s                             a 7 - stares oie", "Avery, Oswald Theodore, 1877-1955", "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, "Avery, Roy C. (Roy Crowdy), 1885-1971", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-pbcp_pkij~chv4", "00000000-0000-0000-9268-5B71487F306F", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Invitation to the dedication of the Avery Memorial Gateway", "101584575X10", "101584575X83,101584575X84,101584575X85,101584575X87,101584575X89,101584575X90,101584575X18,101584575X116", "1965", "1965", "On September 25, 1965, 10 years after Avery's death, Rockefeller University dedicated the Avery Memorial Gateway near 67th Street on York Avenue near the northwest corner of the campus.", "Invitations", null, "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "1", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "The President and Trustees of The Rockefeller University cordially invite you to an afternoon of reminiscence and appreciation of Oswald T. Avery and His Scientific Legacy on the occasion of the dedication of the Avery Memorial Gateway Wednesday, September 29, 1965 at 2:30 o'clock in Caspary Auditorium Speakers: Maclyn McCarty Colin M. MacLeod Wendell M. Stanley Theodosius Dobzhansky Robert W. Holley", "Rockefeller University", "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-saze~pbau~uw87", "00000000-0000-0000-C737-0E65F8E4FFE1", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from \"Report of the Director of the Hospital\"", "101584575X101", null, "1912", "[January 1912-October 1914]", "Avery went to work for the Rockefeller Institute in 1913. This excerpt from the 1912-1914 report of the hospital's director, Rufus Cole, to the Rockefeller Institute, contained a brief outline of Avery's research during his first year with the hospital.", "Official reports, Excerpts", "Immune Sera,Antibodies", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "3", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research Volume III    January 1912 - October 1914 substance in the splitting of sugar which induces this reaction.  The problems relating to intoxication are being attacked from several standpoints, but the above indicates in general the line of procedure. Dr. Dochez has continued his work in relation to the occurrence of pneumococci of the different types, not only in patients suffering from the disease, but also in normal mouths.  While sufficient studies have not yet been made to bring conclusive evidence in regard to this point, it seems fairly evident that the pneumococci obtained from normal mouths are not of Types I and II, but in general behave quite differently from these organisms, which are apparently more fixed in their characteristics.  The definite solution of this problem will be of some importance from the epidemiological standpoint. Dr. Avery's work at present is concerned with methods for the concentration of the serum used in treatment.  This work so far indicates that the protective substances are all contained in the globulin fraction, about equally distributed in the pseudoglobulin and the euglobulin.  The separation of the so-called insoluble globulin by the method of passing CO2 gas through the serum dilated in water has given most interesting results.  This fraction of the protein, which represents only about one-twentieth of the total protein of the serum, contains a large part of the protective substances, so that it is now possible to obtain a concentrated serum containing twenty times the protective power of that of the whole serum, but containing no more protein than the whole serum.  However, in the manipulations almost one-half of the protective substances have been lost, and a study is now being carried on to try to reduce this loss if possible.  Concentration with a loss of only twenty-five percent we would consider very satisfactory, and it is quite probable that this result can be obtained by the present method after the technique has been better developed.  As soon as this work is completed, I hope to have Dr. Avery study further the question of immunity to Pneumococcus mucosus. Syphilis.  During the past quarter Dr. Ellis has had to carry on entirely alone the work of the treatment of patients and, therefore, has been able to carry on very little study outside of the clinical study in the treatment of these patients. The method of intraspinous injection of serum, as devised by Dr. Swift and Dr. Ellis, is now being employed quite widely throughout this country with, in most cases, favorable results.  This work has not only been valuable in itself, but it has undoubtedly stimulated a great deal of work in the treatment of tabes and general paresis by intravenous treatment alone.  The results of intensive intravenous treatment have undoubtedly shown that in many cases excellent results may be obtained by this method alone.  In other cases, however, it is necessary to use the intraspinous method before results are obtained.  In the future, no doubt, the method will consist in treatment, first by intravenous injections, with careful study of the spinal fluid, and second, in the cases where satisfactory results are not obtained, by the direct intraspinous method of treatment.  The intraspinous method of treatment has been employed elsewhere in the treatment of general paresis, and a number of observors have reported excellent results.  All this work is undoubtedly going to lead to very great improvements in the method of treatment of tabes and general paresis.  Dr. Swift, who has studied our cases and has investigated the reports of others, believes that tabes can probably be arrested in ninety per cent of the oases. We believe that it is important that in the future these cases be treated as syphilis by those who are engaged in the active treatment of this disease, and not be treated by neurologists who have little knowledge concerning the nature of infectious disease and are little interested in specific", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", "Scientific Report to the Corporation and the Board of Scientific Directors of the Research Institute", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-5asd.xvv2_ancf", "00000000-0000-0000-3662-6213B35420E3", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Pneumonia", "101584575X102", null, "1914", "4 April 1914", "In this April 1914 report to the Board of Scientific Directors, Cole outlined the research on pneumonia at the hospital in the previous quarter conducted by Drs. Avery, Alphonse Dochez, H. T. Chickering, and Zacharias. Included are reports of Avery's experiments on producing large quantities of Type I serum and his early collaboration with Dochez.", "Official reports, Excerpts", "Immune Sera,Antigens", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "4", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "alll cen mee ne nee Et ru hitesedd aaa yO ne es plete 7 ’  CASta aDpRES® ROKSTITUTE  THE HOSPITAL OF THE ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH, Pye 66TH STREET AND AVENUE A, NEW YORK iyi obe  April 4, 1914.  To the Board of Scientific Directors of the Rockefeller Institute for Medical Research,  centlement- The Director of the Hospital has the honor to submit the following re- jort of the work carried on in the hospital during the past quarter!-  Pneumonia!  During the past quarter thirty-five patients suffering from pneumonia have been treated in the hospital. This ia not as large a number as it was hoped sight be admitted during this season. During the past week, owing probably to newspaper publicity, there has been a marked inorease in the number of admissions.  The atudy of the type of organism ooncerned in eaoh case has been continued, and it has been found quite possible and fairly easy to determine readily the type of organism concerned in the individual oase. During the present winter, of the fifty cases treated, only about twenty percent have been caused by organisms of type I, while almost forty peroent have been due to organisms of type II. Whe- ther or not this indicates a greater prevalence of pnewnonia due to this latter type of organism during the present winter in New York than during last winter, it is diffioult to say from our limited experience.  Our previous experience in regard to the relative severity of the condition in cases due to the different typea has been confirmed. The cases treated with the type I serum have all done well, and the experimental and olinical evidence indicates that this serum is of therapeutio value. ‘The effect of the      type II serum, however, has not been so satisfactory, aince four of the patients _ i fully treated with this serum have died.. The results were diffioult to understand until further study of the serum used showed that ita protective power in animals | was much lower than that of the serum employed last year. All of the type II serum used for treatment has been obtained from the first horse immunized and from which all the serum used for treatment last year was obtained. It is not known why this  horse, which was previously able to yield a potent serum, now fails to do so, but          ol eee ent on A Ur MASaie ccna j it is known that horses yielding anti-toxic sera and also those producing other anti-bacterial sera may gradually lose their power to produce effective sera. The reason why no other type II serum was available for treatment waa because the second horse, whose immunization was commenced last summer, developed a chronic joint in- OY fection and had to be sent to the farn. We also now have another horse in process of immunization with organisms of type II, and also a horse which is being immunized to organisms of both type I and type II. The serum of this latter horse now shows  protective power against both types of organisms as high in each case as we have ever     found with a monovalent serum. This serum is now ready for use.  Dr. Avery has continued his efforts to concentrate the serum on a larga scale, but so far without very satisfactory results. The experimental work has shown b  that the protective substances are largely contained in the euglobulin fraction, as     1a vbtained by the salting out method, and also are present in the so-called insoluble beg  oN globulin precipitated from the diluted serum by oarbon dioxide. However, it has been ‘     ey “ound so far that the loss of immune substances in filtering, eto., when these methods By:  ji] are employed on a large scale, is too great to justify their use. Modifications, t however, are being made and it is hoped that a satisfactory method may be worked out. un     Dr. Dochez and Dr. Avery have oarried on studies concerning the various H: types of pneumococod in relation to the mode of infection and epidemiology of the disease. Han  Dr. Dochez is carrying on a comparative study of pneumococci present in the mouths of normal individuals. A large number of strains have so far been isolated from the mouths of fifteen normal individuals. In only one instance has an organisn of the fixed types I, II or III, been isolated. In this instance an organism of type II was isolated from the mouth of the wife of a patient with pneumonia due’ to an organism of this type. Further work, of course, may show that these fixed types occur in normal mouths much more frequently than the resulta so far would indicate. Rabbits are being immunized to various strains isolated from normal mouths, in order to learn more of their antigenic. properties.  Dr. Avery is studying the persistenoe of the pneumococci of the various types in the sputum of convalescents. The evidence so far indicates that the pneumo- cocci of fixed types can usually be found in the mouth or sputum no longer than a few weeks after recovery. Later only organisms of the type found in normal mouths are present. It is of great importance to learn whether or not the organisms present  in pneumonia are simply displaced by these. latter organisms, or whether poasibly \\\\. ps IQ  & transformation may have taken place, In order to aid in determining this point, the antigenic relationships of/a number of the organisma isolated during the disease, to those found in the sane mouths after recovery, are being studied.  Dr. Avery is also studying the behavior of the various specific type strains of pneumococci in the different carbohydrate media.  Dr. Chickering has been studying the time of appearance and disappear- anoe of agglutinins in the blood of patients suffering from pneumonia. In untreated cases the agglutinins appear only about the time of orisia, though in some cases they may be delayed until a later period. In the treated oases the agglutinins appear much earlier. The presence of agglutinins, therefore, 1s not of much value in de- termining the type of organism concerned, but the demonstration of agelutinins may be of some importance in prognosis. Furthermore, thia study will offer confi rma~ tory evidenose of the etiological relationship of the type of organism isolated in the oases where the organisms are only obtained from the sputum.  Dr. Zacharias is about to undertake a study of the hydrogen ion con- sentration of the blood in pneumonia by the electrometric method. The relation of the reaotion of the blood to its oxygen and carbonio acid content will be studied. The relation of the degree of acidosis indicated by the increased ammonia excretion in pneumonia will also be considered, in order to determine whether the acidosis indicated by urine analysis really corresponds to an inoreased acidity of the blood.  The study of the production of methaemoglobin by pneumooocoi has been continued, and also the study of the toxins produced by these organisms. It has been found that by freezing the pneumococci and drying in vacuum while frozen and then making up in salt. solution, the resulting solution is toxio for guinea pigs: and the same effects are produced as by the injection of the bile extract. Fur- ther evidence has therefore been brought that the toxic substance is pre-formed in the bodies of the bacteria and set: free upon their dissolution. The method algo permits large amounts of toxin being obtained and preserved, so that experiments  may be carried on from day to day with toxins of equal strength.  Diabetes:  The following is a verbatim copy of the report raceived from Dr. Allen. ir-reixtion—te—hte—nerites While in certain instances Dr. Allen has allowed his                      ‘s Pew : atin enthusiasm to lead him to draw conolusiong, not yet, justi ied, yet it is very evi-  dent that he has a definite and meet interesting point of view. The results ob-  tained in the one patient so far treated are most striking and very promising.  Report from Dr. Allen  Work has been continued along the paths previously outlined. The  progress along a few of the principal lines may be summarized aa follows:- I. Produgtion of Diabetes.  A. By simple removal of pancreatic tissue:- The results in a long series of dogs ure constant. The newly observed relation between pancreas and body-weight also holds good, i.e., large dogs have less panoreas in proportion to body-weight than small dogs, and become diabetio from removal of a oorresponding smaller fraotion of the panoreas. Large oats, on the oontrary, have as much pan- creas in proportion to body-weight as amall oats, and similar fractions must be re- moved in order to cause diabetes. The \"apparent\" sugar tolerance is not an index of susceptibility to diabetes. For example, large dogs normally have as high a sugar tolerance as amall dogs. Monkeys have a low sugar tolerance, and in pigs the apparent tolerance is almost nil, as Carlson reported. But if one attempts to maintain a oontinuous glycosuria in these animals, the organism responds by assimilating even the largest quantities of sugar, thus illustrating the difference between apparent and real tolerance. Even after removal of sevon-elghths of the pancreas, a pig now under observation takes 500 gms. glucose daily with only slight glycosuria} but the glycosuria is now continuous and increasing, and true diabetes will probably be the result in such @ predisposed animal. Pressure of other work has thus far prevented a satisfactory series of experiments with partial panoreatec- tomy in species other than dogs and cats, though the monkey and pig promise to be highly important (especially for the problem of acidosia) when time oan be devoted to them. .  B. By diet:- It is now safely established that dogs and cats can be predisposed to diabetes by removal of suitable portions of pancreas, and that carbo- hydrate-rioh diet will then act as an exciting cause of diabetes. When the pan-  creas remnant is of certain size, the animal is glycosuria-free on meat diet, but", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", "Scientific Report to the Corporation and the Board of Scientific Directors of the Research Institute", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-unmv.2fnf.vicb", "00000000-0000-0000-454C-8C75844262A6", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from \"Report of the Director of the Hospital\"", "101584575X103", null, "1914", "June 1914", "In this June 1914 Director's Report, Cole briefly outlined the research on pneumonia at the hospital in the previous year conducted by Drs. Avery, Alphonse Dochez, and H. T. Chickering.", "Official reports, Excerpts", "Immune Sera,Transformation, Bacterial ; Antigens", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "3", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "REPORT OF THE DIRECTOR OF THE HOSPITAL June, 1914. Owing to the brief period intervening since the last meeting of the Board and to the fact that the year is approaching a close, very little new work has been undertaken in the hospital, and the activities of the Staff have been confined mainly to the completion of work reported as in progress in the last report. Pneumonia:  During the year 73 patients suffering from pneumonia have been treated in the hospital.  A complete statistical study of these cases has not yet been made.  The type of organism concerned has been determined in all cases. In addition to the cases studied here, the type of organism has also been determined, by means of immune serum sent out from here, in the Kings County Hospital, in Brooklyn by Dr Lyle, in the Brigham in Boston by Dr. Walker, and in the Pennsylvania Hospital in Philadelphia under the direction of Dr Lewis.  These observer [sic] have had satisfactory results in determining the types of organism in about one hundred and fifty cases.  Sera have been sent also to other hospitals, but reports have not yet been received.  When this material is all collected, we shall have considerable data as to the relative frequency of occurrence of organisms of different types in different cities.  Dr. Lewis in Philadelphia is now immunizing horses to the organism of type I and type II and expects next year to carry out serum treatment in the Pennsylvania Hospital according to the method employed here. The next important problem as regards therapy is the improvement of the method of treating cases due to organisms of type II.  Whether this end will be attainted [sic] by concentration of the serum alone is not yet certain.  Work is being continued on methods of concentration, and while a considerable number of facts have been determined in regard to the protein fractions which contain the protective substances, a practical method of concentration has not yet been devised.  We hope to do this during the summer.  It is probable, however, that an important factor in immunity against organisms of type II and Pneumococcus mucosus must be supplied by the infected organism.  Efforts are being made to determine the nature of this factor. Dr Avery has also conducted a number of experiments studying (a) the rate of disappearance of antibodies in serum; (b) the effect of inactivation on immune serum and the effect of the addition of complement on its efficacy; (c) the absorption of antibodies in immune serum by means of homologous and heterologous types of organisms.  The effect of such absorption on agglutinins, precipitins, protective substances and also the effect on complement deviation has been tested.  Experiments in regard to the bacteriolytic effect of immune serum are now being carried out.  The published reports of these investigations will be made within a very short time. The second important problem which we have had in mind in the studies on pneumonia is related to epidemiology.  It is evident that it is of great significance to learn whether or not the fixed types of organisms are only present in the mouths of those infected with the disease, and whether organisms of these types quickly disappear during convalescence.  Lastly it is important to learn, if possible, whether transformation of the types of organisms ordinarily found in the mouth into the more fixed types may or may not occur.  From Dr Dochez's study it may briefly be stated that organisms of the fixed types are not ordinarily present in the mouths of healthy individuals.  The patients who have been treated here for pneumonia are returning from time to time, in order that it may be learned whether or not the type of organism found during the disease persists.  As previously stated, organisms of the fixed types have persisted for from two to three months following an attack of pneumonia.  It is possibly significant that in these cases where the fixed types have persisted, delayed resolution has occurred.  If it is shown by these studies that the fixed types are not ordinarily present in the mouths of healthy individual, though they may persist in the mouths of convalescent patients, these persons then acting as carriers, it is evident that these observations may have considerable significance in devising means for the prevention of this disease.  At present, however, these question [sic] are not fully answered, and it has seemed of importance that we extend these studies to a considerable degree in order to obtain definite information in regard to these points.  The problem as to whether transformation of the so-called atypical types into the more fixed types may occur in the mouth or not, and whether the occurrence of the disease may be due to such transformation, is an extremely difficult one to solve.  It is hoped that by studying the antigenic properties of the organisms existing in the mouth during the disease and those of the organisms present during convalescence, some light may be shed on this question.  Such work is now in progress. Dr. Chickering has carried out studies on the agglutinating power of the patients blood for the type organisms and for organisms isolated from the patient's own sputum.  This work is now ready for publication.  Dr Chickering is also conducting some experiments to determine whether or not the antigenic properties of the organisms isolated from the sputum correspond to their agglutinating properties.  To do this has required the immunization of a large number of small animals. Dr Avery is also carrying out an extended series of studies on the fermentative reactions of pneumococci, in order to determine whether or not the different types of organisms show any difference in their fermentative reactions. The work on pneumococcus toxins and on the production of methaemoglobin by pneumococci and on the haemolytic action of pneumococci has been completed and very soon will be ready for publication. Diabetes:  The work in diabetes has been continued along the lines outlined by Dr Allen in his extensive report at the last meeting.  So far there have been three patients suffering from diabetes under treatment in the hospital, and the results of efforts to lower the total metabolism have been most encouraging.  All of the three patients are now free from glycosuria and free of acidosis.  They have all been clinically unfavourable cases.  It is intended to continue this work quite actively during the summer, as Dr Allen will remain here during the entire time. Syphilis.  Dr Swift and Dr Ellis are now busy collecting all the material of the work on syphilis since the opening of the hospital, and it is hoped to publish this, probably as a monograph, during the coming summer.", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", "Scientific Report to the Corporation and the Board of Scientific Directors of the Research Institute", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-q96c-fxri.rsyr", "00000000-0000-0000-2857-2A3CDFFDA7EE", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from \"Report of the Director of the Hospital\"", "101584575X104", null, "1914", "16 October 1914", "This October 1914 report to the members of the Rockefeller Institute almost exclusively addressed the research and treatment of pneumonia conducted in the hospital over the previous year.", "Official reports, Excerpts", "Immune Sera,Therapeutics", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "8", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "‘f44  . “t,t  October 16th, 1914,  To the Members of the Corporation of the Rockefeller Institute for Medical Research,  Gent lemon: -  The Director of the Hospital has the honor to submit the following report of the work carriod on during the past year.  When the Hospital was oponed it was stated that one of its functions night de the training of men to take part and aid in the change which was likely to take place in the toaching of internal medicine. It is gratifying to know that tha objact has baon, to a measure, attained, in that a number of the assis- tants in the Hospital have now been callod to full-time academic positions in the teaching of medicine in various univorsities. Dr. Canby Robinson was called to Washington University of St. Louis, to become Assistant Professor of Medicine, and to aid in organizing the Department of Internal Medicine in the new hospital of that institution. Dr. Francis Peabody was called to the Harvard Medical School, and to the Brigham Hospital, Boston, to become Assistant in Medicine, and Resident Physician, respectively, in these two institutions, Finally, Dr, Swift, who has been Resident Physician of the Hospital, has received and accepted an appointment as Associate Professor of Medicine in Columbia University, and Dr. Fraser, who has also been a member of the staff, has been appointed an Instructor in Clinical Medicine in the same institution. All these men are now acting as full-time instructors in medicine. While these appointments add to the difficulty of main- taining the staff at all times on a most efficient basis, yet for the present, at least, such appointments are gratifying, not only because they show that the  Hospital is able to occupy an important place in training such men, but also       rye because they offé6r an encouragement to, the younger men whn are working in the  Hospital with the view of later making an academic career. In addition to the loss of Dr. Swift and Dr. Fraser, the Hospital has also lost Dr. Ellis and Dr. Zacharias, who have joined the English and German armies respectively. It is hoped that at the end of the war ome or both of them may return to their work which was so suddenly interrupted.  Dr. A. R. Dochez, who has been a member of the Hospital staff since its organization, and was previously on the staff of the Institute laboratories, has been advanced to the position of Resident Physician, to take the place left vacant by Dr. Swift. The following named men have been appointed to occupy the other vacant places on the staff: Dr. Alan M. Chesney, who formerly spent a year in the Hpospital and during the past year has been on the staff of the Johie Hopkins Hospital; Dr. Ross A. Jamieson, who is a graduate of the University of ‘oronto, and has been for the past two years on the staff of the Bellevue Hospital in this city, and Dr. Franklin C. McLean, fomerly professor of pharnacology in the Uni- versity of Oregon. An important change in the organization of the Hospital staff tas taken place during the past year by the appointment of Dr. Donald D. Van Slyke to take charge of the chemical laveratory. The organization of this depertuent of the medical work has offered the most serious difficulties, and it has been very difficult to obtain a man of proper training and scientific inclination to act as director of this laboratory. It is felt and hoped that in Dr. Van Slyke the ideal man for such a position has been obtained.  In looking to the future development of the Hospital work, it is be- lieved that, with the discovery of new methods which may be applied practically in the treatment of disease, definite organized efforts should be made by the Hospital, er by the Institute as a whole, to assist in putting these methods into wore Widespread and efficient operation. This can bs 4088, oa rrorts to assist gensral practitioners in carrying out new methods of treatient or by assisting in  the erganization ef other institutions for carrying out special methods of treat-    ey)  \"Fete d  -\\\\-  ment. The development of this idea could «311 be one of the functions of the de- partment of Public Health, or the Third Section 28 suggested by Dr. Prudden.  During the summer months just past, owing to the work which vas go- ing on adjoining the Hospital, ani owing to the fact that tha staff was 30 vaterially reduced, a very small number of patients have been admitted and the work has been reduced so far as possible. It was not thought advisable to close the Hospital completely since, in order to obtain support from the public and uodical prffession, it is necessary that at all times the Hospital be open for the examination of patients and for the reception of suitable ones. The following  is a brief review of work carried on in the Hospital.          Pneumonia. During the period since the last report eighty cases of  pneumonia have been treated. Numerous studies have been made on these patients  trom various points of view by Dr. Cole, Dr. Dochez, Dr. Avary and Dr. Chickering.  The results obtained may be Classified under the following heads:  Infection and Epidemiology. Studies have been made of the types of pneumococci occuring in normal mouths and in the mouths of patients convalescent from pneumonia. In the mouth of only one normal person have pneumocci of the fixad  types I, II, and III been found. This person was the wife of a patient suffering  trom pneumonia due to pneumococci of type II. In her mouth pneumococci of type II.  were found. Studies of patients convalescent from pneumonia have shown that pneu- mococci of the type found during the disease usually persist in the mouths for from one to several weeks. Later than this they are not found. Only pneumococci of the non-specific type IV. are then present. During the present sumer Dr. Lyle has carried out a study of the types of pneumococci present in the mouths of patients suffering from tuberculosis. Among fifty cases, in two, pneumococci of type I. were present. In no other cases were pneumococci of the fixed types found. These stidies indicate that in cases of pneumonia, except possibly those due to Organisms of type IV., infection probably occurs from without, though there are probably contributing factors in etislogy.  In no case has direct evidence been obtained of transference of one organésm of one type into those of another type. It is probable, however, that in the evolution of the pathogenic types such transformations have occurred. That this evolution may have been along different lines in different parts of the world is shown by the fact that in South Africa a type of pneumococci is present which has not been met with here, and this type is there fairly wide spread. A stall  ruoor oF organisms have been sent to us from South Africa. The four types found       ‘\\\\- woe - l.  here are among them and in agaition a fifth type, as mentioned.  Careful study of the organism isolated from each case of pneu- monia coming under our observation has been made. The results confirm our pre- vious report in regard to the relative frequency and severity of cases due to the iifferent types of pneumococci. Stulies concerning the occurrence of pneumococci ot different types in cases of Ensumonia have also been made with our assistance in the Brigham Vospital at Boston, tne Pennsylvania Hospital in philadelphia, and the Detroit General Hospital.  Dr. Chickering has studied the agglutinating power of patients! serum for pneumococci of different types. The resvlts afford further proof of the specificity of the different types and their relations to the disease. In the blood of patients with infections due to organisms of type IV. only the homologous  organism is agglutinated. In the blood from patients with infections due to Organ  :sms of types I. and II. only organisms of the corresponding type are agglutirated.  Acglutins have usally disappeared from the blood within a few weeks after the end of the disease. In the cases where they have persisted there has existed sore specific complication, as delayed resolution or empyera.  Treatment with serum. Further studies in the treatment of cases have confirmed the opinion previously held in regard to the efficacy of immune serum in the treatment of cases due to Organisms of type I. In regard to the serum effective against organisms of type II. out previous anticipations have not been entirely confirmed. During the past winter we had several very Severe cases due to organisms of type II, in which the serum seemed to have little or no effect. A study of the serum then being used showed that it was mech less effective, as tested by protective power in animals, than was the serum which had previously been used. While, therefore, the results were not necessarily dis-  couraging, except as regards the statistical study being made, nevertheless,  EINE NN ES TEN ee           Yhae \\\\  ror vd soled     \\\\ when it is realized that it 1s very difficult to obtain and keep the serum of Le type II. at its highest efficiency it is evident that the complications in the way of carrying out effective treatment with type II. serum are bouni to be considerable.  An attempt had been made to render this serum more effective by con-  centrating it, but the results as determined from experimental studies are not en-  couraging. It is quito possible to cause considerable concentration of the imdane     subs tances since al] the protective substances have been found to be in the glooulin traction of the cerum. Ynen the pretactive power or concentrated serum is tested , however, the possibility of obtaining greater cuative effects from its use does of sean probable. The reason for this is as follows. Serum or type I. protects nice against 0.1 c.c. of culture when 0. 2 c.c. of serum is used. It is found, however, that, no matter how much the amount of this serum be increased, even if ~ c.c. be used instead of 0. 2c.c. , the amount of culture protected against can ever be more than 0.1 c.c. This holds good for concentrated as well as not con- centrated serum and has been found almost constantly trues ina very large number of experiments. tn the case of serum type II. this limit of dosage is still lower. 0.2 c.c. of serum never protects against more than 0.61 c.c. of culture, and no greater amount of culture can be protected against, no matter how laces the amount of serum. Tnpresnians of type III. even the smallest amount of culture can- not be protected against by immne serum, using even the largest amounts of serum. The facts as stated render it evident that, in aidition to the immune body, probably a second factor which must be supplied by the body itself, is necessary. In infections due to organisms of type I. this second factor is probably not required in large ampunts. At any rate, by increasing the amount of immune cody, the demand on the organism probably my be reduced. In the casa of infection  due to crganism of type II. the requirement of this second factor is greater and  “ven vary large amounts of immune body will not supply this deficiency. In the    indicate the following arrangement as regards Pathogenicity and immunity. First, Froup IV. in which the Pathogenic effects are only slight; the Fatients infected Practically ali recover. Immunity apainst these Organisms can very readily be ob- tained but the Practical diffiouty in the efficient use of the serum consists in the fact that these Organisms differ among themselves, each One possessing specific  lismuno logical characteristics. Next come organisms of type I. which are found in  effective. Next come Organisms of Broup II. These Organisms possess a still higher grade of pathogenicity; the infections caused by them are more severe and the mortal- ity is higher. a high grade of active immunity my be obtained for these organisms but the passive immmity obtained by employing the serum of these immnized aninals is conside-ably less effective in protection, and probably in cure, than is that obtained from the serum produced by the inoculation of organisms of type I. lastly,  organisms of type III. $0-called Pneumococcus macosus type, which probably possess    hy a)  » ed, ?  group is entirely lacking, the serum has no efficacy whatever.  The rasults of treatment indicate that much has already been accamplish-— od with cases of type I. and it is hoped that this alone nay make a material reduc . tion in the total mortality from pneumonia. The chiof effort is now being directed to improving the treatment of cases due to organisms of type II. Little can be hoped for, at present, as regards any form of effective specific therapy against  organisms of type III.", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", "Scientific Report to the Corporation and the Board of Scientific Directors of the Research Institute", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-b3bd_imcd~ejas", "00000000-0000-0000-5B97-82FCFDE136B2", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research, January 1915", "101584575X105", null, "1915", "16 January 1915", "Several pages of this Director's Report addressed the research on pneumonia conducted by Avery and others at the hospital in 1914.", "Official reports, Excerpts", "Immunity,Antibodies,Diabetes Mellitus,Heart Diseases", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "12", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "which will imprevo the results in this type of cases. The results of serum treatment          REPORT OF THE DIRECTOR OF THE HOSPITAL.  January 1915,  During the last quarter reer cntients: suffering fre. ene of the fclloving wi.eaces lave “san aucitted to the Hospital for treatuent ani stucrj; acuve lovar Fnew.or.is, Cincetas, anc heart disease. A few of the patients formerly under treat- wont for aypnhilis of the central nervous system have been readmitted for short periods +o receive additional treatrent.  Acute Looar Preusonia: The work on acute lobar pneumonia has been mainly a continuation of that carried on during the past uears, especially along the lines discussed ir our dast report. The number of patients admitted furing the present quartor is avout twice as large as the number admitted last year during the samo Period of time, so it seems probable that quite a large number of patients wili be available for study during the presert year. Unfortunately, so far as testing the value of the method of treatnent by reans of immune scrum i-s concernedg very few cases of Type I. have beer. admitted. On the other hand, an umusually large proportion of the patients so far admitted have been infected with pneumococci of Type II. Fur~ thee experience with the serum treatnent of cases of Type IT indicates that the value of this method of treatiient in these cases is not nearly so great as it is in the cases of Type I. This was to be expected from the experimental studies. Further efforts  are being made to determine whether or not sors nodification in the method can be made  in cases due to Typo I, however, continue to be very encouraging, and there seems  every reason for believing that semm trearment in cases due to this type of organisa is of very great value. “hon one renemters that the nuuber cf deaths in New York,  cue ro pneuvuenia caused by pnercosocei of Type I is greater tian the deaths due to dizntheria, and much greater than the total viecser of weaths due tc scarlet fever,  cerebro-spinal fever, and typhoid fever combined, it is quite evident that we are    Meena nen oe : “cab tai SoF  - R - ' justified in Considoring this type of Pnounonia as an antity, and in feeling much Oncouraged if the méthnd employed is able to materially reduce the mortality in cases of this one type. It is planned during thea coming winter to Supply a limited Snount of sermus to several other hospitals, So that it is believed that by Spring w@ S22) obtain sufficient Statistics to permit of a definite judgement as to the therapeutic valus 0: this serum.  During tho present quarter the studies of pr Dochez and Dr Avery concerning epidemiology have been extended, and the observations praviously reported have been  confirmed. The report of this Study is now in press. Dr Avery's Study ef the dis  ~erui have also been extended and completed, and the report will appear in the Feb- Tuary nusoer ef the Journal of schexlorxaxixet Experimental Medicine. The studies S2v6 snow tuat Practically al) the iczmune substances aré contained in the fraction of protein Precipitated by 38 to 40% armoniun sulphate. Since this Precipitate con~ tains only one 8ixth of the protein, it is evident that a method has been found for reneving a considerable amount of the protein of the serum used in treatment. This is now being carried out on a larger Scale, so that treatment may be made with such soncentrated serum. This work also has considerable pupsly scientific interest in  relation to the distribution of immune bodies in the fprum protein fractions,  & considerable anount of his time to the care of patients, he has had less Opportunity than Previously to carry on experimental Studies. However, he has continued his study cf the nature of the immunity in Pneunococecus mucesus infections, and it is to this eroblex: that his Sxperinental wort will be larsely devoted during the coming winter, The laboratory Studies of the Rirector cf the Hospital have been manhly “8VGCeL to a Stuly of methods of quickly Producing active dimunity and to a determi-  “stlon of the best methog by which such vapid active diamunity may be obatined. It    a  “x  fourd that if even the slightest  . \"Ooo ~ ved  nas c  vitieacy of iunune soru, in such aninni,  Sor  So far tested, hovver, it has besn Lipo active inmunity incide of a period of six  -evised for dzcrsasing this periou, in ord  for conoinins vaccination ith Passive inmunization  soncemin~ tha nature of active immunity i i-portencs in order that the specific trs-  due te pneumccocei of Typs Il. ye  tinunt of patients  wighin 3 8     of active immnity is produced, the  ousl; incrcasud. With the methods  bis to produce even thisslight grade cf  days. It is hoped that a nothod may te  er that we roy have experim:nt2l justification  in our paticrts. Further knowledge  h pneumococcus infecticn is esvscially of  Q  uffering from infsction  bu rendered ners offieacious.  Dr Chickering, in addition to tho caro of pationts, hae boon working with  Dr Gay, and tho following is tho report by Dr Gey of the results obtained in their work.  Rupore by Dr. Gay,  Through tha courtesy of tie Board and of Dr Cole, the facilitior of the  lvtoratctviss of ¢  for ‘chich he wishes to make grateful achno: lodgene  with the assistance of Dr Chiskerinz, “ho  Th: study first undertaxen dealt with tho     Sutipneunioceccus serum on the addition of  such a precipitetes, which could be producsd rapidly and  watereclear extract of washed, alcohol~precipit  therapeutic uses of either Plain or sensitized vaccines.  atanioned, owing to the greeter interest awakened by another observation.  «ie Hospite] heve been oxtended to Dr Gay for the past two months,  His wort: has been carried on  + ve  will share in the publication to be mads.  sss  possibility of trsatment by mears of a com  vination of serum and pneunococcus vaccine, or by means of a sensitized vaccine. The experinental conditions of pnoumonoccus infection in the aninals that have been tested  have appanently not lent themselves particularly well to a demonstration of possible  Certain suggestion have been  cttained which might aventuelly lead to something of more practical significance.  During the last. month, however, experiments along this line have been largely  It was thought  worth while to try the possible curative effect of a specific precipitate produced in  an extract of pneumococci. It was found that  in large volume by adding a  ated and ground pnevmococei, is apparently    as protective against prueunoceceus infection in mice and rabbits as an aliquot amount of the oririnal ser. This Precipitate nay further te washed in saline ang still retain its curative Properties, and in Sone instances, when freshly produced and dis- Selyad b a scall amount of alkali(maoH) wilt Stijl retaia as much curative value as vhe original senz: from which it is derived, The addition of teo Great anount of ale sli, even if it be Subsequently neutralized with HCl, may destroy the curative value. Te Sanie efiect, however, is also rpoduced by tho addition of large amounts of alkali to tho original diluted sorun. This curative precipitate contains a ver; small amount of preteins as determined fron tne total nitrogen. Vhereas the Original serum cor- tains 6 to 8° of Protein, various Speciziens of the Specific Precipitate, restored to th original voluue of Serun, have been found to Contain from .18 to ,34%, From the ob- Servations hitherto nade, it would seen that the strength of the protection afforded cy this Procinitate is not Froportionate to the Percentage of protein Present,  The concentration of the precinitate in smaller voluns apparently renders it Possible to protect mice against sonewhat larger doses of the Ppneumoceccus than can be rrotected against by the whole Sérum. Tho serum fron which the Precipitate has been revoved contains much less Protective value than the original serum, although it usu~ ally contains a berceptible amount. The method for producing the maximum amount of pre:- cipitate, the Possibility of putting this precipitate into solution without destroying its protective value, and many other Problems remain to be worked out in more detail, Ye do not know, for instance, whether the precipitate itself is the protecting substance  or sone other combination of antibody adherent to it. It seems evident that the pre-  equally well when dissolved,and also owing to the fact that the undissolved precipitate does not protect against a type of pneunococcus other than the one against which the sorun eriployed is active,  It would seen that there observations open a field of considerable interest for investigating the nature of immune bodies of various sorts, and indicate a means: of con-  centrating immnne codiss ina Solution of low protein content which may be advantageously in tréatuent, not Only ef pnermonia but possibly also a? ctner infections,       Dec. 4th. Curative Experiment  With varyins doses of serum and Gissolved precipitate and a Tixed doses of sulture.  Volume = C.5 CeCe                      ae Diluted serum 0.1 + Culture Preumococcus I 9g 0.05ce Survived 2 \" \"9,95 f  Be \" \"0.025 n  kg mn \" 6.0125 . \"  De \" \" 0.00625 + 14 days 6. Dissolved precipitate O.2 +. n hoon \" Survived 7. n n 0.05 .  g. i\" n 0.025 \"  o i\" n 0.0125 mn  10. on q 0.00625 0  ll. Exhausted Serum O.1 \"  le, n \" 0.05 . + 360° 13. \" \" 0,025 + 36° 14, Contrel + 0.05 + 20° 15. 8 + 0.0002 + 18° 16, 0 + 0.00001 + 40°  17. \" + 0.000001 + yge { “*&-  Diabetes: During the past quarter the efforts of Dr Allen and Dr Stillman have teen mainly directed to the treatrent of patients suffering frem diabetes, along the lines discussed by Dr Allen in his report to the Board,last April. It will be re- called that the studies on animals suffering from oxperimental diabetes led Dr Allen to believa that for the successful treatuent of dinbetes it would be important to keep sha r.tient on as low as caloric diet as possible, in the hope that the demand then nage upon the assinilative functions of the body would be reduced as far as possible, the theory being that any increased demand made on these functions, over that absclutely necessary for the supply of sufficiert energy to carry on the activities actually necessary for life, would cause a strain on the organs functioning in assimilation, in- Cluding the pancreas, and so reduce the functional power of such organs. It will be re- called that Dr Allen showed that if two dogs be each rendered diabetic fia the same grade, and that if one be fed on a high caloric diet, and the other on a low caloric viet, the one receiving the high diet would, in spite of this, lose weight, and would also become feeble, and finally die. The other one would also lose weight, but remain aprarently active and ina comparatively normal state.  Twenty seven patients have now been admitted to the Hospital, suffering frou  diabetes. Sixteen are still in the Hospital. The study of these patients has shown that, by the withdrawal of all food except alcohol, all patients suffering from diabetes can rapidly and completely be relieved of Slycosuria. It is also of very great impor- tance that, at the same time, the excretion of the abnormal fatty acids is also reduced to a nininum, so that, instead of the withdrawal of food causing an intensification of acidosis, it is the best means of relieving the patients from threatened acid intoxi~ cation. In order to obtain this freedom from glycosuria and acidosis, it is sometimes necessary to withhold food from the patients for a considerable length of time, in cer~ tain cases as long as @ight days. After the patient is free of glycosuria and acidosis  it is then necessary to get him back to a living diet, containing a sufficient amount  of the variitts kinds of food. No effort is made to have the patient increase in weight       en  \\\\ . however. If he is emaciated, however, it is thought important that the weight should  revain stationary. By carrying out these Simple measures, it has beon possible ina large number of the cases, to keep them free from glycosuria and acidosis and on a caloric diet of sufficient size to maintain life.  It will be rorenbered that in his previous repart Dr Allen stated that he thought that by this nethod, the total metabolisr night be reduced. To test this hypothesis 2 series of calorimeter experinents have been carricd on with Dr Lusk and Dr De Bois, the patients being temporarily rerovei to the Bellevwe Hospital for this purpose. This study is still in process, but in one case, at least, a reduction of 20% in the basal .ttabolisn tas been observed, followin; treatuent as outlined. Most of the patients have shown increased tolerance to carbohydrates and protein following the procedures tescribed, though it is still impossible to say whether it is essentially greater than that observed when patients with diabetes ara made free of glycosuria and acddosis by ths Slower methods previously employod. It must be renembered, however, that with the ..thods previously used it has been impossible in many cases to render them free of ‘lycosuria and acidosis. Only further extended studes can show how great prolongation cf life may ba expected by the method employed, as coripared with that obtained by cther wethods. It is generallt assumed,however, by clinicians that in the treatment of dia~ betes it is of great importance to render patients free of glycosuria and acidosis,and that thereby tolerance nay be increased and life prolonged. It seems that the msthod proposed offers an improved way of doing this. That the total caloric values of the diet is of importance, and not merely the actual anount of slucose~producing sugars ana proteins, &s shown by the fact, now well established by these studiss, that where a patient is free of glycosuria and acidosis on a given diet, containing proteins and fats, with of without carbohydrates, the addition of a given amount of pur fat, without any change whatever in the amount of protein and carbohydrate, may cause such a patient to asain become glycosuric.e This fact together with the fact that pipaemia so often cecurs in diabetes, has suggested the importance of further study of the fat metabolism  in diabetes, especially a study of the fat content of the blood. Consequently it is          -%- 3G raanned that during tha coming quarter Dr MeLean and Dr Stillman will carry out furthor studies dealing with glycaemia and dipaemia. Dr Allen is also engaged in the study of the pathotogical material obtained frou experimental animals and cases of huxan dia~ betes to study further, by means of more delicate stain reactions, the finer changes in the pancreas occurring in diabetes.  heart Tisease:- Studies concerning the mode of action of digitalis have been continued by Dr Cohn withthe assistant of Dr Jamieson. The studies made last year of the action of digitalis in patients with normal cardiac rhythm wibhout oedema, and with bleed pressure within normal limits, are now in press. During the present year studies are being made of the action of digitalis in patients with abnormal cardiac rhythm,and in patients in which oedema, and abnormally high blood pressure are present. Special Studies are being made of the mode of action of digitalis in patients with fever,especi-- ally in cases of pneumonia,  In previous studies,in order to determine that digitalis was actually active in an individual, it has been necessary to push the administration to the point of alter- in tho heart's rhythm(heart blcok)or of inducing gastro-intestinal symptoms. These wery the nost cortain tests. In reviewing the electrocardiograms made during these studies it has been found frequently almost uniformly - that before these signs became manifest the shape of the sc~called T wave in tne electrocardiogram became altered. With greater attention to the details of calibrating the curves, it has been found that the beginning of this change can be detected easily - in two to three days ~ after giving the drug. This chage, together with a change in conduction between auricles and ventricles,which also often occurs early,offers two new,sure criteria for deciding early that digitalis is acting on the heart. By observing these signs,patients may be saved from the dis-~ agreeable effects of this forn of treatment. Ina Single instance abnormal primary negative T waves have been seen to become less negative, and later positive. These chanses persist varying lengths of time, and the curves return to their initial forn after about ten to fourteen days after stopping the digitalis.  Tests of these signs in patient§ with fever(pneumonia patients) have been nade.    we ba ay a , + meee  -\\\\- i vad  The failure ef digitalis in these circunstcnces to lower heart rate is familiar, and its use by many persons has been discontinued. In wany €& these patiants the electso- sirdiograrhic changes alrealy :ention -lse ocevr -ftcr the accinistration of digitelic provatly after the saxe loneth of tire, ond with the soe cosage, 28 in cases of cardiac diceass. If further enveraenes berss out these observations, we cncll have velusdle ne. wethods of Jucging the value of certain stiiulants in these conditions.  It nas previously been Supposed that for digitalisprep-raticn to be effecient  LSeS With  be. e tS LF) .  faver itore than the usucl dose «as reqvirei., Dr Jamieson hes therefore couvpared the lathel dese cf evystalling strezhantiiin in nersel cets ang in cats with eperinentsl pneuucuia, induced oy insufflation. He hes feund thet in both series of onicals death cecurs aftor the sane S-otnt of the ding lias been injected;that there is no disference in the action ¢ Svropanthin in the infected and in the non-infected ani- undSe This result Pargllols the o>vservations x2do in Frvients. However,vnile the in- fectel cats Scwed well-arked consolidaticen of the lungs, tney failod to develop fever. It is possitle that the absence of fever indicates that a true intoxication, interfering with the action of digitalis,was absent,ana that,had fever been present,tha result vould lave been different. It is planned, thersfore,to rezeat these exporinents in dogs.  Human hearts in which electrocardiograns have supplied evidonce that the swaller branches of the auriculo~ventricular conduction syste: had undergone pathological changes (cundle~branch lesions) have been studied in Serial sections. Structural alterations were found, but it was impossible to say definitely whether these cro to be correlated with the alterations in the electrocardiocrans, The nearts were supplied vy Dr Thonas Lewis in a state of dissection designeé for weighing the ca ities scparately, and were not quite fitted for the vUrpose of the histological Study Decauso the continuity of vce endocardium had been Scrificed. Tha failure to fing an adequate lesion tc account for the change uay lie in this circuustence.  Furtuer experiments on tie distrisetion of the vagus nerves and more eszecially  ci the acceleretor nerves are in Progress. It nas basn found thet the sins node doss       ee Picante. etn ea ; ; ie tii a     . mo ~ TK BS, net form a relay in tha path of the agus norves. The new experiments are based on the iuca that during left vagus Stisulation, th> accclerator nerves espocially are rcspon— sible for eontinucd auricular activity. Chouical Laberatory:- Tho foilvoing is a report of tho work carricd on by Dy Von Clyhe and the physicians working undsr his irmediate direction. uo Study of tho fate of the products of protcin digestion, begun tuwc years ago with Dr Moyer, and intcrruptcd last ycar in ordcor to develop reliable methods for urea  deturcination in blood and tissue extracts, has becn takcn up again.  fae  ir Cullen and Dr NeLean have investizated the qucstion of urea formation in the  livir in etheorisud dczs. Perfusion of excised livers by tho Folin school failed to  yy wy wu  revial an ability on the part of this orzan to transform amino acids into urea, and throvm doutt on the former vicw that the liver is the chief saat of urea formation. “orking with livo aninals at varying periods aitor rcat feodingy;howover, it has boon found that the henatic blood in every caso contains urea in greater concentration than tho portal,the increase in the urea rosulting from passage of the blood through the liver veryins in diffzrent oxperinents from 3 to 15 &. Passage of the blood through the muscig causes relatively siizht increase,usually none,  Mr Eodling of Dr Carrel's department, is experinenting with portal vein fistulus in dcss. Then the experinents are successful, as promises soon to be the case, it will be pessible to follow the tire curve of the rise of anino acids in the portal blood after the ingestion of protein; by deternining at the sare tine the curve of the blood urea, the two curves may be related to the progress of the food proteins along the alimentary canzl, as shown by X-rays. It will be of interest to ascertain how soon after the in- gestion of proteins it is possible to detect the presence of their products in the blood and how scon after this the formation of urea begins. It is also of theoretical interest to ascertain whether the body begins to break absorbed amino acids into urea as soon as their ebsorption is under wey,orwhether it waits until the tissues in general have be~ cone charged with amino acids in excess.  With Myr Sbeling it is also plenned to oxperinent with dogs having thoracic duct fistulas, in order to determine how important a channol the lymph is for protein ab~ sorption.  Mr Cullen has assisted Dr Avery in studying the effect of immunizing horses against xtxxgz pneumonocci on the relative propostions of the different proteins in the bleod. It is of interest to determine wether the protein fraction containing the iucune bodies increases during immunization, and whether such increase in any way para- llels the protective power of the serun.  Dr NcLean is making a study of kidney function in diabetas,and, in conjunction with Dr Cohn's dupartment,in heart insufficiency. The ratios of the concentrations of urea and chlorides in the blood to the rates at which these substances are elininated ty the kidney has been mathematically expressed in equations by Anbard,and Dr McLean's studies indicate that these ratios are very delicate indicators of the excretory power  of the kidney. The effect on these ratios of the administration of digitalis to edenatous heart patients is striking, and it is hoped that a comparison of the results of this study with electrocardiograms nay decide the question as to whether the prinary sffect of digitalis in such cases is on the heart ot the kidney.  Some of the diabetics show a pectliar,abnornally increased permeability of the kdineys to chlorides,the significance of which is still uncertain. This condition is also being ‘studied.  In order that sufficiently numerous data on the blood chlorides night be ob- tained in the above work it was found necessary to devise a method which would per= nit accurate determinations with much less material than the 20c.c. required for the usual Volhard titration. Dr Van Slyke and Dr McLean have devised an iddonetric method which pernits the determination of the chlorides in ome or two c.c. of serum with an error of less than 1 part per hundred.  The study of the chemical constitutidh(amino acid content) of certain food proteins, begun by Miss Vinograd last spring in conjunction with Dr Osborne of New  Haven is being continued.            Miss Vinograd with the collabcratiox of Dr Losee of the Lying In Hospital is oncaced in a thorough study of the Abdernalden serum reaction, using the Van Slyke enino nitrogan s:ethod in order to obtain a guantitative reasure of the extent of pro- tiolysSis occurrirg. The technique has teen so developed that an active serun usually “ives an increase of 100% or more in ariino nitrogen; so that there is no longer doubt 2S to either the occurrence, or the de-sree,of proteolysis occurring under the conditions ct the reaction. It does not appear that the reaction is very specific; but a final decision 7s to its value has not yet been reached.  Recently several European writers have announced that in the most dangerous tyres of diabetes the organism loses not only its ability to burn sugar, but also its wtilaty to metabolize the alimentary amino acids. In consequence an abnormally large proportion of tho urinary nitrogen is in the form of amino acids. The determination of a..ino acids in diabetic urine is subject to peculiar pitfalls, and it appears un- certain that any of the clinicians reporting the above resulté have beon aware of them.  It is planned therefore to take advantage of the diabetic material in the Hospital to  octain data on this important point.  RI. Cole  nv", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-qs56.fd4u.5g7c", "00000000-0000-0000-645D-5B0D088CF42C", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research, April 1915", "101584575X106", null, "1915", "April 1915", "Several pages of this Director's Report addressed the research on pneumonia conducted by Avery and others at the hospital in the first quarter of 1915.", "Official reports, Excerpts", "Bacterial Vaccines,Serology,Heart Diseases,Diabetes Mellitus,Nephritis", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "15", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "Parra an ey * eons sate 2 aman iat Set teas  Sn eee ON NENEREINE IE = mae A  ETE meee        REPORT OF THE DIRECTOR OF THE HOSPITAL OF THE ROCKEFELLER IMSTITUTE, April 1915.  Acuts Lebar Prewnonia, During the quarter forty-three patients sufforing iror. acute lobar pneumonia have been adiitted to the Hospital. This numbet 4s somew saat larger thar. the number admitted during the same period last year. Of these ¢ vatients ten were dus to pneumococei of type I, All the pationts of this type were treated and all rooovered, _ No observations have been made to change our former con= clusions as to the efficacy of serum of type I. in cases due to type I infection. Curin> the present winter only a part of the cases due to type II have been treated with irrune seruz. The resukte have not been Satisfactory. There has been no evi~ sou.ca that the seri: has had any very marked effect, At the present time we are 0% treating cases 2ue to thie type of organism with immuno serum.  Small amounts of the serum of type I have been sent to tne Brigham Hospi-~ ‘al in Boeton, the Presbyterian Hospital in New York, and to Dr Hanes of Richmond. ports on the results obtainad have not yet been received. te the type I serum is to ts sent out and to be used more or less indiscriminately, an important question is as to the use of preservatives. No preservatives have been employed in any of the serum so far used. It is obvious that the use of such serum is accompanied by considerable danger, Considerable study will have to be made te determine whether the use cf a preservative is practical, where such large amounts of serum are ade tinistered, A practical method of concentration of the serum would, to some extent overcone the difficulties. The study of methods of concentration by Dr Avery and by Dr Gay and Dr Chickering has been of great theoretical interest ,but,so far, these studies do not seem to be of practical applicatione The method employed by Dr Avery requires vary exte:aive handling of the serun, with corresponding danger of contami- cation, and a doncentration of only about four times is obtainede The concentration  cf serum by ths method deseribed by Dr Gay and Dr Chickering has demonstrated the ~2-  sbility of obtaining the inmune \\\\odies in very small amounts of protein,but the ‘ts so far,owing to untnom factors, cannot be constantly obtained, and in many  .3 where such results are obtained there is considerable loss in the immune sub— 12, 86 that the method practically would be verz expensive. Important principles, ‘Ty, have been demonstrated by these investigations and it is quite possible that --ical application Hay de mada of them. The important fact, however, has been de~ ined now beyond any aouht that the limit to the practical application of this form -.2Tapy ds dependent upon the degree of infection. If the infection is of a certain ~s no amount of immune serum will protect, and concentration of immune bodies is -< Practical advantages Observations which ve have made, however, indicate that, -. tie concentration of imcune bodies in a given serum may not furnish better pro- sn than ds obtained by the unconcentrated serum, it may be possible to produce  - of nigher grades of protection, possibly depending upon the so-called avidity of «10 Dodies for the bacteria,  The therapeutic treatment of cases of type I now seems to have reached a  tical stage, while the treatnernt of pneumonia due to other types is still in a -¥ experimental condition. The New York Board of Health is now preparingmserum of ‘3 IT and serum of type I is being prepared vy Dr Lewis at the Phipps Institute, Phila © -Falae Commercial houses have desired our compperation in the preparation of serum - “6 have discouraged their taking up this matter at the present time. A more de~ -tsd account of the exact nature of the work on pneumonia follows.  Observations dealing with the occurrence of Pneumococc’d of various types in -touths of healthy persons, and in those of persons sick and convalescent from pneu a are being constantly carried on by Dr Dochez and Dr Avery. Twssty families in sh one or more members have been sick of pneumonia have been quite thoroughly studied. “: olght of these families one or more healthy members have been discovered to be “ters of pneumococci of a fixad type. In all the so-called carriers so far de-  -néd the type cf organism has been the same as that responsible for the case of       ~3- an cieunonia in the family. These carriers nave been followed wherever possible until the fixad virulert type of prnaumococcus Sisappeared from the sputum. The longest  rériod during which healthy persons have so far been found to carry the fixed type of vrsanise has been forty days; the shortest twenty-one days; the average approximately thvity dayse In none of these carriers that have been observed has pneumonia devel- enede Three inetances of pneumonoccus infection occurring in individuals in contact vith cases of vreuxopia, however, hhve been observed and studied. In each case the  tyne of organism isolated corresponded to the type found in the case considered as the souree of infection. urther studies have oeen made concerning the length of time during somvalsscence, during which patients with pneumonia harbour in their mouths the ifgad virulent types of pneumocozcd. The longest period during which the patient has savvied She pnaumocoscus of the fixed type has been ninety days, and the shortest period nas de2n trelve dars;éhe average has teen about twenty-nine days.  Strdies concerning the mode of action of antipneumococcus serum are being made. Dr Avery has shown that when a mixture of pneumococci and anti-pneumococcus serun is made no dbacteriolytic or bactericidal action could be observed, When,however, raites are made from the mixture at short intervals of time, there can be demonstrated a weliedefined inhibition of growth during a period ef about six hours. This retarda~ tion of growth is not dependent upon agglutination, since it may occur in dilutions of serum in which no agglutination of the organiem occurs, and it is not completely speci- “| fic for the different types, while agglutination, on the other hand, is very epecific. 7 This phenomenon has suggested that part of the action of the serum may be due to ine nibition of certain metabolic activities of the bacteria. We had previously found that iovune serum when mixed with pnewmococci did not inhibit the power of the lattsar to produce methemoglobin. Later observations, however, have shown that if the bacteria | and serum are allowed to remainlin contact with one another at 37°C for two hours pefore tne hewoglohin is added, no methemoglobin formation occurs, so that the serum apparently  doss possess tue power of inhibiting this functional activity of pneumococci.    neni ena          Dr Deenss and Dr Avery ars making observation to determine whether or not the serum izhibits certain digestive and feruentative properties of the pneumococeus, especialiy the formation of amino acid from protein, and the fermentation of various carbohydrates. It has been found that when the normal curves of these types of acti- vity have been determined, when immune serum is added to comparable mixtures there is either complete or partial innibition. It is possible, therefore, that a part of the action of ixmune serum may be due to the inhibition of fermentative processes, which tacteria carry on in the medium immediately surrounding them, which processes are neces= sary for the life of the bacteria. Experiments indicate,however, that the inhibition of these activities of the pneumococci are not absolutely specific as regards sera of the different types in relation to the corresponding orzanisms.  Dr Avery has made a study of the relationship of the fermentative reactions of the differert types of pneumococci to the antigenic differences. A large number of strains of the different types have been studied, but no welledefined differences in tne fermentative activity of the members of the different groups could be determined.  Dr Chickering has continued the work comrenced by him and Dr Gay on the speci- ile precipitation of antitodies from antipneumocoscus serum by tacterial extracts.Their conjoint work has already been published. Various methods of producing the bacterial extracts have been tried, but the use of large amounts of acetone as employed by Dr Yan Slyke and others in the precipitation of ferments, has proved to be of the greatest value. Dried acetone-precipitated bacterial substances go into solution much more easily than the bacterial substances prepared in other ways. The fractional employ- nent of bacterial extracts in the precipitation of irmune serum has been found to be sfiective in completely removing the protective substances from the serum. To remove the bacterial protein from the serum precipitate, the best method has been found to consist in extracting the precipitate with normal saline, after the addition of weak a.xalie, as sodium carbonate, heating and shaking. The supernaten’ fluid after cen-  trifugalization contains onlyia very small amount of protein and apparently protects Stirals as well are ths wholg serum. fush extracts not only contain the protective  susstance, out alse acclutinin and précipivins, Studies nave been carried on to de-  vercine the toxicity of the extracts in various aninale, under various condations. No  Mcinistration of such extracts has yet been made to patients, but the experiments ine  -icnte that there ig little denger ef sheir Froducing toxic effects. Studies have also  seen made te detenning tne duration of the inmunity following the adainistration of  and also the possibility of produsin-~ 313  extracts, & Siigat active, as weil as passive, im  ‘Unity, by means éf suck. extracts. Apparently passive irmunity, whether obtained by  vhole serum or precipitates, kas disarpeared at the end of about five dats, but aftor  “-& days when the extracts are used Blisht srades of active immunity are seen,  Studies are being sarried on by Dr Cole with the assistance of Miss Stryker  sith have for their objects first, the determination of the differance between active  “Ld passive pnsumoceccus immuhity, and Secondly, the production of mors effective ime  “be Serum. By careful qualitative studies 4: mice it has teen found that by the pro-  “sstlon of very slight grades of active inmrity the cegree of effect obtained from ine  tums serum is very markedly increased, mich more markedly tiian can be accounted for by  . Simple aidition of the two factots. Second, ty traating animals with @aily doses of  -iving bacteria, combining these doses of bacteria with an amount of immne serum suf-  zistent to protact tha animal, it has teen found possible to produce within three or  cur weeks such immunity in rabtits that the serum will protect mice against doses of  virulent culture as high as 1 to 1.5 c.e. » whereas horse serum, obtained by the ordinary  rethod of immnization, even after six months, or even after one year's treatment, is  able to protect against no mors tian sl or at most .2 ¢.c. of virulent culturo. A  reat is now in process of immunization, and if the serun of this animal proves sitfica~  cious, we shall proceed with the di:rmumization of horses by thic sew method.  Dr. Conn, Heart Disease,  It is seusrally Helisved that the form of the electrocardiogram remains con  ‘taut in human beings for lonz periois of time. In experiments on animals it has been    =~ 6 = found that its form can be altered by the application of heat to the apex or base of “© ventricle, by the application of muscarin to tha surface of the heart, and by the tinulation of the vagus nerve, In studying the curves of patients to whom digitalis is given we have found that under the influence of this drug the form of the electro= >réio-ran is also changed. The change takes place in that part of the curve in cluddd between the R and the end of the T wave. The T wave flattens and later becomes -nverted in the greater number of our patients. It is a general rule that inversion c: the T wave depends on the fact that the apical portions of the ventricular muscle ire, relatively, electrically more active than the base. Digitalis evidently has the rover of bringing about this state. Ye have discovered this effect of digitalis in 2 day and a half to two days after beginning the administration by mouth. It outlasts “32 adrinistration by five to twenty-tyvo days. If the presence of the electrical hance actually means that the drug is still present in the heart muscle, it bacomes ‘lear why digitalis action in patients comes on so promptly when a second course of treatment 4s ocegun ten days after the first ended, for after so long a period complete :cinination ia usually believed to have taken place.  Ye have utilized this Sisn of change in T waves in studying the question of sianlation of failing hearts in pneumonia. In pneumonia, especially among acute in= | “ctions, the value of digitalis as a stimulant has been much debated. The first 0int we though, which should be decided, was whether there was a way of determining  ~at digitalis was exerting an influence. It was clear, from our own and from the ex- periences of others, that te judge by its effect on the rate of the heart was valueless. “e found in the electrocardioprams of pneumonia patients what the T wave changed just sit does in the non-febrile. The changs takes place in about the same length of tine, and it requires about the same amount of the drug to produce it. Te have, therefore, Aueans of telling that digitalis does produce an effect. Inthbe group of patients  42 avélop auricular flutter or fibrillation, a rhythm of the heart, which develops  -Oitaneously in pneumonia, digitalis is beneficial. The rate of the heart, when    an saese rbthus occur, usually rises to a great heicht. Disitalis has the power of re# icing the ventricular rate in them, by blocking the auricular impulses, just as it is in non-febrile patients having these tyythms. Whether it is beneficial when t.¢ chythms is normal, we are not yet able to say.  Dr Jamieson has attempted in another way to find whether there is a iiffer- -see between the action of digitalis in pneumonia and the non-fetrile state. Hoe in- ‘ceted crystalline strophantin(ouabaine) intravenously into dogs and cats, to find the -inimal lethal dose ger kilogram. In this he followed the method of Hatcher and Eg- ‘leston. Then he injected a number of dogs and cats with virulent cultures of pneu- zeesd by the mthod of insufflation, hen the animals were at tho height of the di- sease, he injected strophanthin into thom in the sare way as in the control. He found that the minimal lethal dose per kilogram was the sane in the infected as in the non- infected animals, 6.1 mgm. in cats and 0.12 mgm. in dogs. Post nortem the lungs © cf the animals were seen to be consolidated,  Tae conclusion ie dravn from the observations in patients and from the ex rerimonts in infected anirals, that digitalis or strophanthin, may be expected to act in the samé waye  Dr Cohn carried out experiments, sone time ago, in dogs which showed certain quite definite differances between the action of the right and the left vagus nervese Tae main difference was that stimilation of the right acted more especially on the pace-naker, while stirmlation of the left acted nore especially on auriculo~ventri- cular conduetion. Experiments were next carried out to see what influence the sino- auricular node, usually accepted as the pace-maker of the heart, exerted on this dif- ference. The sinus node was therefore destoryed in the jaws of a clamp. It was found that stimulating the vagus nerve still showed about the same differences as tefore, except that stirmlating the left vagus nerve caused a greater effect on the vace-razing funetion than it did before destewying the node. This effect differed  , vefere clamping, the auricles continued to peat during stimilation, after  slanpins they ceased. It appeared, therefore, that an influetice which caused the       -Se= “.534e8 to sontinue to contrast suring leit vagus stisulation had been destroyed. The s.nnt vas therefore rade to discover whether the accelerators provided this stimulus. os? 7308 accordingly sut ina number of experiments, sometimes the ri¢ht, sometimes leit, and sometines toth, Cutting ther produced no influence on the usual differ *; Eotuveen the vagus nerces. What mechanisr it is which is affected wen the S-A -‘¢ ib destroved, has not ye. been ascertained,  The study of the action of digitalis in pationtsa suiforin;; from heart disease csing continusd. Tne action of the drug is teing studies on rate of the beats,on suction, blood pressurs, diuresis, and its effect on the electrocardiogram. Former  - [6.08 have dealt sith its astion in payients having simple forms of disease. Ye are . 20llseting data in nore complicated forms in which abnormal rhytims, high blood Ssure, o@dera, and dypsnosa are found. Experiments in aninals are going on, plane - t@ correlates the chan326 which take plave in the size,shape and weicht of the heart “r various conditions with the eiectrocardiographic curves. The shape and size are \"dled by X-ray observation = the weisht, post mortem. Those animals are to be  “ated under the conddtion of strain, as when dogs run on the tread-mill, and also in  Fvescnce of artifielal valvular lesions, The influence of digitalis on them is  -0 10 La studied,  Study of Diabetes. Up to the present time forty-one patients with diabetes ve been treated. in this hospital. The results of treatment of patients, tp to the :vesent, are as follows: Total nunber of patients treated, forty-one. Number of xaths, five. Of these, two wers deaths in coma shortly after entering the hospital;  “<t patient with a sever cardia leston died of heart failure, shortly after entering  3 hospitals one boy died following a break in diet; one man recently died after  “sral months of declive in spite of traatnent, and autopsy showed caseous{ tubercular) -\"snnda. Two cther patients treated 4n coma were brought out of coma. Of several  #8 received sless +o coma, rene ent into coma. Two patients have left the hos-    ws EN ee  ~ vithout permission cr instructions, aitoar several months of treatment and improve- . (One of these left because he imazined himself well.) Twanty patients have been rz24 in satisfactorg$ condition, free from glycosuria and acidosis. Several of “ere childrec, ranvinz from seven to seventeen years of ase. Two patients have ~-d eving to treaking diet, but have re-entared the hospital in better condition 1 first, and have teen cleared up more easily than at first. One other patient  as +  olisht relapse owing to the advice of another physician that he eat bread. Other-  J reports show continued freedom fror. glycosuria or symptor's. This includes  ‘crotreet, the first patient, «hose case was one of the severest. The remaining  ml: val 7 ‘ ‘ae i  PS  .-G in the hospital are kept sugar-free and apparently improving. Tie reutine study of these patients, involvingas it does a very large number . ical enaiyses of urine, requires mush tine and labour. Under the section | ' Go irom the Chemical Laboratory\" is siven a description of the studies of aci- :vhisn are being made. Ds Stillman is alse studying the blood sugar in a series of cases, especialiy lcrense to the relation between the concentration of sugar in the blood and rate -ssreviox in the urine. | As will be remembered, the theoretical. basis of the treatment employed is . pancreatic function may be spared by diminishing body mass and metabolic activity. ‘s vas based on the changes observed ih depancreatized animals, under varying condi~ . +8 The studies in animals are being continued, parralel to the studies in patients. tially depancreatized animals are being subjected to conditions which alter body : oy 3s or metabolic activity. It is hoped by such observations to obtain evidence in “ort of the theoretical deductions on which the treatment is based. Dz Allen has previously brought experimental evidencx in favour of the view . blood sugar exists in a differe:t physical state in diabetic and non-diabetic ani- ‘&. Experiments are now under vay and observations are being made having for their.  pose the demonstration of sush physical differences.    a. —=—=— CU  nA Ma  Cory                 eee Beier:  edie 2 eee  A a acest nei nerd tlle Nel nei lan lt +. see ge oo i aad a vu ss oe ro ore ee  eee ae oT  Tne study of morphological changes in the Islands oi Langerhans occurring  @ ee  Liletie animals and in patients is being continued. The large amount of material  wdivals in all stazes of the disease, as well as o? fresh huuan material, affords  .settioual opportunity for the study of this problem.  ph roseiienrirreey  Pe et eI ge ne LR at a  % is nov planred during the sumer to publish the naterial sc far obtained  - 2orn of a “ienograph. This vill include (a)a report of the animal exverizrents .      _ Tort, the sasis for the propoesd modification in the treatment of diabetes; (bd) s.irtior of the rthod of ‘traatmert and reports of clinical cases treated. Studies concerning tie Nature of Acidosis and especially Acidosis ir Diabetes rv Van Sly:e, Dr Stillman and Mr Cullen have undertaken a study of acidosis 1 UDTe6. Pralininary work by Dr Van Siyke and Dr Stillman has indicated that the  waiiuation of the carbon dicxied tension of the alveolar air is one of the most     vile wethods, if not the most vaiuaole method, of determining the actual condi~ .¢f the patient, as rezards acidosis. A very large number of observations of al~ as COg vension in the patients with diabetes has been made by Dr Stillman, by a  wrod thiol is quite sinple, but seems to yield accurate and comparaple results, at  -:$ in patiants not comatose, and who can be daught a very simple procedure. Dr Van ze and Mr Cullen then wndertook the problem of determining the changes in the bloo# ish corresponds to the changes in alveolar carbon dioxide, in order that the inter- “tation of the latter determinations might be put on a firm scientific basis. Mr “len has gone on with the work commenced by Dr Zacharias last year, the direct de- -urdnation ef hydrogen ion concentration in the bloo by electrometric methods, and  . devaloped the wethod for the present purpose. It had already been shown dy  “saslia and ethers that in acidosis the actual hydrogen ion concentration of the biood _l3xgoes little or no change and Mr Cullen nas confirmed these observations. It ther 3d that it might be possible +o determine by this method whether or not the reserve  +y of the blood in such cases was decrea@ed. To determine this two metaods  “3 beer employed and by. these methods i+ has been possible to determine that such de--  ¢                  wken normal plasma is treated in the same way. As an example of results,the hydrogen  1h ual                                 cle ae ne ee ee eee reg ae ee ee eee nem”  W~1ll- rreaco in reserve alkalinity is actually prdsent. First, when a given amount of 4C1 is added to the plasma of a case of acidosds, the acidity developed, as measured —  “y the hydrogen ion concentration with the electronetric method, is mich greater than  ion concentration obtained after addddg one volume of “As H Cl to the plasma from a severe case of acidosis wae one hundred and fifty times as great as the hydrogen jon  concentration of ‘normal Plagne similarly treated. Second, Dr Van ‘Slyke ‘has béen able to show that the ability of the- placma --  to fix carbon dioxide which, of course, depends on the amount of reserve alkalie, in  Sa a ae a Fatt ee a Rr rer Pe pn ; ae  such torus ‘as s alkaline + pheno and ‘carbonate, ‘4s reduced in acidosis.” In order’ to                Hiden owe te  y towering the leveling wb | velow the o etch of ‘abe          yw - ~ 12 - UAE oon see  a3 the result of which the COs gas iumediately leaves the solution. The mercury is .  shen let back into the pipette, and the volume of CO, read off on the upper stem,on. |  “nich a scale is etched. Duplicate resuits rarely vary by more than one per cent. . Normal plasma binds 75 per cent of its volume of COo. In cases of acidosis  -2 fijure may be as lew es 20 per cent. This new and simple technique for determining     th3 co, capacity of the plasma provides a clinical method for the measurement of aci- x _  cosis direstly in the blood itself. Ina series of cases there are now being deter=     vined iitenecusiy, firet the alveolar * C035. second, the alkaline reserve of the plas :  sau 8 studies: arried ere the nemical taborate “under the  rik state me           ai                                       eee Om aye jr wo dit Aa, Se bade fe & peer gt  e13= .. - ae . a Ae     resuits obtained have not indicated that through immunization, at least with pneuno~ socci,one or other of the protein fractions 4s increased over the normal, Whether a sindlar erltical study of the effects obtained by immunization of horses to diphtheria toxin will yield similar results,contrary to those now generally held true, cannot be stated but these observations throw some doubt on present conceptions. i The study of the ‘enino acid content of certain proteins, which Dr Van Slyke and Miss Vinograd becan, over a year ago, in collaboration with Professor Osborne of | New Haven, has reached a satisfactory conclusion. One point of interest which has finally been settled concerns the content of cliadin, the principal wheat protein, in | lysine, one of the amino. acids which the work of Osborne and Mendel has shown to de- : LA absolutely escential for, srowth. , All work except that from this laboratory had “pros viously indented that this wheat protein contains no lyeine. Nevertheless Osborne” and Mendel found, that they, were, able to cat least maintain rate in quilibrivn with tne’     as their so16 protean, fot  Se, re                of the dying Tn Hospital, “has. boon. ¢ contimaed. ‘The nethod exployed, has ‘consisted. ‘ta     de termining the, Ancrease ta aminoen! ‘trogen in. the ‘mixtures as indicating degree’ of  oe  proteclysise - Piret, proteolytic ‘enzymes ocour, dn measurable amounts in every. normal -  BerUwie | ‘They are by no means characteristic of | prognant serae _ Second, such proteo~    ~i4'~ tic enzymes digest not only placenta protein, but other proteins as well. Third,on  : average the enzymes in the sera of pregnant women are somewhat more activexx than 338 in the sera of noi -pregnant woren and of normal men. The individual variations, -cvor, in the latter cover nearly, if not quite, as great a range as those of the  cicft precnant women. In certain pathological conditions,morsover, as in pneunonia, - ar..tly the most active disestive actior is seen. No evidence, therefore, of the  c .to of a specific enzyme in the serun of prepnant women has veen obtained. The coat quantitative variations observed in the amount of the non-specific ferment pre-  t in normal and pathelogical sera, as well as in the sera of pregnant women, indi-=- vig that the Aoderhalden reaction is unreliable for the diagnosis of pregnancy.  4th the assistance of Dr Losee at the Lying In Hospital, studies on the tox ‘4a of pregnancy have veen carried out. The most concrete theory corcerning these ‘oxemias has been that of Eving,according to which the toxenias are due to liver insuf- ficiency. The cherical evidence for this was the increase of non-determined nitrogen in the urine, which Ewing attributed to excreted amino acids, which were supposed to -ave escaped normal catabolism because of the inefficient liver. Direct determinations  ' «he amino acids, aa well as the other nitrocenous constituents, not only in the urine ‘yt also in the blood, have teen made, and the amino acid content in both has been four to be absolutely normal. The results make Ewing's hypothesis untenable. The abnormali ties of the urinary nitrogen appear to be, as surmised several years ago by Underhill of New Haveny the abnormalities of hunger, and nothing more.  Studies in Nephritis. Dr McLean has continued his etuay of kidney func= tion, and has used for this purpose cases of cardiac disease admitted to the Hospital ‘or study and, in addition, a small number of cases suffering from nephritis of various types have been admitted in order that studies may be carried out cn them. Dr McLean has continued his study of the ratios between the concentration of chloride and urea ir the blood and the rates of excretion of these subs* bances in the urine. The method  5: sstimating the excretory afficiercy of the kidne? in this manner has been reduced          > -1  ey  ,  tu a yracticai basis Ly Dr IisLean, so that the resuits are expressed in per centage of norral efficiency. The labour of calculation has been reduced to a minimum by ivisiny a slide rule for the purpose, which enables one to perform the otherwise -.ovhae prolonzed calculation in a few seconds. The rethod for determination of calsriles in small a.ounts of plasma has teen further simplified by Dr McLean so that results can readily te obteined within a half hour after the blcod has been drawn. Tt is plaraed to continue this study of nephritis, employing this method in compari-  “vi 2th other methods for determining kidney function.", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-yjw7~h8qc~5cpe", "00000000-0000-0000-C3DB-3D3463F26B00", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research, June 1915", "101584575X107", null, "1915", "12 June 1915", "In this except from the June 1915 report to the Board of Scientific Directors, Cole outlined the research on pneumonia at the Rockefeller Institute and Pennsylvania Hospitals.", "Official reports, Excerpts", "Immunity,Pneumococcal Infections,Diabetes Mellitus,Heart Diseases,Nephritis", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "9", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "June, 1915,     To the Beard of Hclentific Directors of the Rockefeller Institute for Medical Research,  Gentlemen:  The Director of the Hospital hag the honor to submit the following report  of the work Carried on during the last quarter year,  with com: ietior of a number of the studies which are nov ready for publication, Aeu39 Lobar Puowionia, The type of pneumococcus concerned has bow been  Je-:3rmined in 237 cases of Phoumonia admitted to this Hospital, Tn addition,  Jv Dishardson has determine? the type of pneumococcus concerned in 195 cases of  -Honia adnitted to the Pennsylvania Hospital. te have thus quite satisfactory  Sos3rvations dn 432 cases, The results are shown in the following tables:  Casas ix the Hospital of the Rockefeller Institute,  “tiological Agent No. of Percentage Mortality in Cases not Cases Indidence treated with Serum *roMocogCUS type J 78 33% 254 n n Ty 75 324% 29% fn © rrr 22 9% 45% \" \"ow 48 204% 12. 5% Vrer Bacterda . Influenza Bacilius 8 Streptococcus 3 Strenteceecus mMicesus 1 Staphylococeus 2 Pasiitus Priedlender2 Scrertecoceus, “vararlesseons ang , Iniluens:, Bacillus 2 l¢ 6% hasten nt     sentabets             AOINGS a4 sa  5 wri ‘  woe  Cases in the Pennsylvania Hospital.  Etiological Agent No. of Percentage Mortality in Cases not Cases Incidence treated with Serum, Preumococcus type Y 60 31% 30% 7 \" TY 39 20% 25% n \" IIY 13 6% 50% 8 \"ow ~23. 43% 12g  The Zigures indicating percentage of incidence in the two series are not exactly comparable since Dr. Richardson has not included in his list the cases due to or- ganisne other thas pneumococcus. The results indicate, however, the correctness of the conclusions previously drawn from a emall number of cases,  At the last meeting of the Board Dr. Janeway,drew attention to certain races of pneumococci isolated at Baltimore, which, while agglutinating in undi- luted sezum of type II, nevertheless did not agglutinate when then diluted serum was added to them, and when protection tests were made with them the results were not clear cut and unequivocal. or. Bloomfield kindly sent us these five races end these have been shoroughly studied by Dre Averys They were all found to be of low virulence and, a3 has before been noted of the type strains of low virulence, the svotection afforded by the sera of different types was irregular and inconstant,  in cortain instances eyen normal horse serum was able to protect against them.  Naeem mt Ng Snag te cae  4ll these strains were rendered highly virulent by frequent p passage through hice. Tots} on testing protection against these virulent strains, . it was found that type ITZ : serum protected against all of then in a typical manner, while type I serum and  ormal Horse Serwa had no protective power whatever. The agglutinability of these  Strains, however, has in this manner not been increasedy It seems evident there~  fore that these are true typs If strains, but which have relatively slight agglu- tinability. Tris is qucta analogous with what is known of B. typhosus, certain  Strains of yvhisn ara tligibly agzlusinatle. I< is not thought that these exper=  iments effer eny evidence thet the atypical strains are changed into fixed types.    PN  uO. .  The Cccurrencs of sueh  strains, moreover, need not necessarily eause any confusion  du the mathed of diagnosis of type employed by uss  Esrum Treatuent of Cases due to Pneumococe 4 of type Y. Forty-eight cases  236 46 Dneumososed of type I have now been treated with immune serum of these,  tour ified, contrasting with the mortality of 25 = 30 per cent. in the untreated  cases c2 this tye. Of the four fetal “aes, however, one died suddenly during  convalescence fron pulmonary embolism; two of the cases were only treated when  “18y Vere almostMiy ®xtremis\", a few hours before death. This leaves only one  case that can be considered to have been treated with any hope of success, tuds  and a8 treated only on the fifth and sixth days, the patient dying on the sixth. jaye  Epidexiology. Dre Dochez and Dr. Avery have completed their study  oO: carriers. he types of pneumococci occurring in the mouths of 113 normal ine  dividuals have been studied, Paosumococe: of the fixed types were found 11 times,  and these were always present in the mouths of persons in close contact with pneu~  nonia patients, the type found jn ead¢h case being that present in the case of pneve  =tnia with whieh the  porsonstudied was in contact. In 55 other persons Pneumococes  weve 2iso pressnt, but these were all of the atypical group, Group IV. The per=  Bistence of fixed types in the mouths of pneumonia patients and of carriers has also  veen studied, the final results agresing roughly with those previously reported.  Anablastic dmmynity. Dr. Doches and pr. Avery have completed the study  of anablastic immunity, and the results are ready for publication,  Extraction of Immune Bodies from Serum Preci itates. Dr. Chickering has neem OM Serum Precipitates,  continued the study commenced by Dr. Gay and Dr. Chickering and has so developed  the method as to obtain constant results, The best method of extraction has been  found te consist in dilute sodinn carbonate at 42° C. One patient suffering fron  e% intection with a type IT pneunococeus has been treated with such an extract given  suvcutaneously in irexcently repeated doses. In this case the clinical results  Were excellent, and the tests of the pationtts blood sseried to indicate that this     7 : a3 - i: ic ie |  a a hats ie Lo i t ¥ aS \\\\  cnt rns Re Bey we Pare acres:  if re i a i     a ae  ar een tena are    ron * ~g St x &  tf ag  SE  om  vreatment caused a greater increase in immune bodies than had previously been ob-  served. The work of Dr. Buil also suggests that this may possibly be the method  A: ere ie op ew  of  snoice as by this method the immune bodies may reach the blood more slowly. tf Tis method will ve tried further in cases of type IT infection. In the study of it  71353 Olracts a nuuter of observations of theoretical interest have been made.     1  a3 Tecan by Or, Caickering dealing witb thaso points is ready for publication. |  Jve Chistsring has een granted leave of absence for six months. He goes to work  a  ‘ith Dr. Gay and W222 cooperate with hin in treating a series of patients with  typnoid favor by an analogous method. He will return in November to continue his  vork here on pneumonia.  Pr RT Re rent: Fite vie alee? acon  Effast of Combined Injection of Optochin and Immune Serum. Dr, Moore  igharnmtl  “a8 worked in the Hospital during the past six veeks and has extended his studies  wipe.  of optochin, commenced with Dr. Flexmer, in an endeavor to learn whether the ef-  MOR EEO  fectiveness, of irmure serum may be increased by the addition of this druge The  SOT Met  results have boexn quite clear cut and show that the maximum dose against which im  ‘¥  eres  ane serum will protect is definitely increased when the animals are treated at the  fame time with .5 gm of optochin given simultaneously, or better, when 0.5 gm. is ; riven the first day. 0.45 gm. the second day, and 0.4 gn. the third day. These | #0368, Lovever, are railatively very much greater than the doses that can be safely { ministered tc man, Experiments are now in progress with larger animals of other . .  cpsciss to see whether it is possible that a relatively smaller dose may be effective  -2 larger animalse  Methods of producing Active and Passive Immunity, Studies carried  cut in large series of aninals show that the development of immune bodies in the  Serum of immunized animals is inhibited if very large doses of dead culture or  antigen prepared in various Ways are employed. Eighteen rabbits go treated failed to shov any durnne bodiss in the blood whatever. They all showed some active in- uunity, hovever, 4 certain iustances of considerable grade. These experinents |  ring further procf shat there are certain features in active immunity besides the    presences of igzune suostancos in the body fluids. We have called the former type  s  Os immunity somatic immunity in contrast with humoral immunity.  All the studies indicate, however, that pure humoral immnity may be effective af 23 infection ve not oxtrene, and if the concentration of the immmne bodies in wc Dleed be of sufficient degree. The attempts to increase the effectiveness of whe ivuoral factors vy other methods of immunization have shown that this may be dows ur Ireguently repeated small doses of dead cultures or probably better by give ing frequently rersated dmall doses of live culture, combined with immune serum, From thea studies on rabbits and goats it seems that a very definite advance has been made in the practical method of immunizations ;  Hoart Disoase Dr. Cohn and Dr. Jaméeson  Tho records from cases of pneumonia have been collected and are being studied; first, to see, in properly standardized curves, such as these, what changes occur in the electrocardiogram as the result of the disease; second, to see whether digitalis causes changes in the curves from these patients similar to those seen in “ne curves from patients suffering from heart disease. The studies indicate that Cems 2 than jer le oscur as the result of pneumonia, and these changes serve as a svi fov devte:udliing the degree and nature of cardiac involvement due to pneumoniae In regard to the second point, it has been found that digitalis does induce in pusvnonia patients changes similar to those seen in patients with non-febrile heart disease, as indicated by electrocardiographie curves.  The problem of the reliability of the electrocardiogram as an indicator of cardiac hypertrophy is being studied. Comparisons are being made with the X-ray Shadows, and also with the weight of heart muscles Dr. Jamiesen is trying to Produce aypertrophy in dogs by work (on a treadmill). Dr Cohn is making a similar effort in dogs in whieh artificial valve lesions have been produced.  the study of patients with chronic heart disease is being contimed along the idnes previovusir indicated,  Studiss corcerui:.; rhesonena of contraction and conduction are planned. To       (eae ~b— TE vender these studies possible it has been recessery to install a second galvanometer, so that simultaneous records from two points on the surface of the heart or on other contracting organ may be madée This second galvanometer was andered last autumn ‘ut, owing to delays incident to the war, has just been installed. The installation 92 this galvanometer is 0: importance in the work, not only because these special stulios are rendered possible, but also because the new instrument is of much great— -v ageurasy than the old one, and 80 much more exact studies are rendered possible. > 3 rew instrument vas mainly constructed in this country according to design corpished by Dre We Be Williams, and the tests have shown that the instrument en- Sisvcly fulfills the expectations.  Diabetes. Dr. Allen is contiming experiments showing the effect of voising and lowering metabolism (food, temperature, exertion, thyroid, etc.) upon oxperinental diabetes, observations thus far supporting the idea that increased metabolism increases, and diminished mesabolism diminishes the strain upon the  internal pancreatic function. The coming of Dr. Palmer has made possible a series  seers ie Mee a  of experiments to test the hypothesis of combined sugar. This series of experiments jeais with feeding and injection of sugar in normal and diabetic animals, with tests of the blood for sugar, estimation of hemoglobin content, and estimation of various physical properties, (viscosity, surface tension, freezing point, conductivity)  and parallel observations concerning the urine, lymph and tissues.  Dr. Stillman is carrying on the treatment of patients on the lines pre= viously laid down. The number of patients at any one time averages from ten to *4fteen. Ha ia studying particularly the question of acidosis and blood-sugare In regard to acidosis, the analyses made are for the three acetone podies and ammon~ ja in the uriga, and © Og tension in the alveolar air, in parallel with the blood studies of Drs. Van Siyke ané Cullen. The C 02 determinations in the alveolar air, and blood seem to offer the most satisfactory index of acidosis. Patients with slight acidosis or nowe may, during the initial fast, show the same slight acidosis  as a fasting normal person. Cases with severe acidosis, however, invariably show       om Ton a deoreass in acidosis during the fasting. All tests confirm the clearing up of acidosis under the treatment. Sugar is determined in the whole blood and in the sineua, with some idea of observing the permeability of the corpuscles, but chiei- ly to learn whether the treatment reduces the sugar conteht to normale This has rroved to ts so in some very severe cases, but in some older patients it may per= haps not de necossaryrto attempt this, and the question whether it is possible in uvery case cannct be answered at present. €tudies carried on in the Chemical Laboratéry.  Much of the work reported in April is still being contimed, but some of tne problems have boon brought to a sufficiently definite conclusion to prepare ior putlication:  Studies of the Abderhalden Reaction by Dr. Van Slyke and Miss Vinograd Sufficient data have been collected with our quantitative method on the pro=     +aase content of normal and pregnant sera to justify considering the problem as closed, and tha Abderhalden reaction for pregnancy as worthless.  Miss Vinograd, with material furnished by Drs Isaac Levin, has had similar results in atteupting to apply the Abdernalden reaction to cancer diagnosis. As only one preparation of eancer tissue was used in the tests made, however, the usslessness of the reaction for cancer diagnosis cannot be affirmed with the same degree of finality that appears justified in reporting the results of the pregnafc teste  Diabetdc Acidosis. Dr. Van Slyke, with Dre» Cullen and Stillman, is making an extended study and has obtained sufficient data for publication on the first points of attack{ Data have been obtained covering, in some cases, months, with almost daily analyses of the urine and determinations of the alveolar carbon dioxide, with, for comparisor, determinations of the actual reserve alkalinity of the plasma by our new v.ethods, viz. the determination of its carbon dioxide capa=- city, and of its c:ility to maintain a neutral hydrogen ion concentration after  2odstion of seid. ® 2386 tacts show the actual condition of the blood, and are  mg See mime  J     Vas FRET eae  plein tahiti ape OS 3       -8- co iglicate that they readily show even the slightest physiological variations in reserve alkalinity. The alveolar carbon dioxide determinations run fairly parallel -ith the blood vesults, and we have not yet observed a pathological change that was -o4 chomm simultaneously by both. After the direct blood tests, it appears that “6 elveotar carbon dioxide tension is the most sensitive indication of a state of  aciiests. Urine analyses of any kind are much less reliable, although a gross  change in the direction of acidosis is usually accompanied by an increase in the  i A a HY 1  aunorza cutpute ™:3 acetone bodies, on the other hand, may give no warning, even cui the verge of comma.  One point in which the blood and alveolar air tests promise to be of aid in the Allen Treatment of diabetes is in controlling the condition of fasting pa- tients. Some develop no acidosis, or very little; but in other cases acidosis doses develop rapidly, becoming marked after a single day's fast . A control by quisk and sensitive methods for detecting the presence and degree of acidosis seems ‘roortant, for as yet there is no way of telling which cases will develop acidosis an the treatment, and which will not., The sensitiveness of the methods developed “23 uncovered a physiological problem as an offshoot of the acidosis work. It has seg saldeved by some authors that during digestion acid products were ppured into she blood, and that these were responsible for the increased general metabolism and 233% production noted after a meals By others it has been pelieved that, on the contrary, the alkalinity of the biood increases, because of the secretion of gastric hydrochloric acid. Experiments on men which wé have already performed to test this point have already shown that an appreciable increase in blood alkalinity usually follows a meal. ‘The work will be contimed on both animals and men, the effect of digestion of carbohydrates, fats, and proteins being studied, and also the offect of stimulation of gastric secretion in the absence of digestible foods, so that the re~ lative effects of H Cl. excretion and of absorbed digestive products of various nature of the bleod alkalinity can be débermined. |  Tha work of 1438 Vinograd with Dr. Osborne of New Haven on the amino       acid soxosttion ef 2ocd protoins is now boing propared for publication. The basic constituonts of rico protoin, whcat gliadin, and milk albumin have been dotormminod By entirely differont methods in the two laboratories, so that errors by vach could be eliminated. Consequently these determinations are probably the most thoroughly controlled that have evem been thada in amino acid determinations cf proteins. Tne results have a direct bearing on the interpretation of the im portant work of Mondel and Osborne in their \"Growth Studies\", in which lactalbue min and gliadin hevo beon used as ropresentative proteins.  Nephritis. Dr. Mc Lean's studies with tho sensitive functional tests ¢Z cvoa and salt excrotion, are boing continued, with a gredually accmmlating moss of data which promises to be of definite value in the diagnosis and prognosis 22 dicferent types of nephritis.  One case of post-pneumonia nephritis wos perticularly interesting as  so nophritis was accampanied by an exceptional type of acidosis. Plasma tests  saoed a degree of acidosis which, at its height, was nearly equal to that seen ix, diabetics threatened with cam, but the ommonia excretion, instead of being  iucroased, was abnormally lov, boing less than one per cent of the total nitrogen, Daring convalescence the return of blood alkalinity to normal has accompanied the  restoration of kidney fumction, and the ammonia, excretion has risen gradually to  normal, Henderson and Palmer, of Harvard, have reported nephritics in whan a  ‘  low ammonia excretion accompanied an acid urine, but could not demonstrate exist-  ence of actual acidosis, as has been done vy the plasm, tests in this case.     epee < 3 Prey ods Se esi seinen gie ct: 7 ee wee Tage er Po ne SE RU ee aL: eae ET TN Sree y  ; Reset anor NN I", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-fj7j.i22p_kkxn", "00000000-0000-0000-561E-01E09DE2F2F7", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research, October 1915", "101584575X108", null, "1915", "16 October 1915", "This excerpt from the October 1915 Director's Report, Rufus Cole addressed the research on pneumonia conducted by Avery and others at the hospital in the preceding months.", "Official reports, Excerpts", "Immunity,Serology,Diabetes Mellitus", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "10", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "' REPORT  OF THE  DIRECTO? OF THE HOSPITAL  7 THE CORPORATION OF THE ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH  ' OCTOBER, 193%       THE HCSPIPAL  The Director of the Hospital submits the following brief report of the work carried on in the Hospital during the past year.  At the beginning of the past year the staff consisted almost entirely of new men. This was due to the fact that sev— eral of the old men had been calied to positions elsewhere, and others had left to take part in the war. The new men, however, very quickly took hold of the work, and all have proved to be Capable workers. The work in the chemical laboratory has been reorganized under the direction of Dr. Donald D. Van Slyke.  This has resulted in a marked improvement in the work being car-— ried on in this department.  During the year several voluntary workers have been granted the privilege of working in the Hospital and assisting in the reszarches being carried on.  D>. Gay, Professor of Pathology and Bacteriology in the University of California, acted as a voluntary assistant for several months, aiding in the studies concerning lobar pneumonia. ° Dr. Henry F. Moore, (working under a grant from the National University of Ireland) has worked for several months in the hospital laboratories studying the effects of optochin in experimental pneumococous infections in animals.  During the past summer Dr. J.T. Halsey, Professor of Pharmacology in Tulane University, has worked in the Laborator- ies ctudying the effect of amyl nitrate on vagus tone.  At the close of the pneumonia season, Dr. Henry Chick—  ering was given leave of absence for six months to work in the       -2-  Departinent of Pathology ard Bacteriology in the University of California there he had been appointed a Research Assistant.  ie is assisting in studies concerning typhoid fever, and it is tLooved that he will bring back new methods of value in furthering 4.2 cork or. pneumonia.  Very few changes are to be made in the personnel of the ata‘: fort the coming year. Tro new Assistant Resident Physi- ciais have reen appointed: Dr. Walter W. Palier, formerly Resi- deat Physician at the Massachusetts General Yospital, Boston, Mas- sachusetts, and Dr. Reginald Fitz, formerly of the staff of the Erighaa Hospital and Harvard Medical School, Boston. Both of these men have already carried on investigations elsewhere, the former on the question of acidosis, and the latter on the subject of nephritis.  A brief report on the progress of the work along the various lincs of investigation follows:-  PNEUMONTA  Epidemiology- Further studies of the pneumococci in the mouths of normal persons and in the mouths of patients during con= valescence from pneumonia have shown that pneumococci of the fix- ed types I, II and III are found only in the mouths of those per- sons who have lately suffered from an infection with an organism - of similar type, and in the mouths of normal persons who have been closely associated with infected persons, in which case the type of organism found is the same as that causing the infection. The organisma of the fixed types persist in patients, following . pneumonia, only during a period of from a few days to three months. The organisms of the fixed types also spontaneously  disappear from the mouths of contact carriers after a few weeks.       These new facts are apparently of zreat importance for our know— ledge of the epidemiology of acute lobar pneumonia, but at pres— ent it is felt that much care must be taken in interpreting these facts, and at the present time no final conclusions should be drawn. They sugzest strongly, hovever, that in the spread of acute losar pnevmonia, contact infection, either direct or trrough the intermediation of carriers, plays a very important rele, and they Give hope that the prevalence of the disease  may be diminished by more strict isolation of the patients, taouzh the probable large number of carriers may make this  very difficult.  Frequency of Pneumonia due to the Different Types of Ore@nisms- The type of organism concerned has now been deter- mined in about five hundred cases, including the cases in this and other hospitals. The larger statistics confirm the prev- lous observations that about one third of the cases are due to organisms of type I, about one third are due to organisms of type II, a considerably smaller number, 6 to 10 per cent. are due to pneumococcus type III, while the remainder are due to organisms of type Iv. The mortality in the Cases due to organ isms of types I ang II is about 30 per cent. In our experience cases due to pneumococci of type II are a little more severe than those due to pneumococci of type I. The mortality in the cases due to pneumocosci of type III is considerably higher, from 45 to 50 per cent., while the mortality in the cases due to organ- isms of type IV is much lower, 10 to 12 per cent.  The method devised for the rapid determination of the  type of organism Concerned in any individual case has been de-     eee ge re eR  ‘ apni ro settee te Pa [eae UE  ee ee ee  a OnE ECE    veloped so that it can now be carried out in any laboratory. The only Gifficuity which has arisen has been associated with the. determination of type of certain organisms, small in number, “hich give an atypical or incomplete agglutination reaction with Serum of time IT, Ten such strains have now been found and have sormed the basis ofa thorough Study made by Dr, Aver;. His study has shown that all these organisms unaouvtedly belong to typ? II, and all are acted upon ty the type ITI Serum, However these Organisms differ somewhat in their immunity reactions from the typical type II organisms, and also differ among themselves, SO that it has been possible to subdivide these orgarisms into Sevoral grcups.  Specific Treatment.- The results obtained in the treatment with immune serum of Cases due to organisms of type II have not been as good as we had hoped for. The serum treatment cf cases due to pneumococci of type I, however, has proved very Satisfactory, Forty-eight such sases have been treated with immune serum, Of these, four died, Of the four fatal cases, however, one died suddenly during Gonvalescence from pulmohary embolism, an accidental complication, and two of the cases were only treated when they were almost rin extremis\", a fey hours be- fore death, This leaves Only one case that can be considered to have been treated with any hope of success, and this was treat- ed only on the 5th and 6th days, the patient dying on the 6th aay. The results obtained, therefore, are in marked contrast to the results on the untreated cases due to organisms of type <, in which the mortality is 25 or 30 per cent. While, there-  fore, the Tesults obtained in the treatment of Casés due to or—             ganisms of type I have been encouraging, nevertheless efforts are being made to improve the efficacy of the serum in this type cr cases, and also tc render immune serum effective in the cases jue to other fixed types of organisms.  Concentration of the serum has not proved, at present, of practical importance. The attempts at concentration, how ever, especially the studies carried on by Dr. Gay and Dr. Chick— 2ring, have yielded results of great theoretical interest. By -.is methed it has been possible to obtain the immune substances of the serum in a fluid containing an almost negligible amount of tne other serum constituents. The preparation so formed also cortains some of the bacterial substance, and may, therefore, be of value in stimulating active immunitt, The therapeutic value of this preparation is not yet determined, but it is being tested in a small number of patients.  The attempt to produce a better serum by employing a different method of immunization of the horses seems at present to be more promising than the method of improving its quality by concentration, By injecting smaller animals daily with mixtures of immune serum and bacteria, better sera have been obtained than could be obtained by the old method, These results are similar to those obtained during the present year by Dr. Flexner and Dr. Amoss in the preparation of antimeningitis serum. The method has not yet been tried on horses, but it is planned to immunize norses according to this method during the coming winter and to test the serum so obtained on patients.  Optochin.= Experimental studies concerning optochin  ones  (sthylhydrocuprein) have been undertaken to determine whether,                by combining this drug with serum, the results would justify  the employment of this combined method of treatment in the  ceases due to organisms of type II in which treatment with  scerua elcne has not proved satisfactory. This drug is a de- rivative of quinine, was discovered by Professor Morgenroth, and has been eimiployed in Germany in the treatment of a limited num ver ot patients with pneumonia. Judging from the reports, the results octained from treatment with this drug have been suggest-— ive, Sut not striking. The drug, however, has been said tc  have a definite specific action on pneumococci, both ir the animal tody and in the test tube. To control these observations Dr. Moore has carried on studies of this drug, first in the lab— oz~etory cit Dr. Flexner, and later in she laboratory of the Hospi- tal. He nas found that the drug has a definite specific bac- tercidal action for pneumococci in the test tube, and that when administered to infected animals it has a definite curative val- ue. This curative effect is not very satisfactory, however, since the curative dose is very close to the toxic dose. When  the treatment with this drug is combined with treatment with im- mune serum, the drug is able to increase the curative value of the serum, i.e. a more intense grade of infection can be suc-— cessfully treated when the drug is combined with the serum than when the serum alone is employed. Dr. Moore has studied var— ious preparations of the drug and has made us quite familiar  witn the effect of the drug, so that now we are prepared to go aheai and test its value in inoreasing the effectiveitess of im- mune sérun of tvpe IT in cases of infection due to organisms of type IT, and it is planned to do this during the coming winter.  *     a          The pneumonia studies also have yielded other results  which are at present of only theoretical importance, though it  is hoped to make practical application of certain of them. In the investigation of the mode of action of the immune serum, for instance, Dr. Dochez and Dr. Avery have shown that the immune serun has a depressing action on the metabolic activities of the bacteria. Tais is one of the actions of immune serum which has been little studied. tut which may be ot considerable importance. They have named tais form of activity \"antibiastis,\" Dr. Ches— ney has made studies concerning the rate of growth of pneumococci and has investigated further the question of the so-called \"bac- terial lag\", or the delayed growth which occurs when pneumococci are transplanted to new medium. These studies are still in progress, but resuits have already been obtained which may be  of general biological Significance. Observations have also been made which indicate that inflammatory exudates induced by pneumococci have an inhibiting effect on the actiori of immune  serum, These studies are also in progress.  SYPHILIS,  It was hoped that before this time a complete report of the studies concerning syphilis of the central nervous system Could have been published in the form of a monograph. The de- parture of Dr. Ellis for the war, however, and the fact that Dr. Swift severed his connection with the Institute staff, have necessitatec a delay in its appearance. However, during the  past year the patients previously treated have been under ob-  servation, either returning here to be studied by Dr. Stillman,          -~8-  or beins admitted to the Presn;terian Hospital for study by Dr. Swift. The study of these patients, therefore, when it does appear, will be more Complete than would have been the case,  had it been published a year aco. During the year, Dr. Swift  had little time to devote to the preparation of the material, but during the summer he spent most of his time at this Hospital, get~ sing the material ready for publication and it is hoped that the monograph may appear this fail.  The method of sreatment of tabes oy intraspinecl injec- tions of salvarsanized serum has been quite extensively employed elsewhere. Many of the observers have been enthusiastic as to its value; others have been doubtful. It is felt by those tho nave carried cn anc. observed the work in this Hospital that the reason for the adverse opinions which have appeared has been that the method hag been very inadequately and unwisely employed. Inu stead of employing the method as described here, many have er ployed modifications, which have rendered it impossible for the observers to Judge of the value of the procedure, It has always been realized that the method was technically difficult, and that it was necessary to Carry out the treatment over prolonged per— iods of time, in order to obtain results. When the method is rightly carried out, the experience here indicateg that the meth- od is of very &reat value in the treatment of locomotor ataxia, It is believed that the publication of the monograph will permit an accurate estimate of the exact value of the method, and furnish  a reliable guide: for those who undertake this form of treatment.  DIABETES,  In the report to the corporation one year ago, it was    AY ne meen  stated that the work on diabetes had consisted mainly in experimental studies on dogs, carried out by Dr. Allen, and in the treatment of a limited number of patients along lines suggested by the experimental studies. During the past year the number of patients treated has been largely increased. The direct care of the patients has been mainly under the dmrediate supervision of Dr. Stillman, while Dr. Allen has devoted his time largely to the continua- tion of hie experimental stuiies. The methoi of treatment introduced by Dr. Allen has received very wide attention by the medical profession, and is being widely employed. Among those men in this country who have had the largest experience in diabetes, and are best able to judge, the concensus of opinion is that an important contribution to the method of treatment of diabetes has bem made. ‘The only question that has been raised in regard to the method of treat- ment devised here is as to whether or not the principle upon which it is based is new. This is of little importance, however, aince, through the studies that have been made and through the new facts determined, it has been possible to apply the principle in a way that has not been done before, and in a manner to greatly improve the treatment of persons suffering from this serious disease. The patients admitted to this Hospital for treatment have been only those of the most severe type, mainly young people and children, and the results obtained have been very satisfactory.  Dr. Allen has continued his study of experimental diabetes in dogs in an effort to learn more of the essential nature of the condition. The effects of cold, exercise, feeding, etc. on the utilization of sugar, both  by normal and by depancreatized", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-kpbw_9p56~jmi7", "00000000-0000-0000-0AF1-F2CFAA8216F2", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research, January 1916", "101584575X109", null, "1916", "January 1916", "Several pages of this January 1916 Director's Report addressed the research on pneumonia conducted by Avery and others at the hospital in the last quarter of 1915.", "Official reports, Excerpts", "Diabetes Mellitus,Nephritis,Genes,Serology", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "15", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "REPORT OF THE ! : DIRECTOR OF THE HOSPITAL.  OF  THE ROCKEFELIER INSTITUTE FOR MEDICAL BESEARCH,  January 1916.  PHY  : Rari? Rene NNT eee ALR Aap ARERRNRERS <t mercen ee  2 a te  Rec  To the Board of Scientific Directors of the Rockefeller Institute.  Gentlemen: The Director of the Hospital has the honor te sub~  nit the following report of the work carried eh in the Hospital during the paat quarter  years Sineathe opening of the Hospital five years age there has been a steady in~  crease in the number of patients requesting admission to the Hospital for treatment, Tats has been especially marked during the past months. A hospital several times larger could have been kept filled with patients suitable for the studies being carried on. Oux experience more and more indicites,however, that our work is much more pro= ductive when the number of patients is kept low. This makes the expense of the Has»  pital per patient very high, but materially increases the quality of the work.  Za November a clinic on Fneumenia and diabetes wis held at the Hospital. To this were invited physicians who have referred patients to the Hospital. The plan cf sending gut bulletins from time to tics describing the kind of patients whtth are alnitted haa been continued. We were cratified that the last Builetin was copied in the Journal of the American Uedical Association, and alse in the Bulletin ef the New York Board of Health, which I understand is sent te all physiciang in the city. This  was done without request on our part.  tt is evicent that the hospital is becening better know Ioang the people, and the general medical profeasion, and also that any feeling of distrust which ex= isted in the mind of the public is rapidly gisappearing.  Under these circumstances it ia possible that the question of ebligatery Wiwpilea May new be again seriously considered. When the Hospital opened I censidered “0.3 tnaiviseble, but it 4s now probable that we could ask every patient eon admission «9 the Hospital to sign a permit for an autopsy in case of death. ‘The opinion of the ‘eciérs of the Board on this subject would be welcom,  Cancar: stu P effe LP Xe tients with cancer.  It was hoped that the admission of canoer patients micht be undertaken          x January first, but delay in obtaining a satisfactory man to care for these patients > rendered this impossible. Several men are now under consideration, and it is -pad to begin adritting these patients very soon. ACUTE LOBAR PNEUNONIA: During the past quarter the work on pneumonia has rreceeded largely along the lines indicated in our previous report.  The nutor of cases for study has been mest satisfactory. From October first  Jamnary first 41 cases of pneumonia were admittod. This is to be compared with  . 0  cS cases aimitted during a similar period last year. Unfortunately for the progress >! our main study only seven of the patients have been of Type I. All of these have teen trated with serum. One of those cases died, but this was a case admitted only sc. the 5th day, and treatment could be very inefficiently carried out. Death oc- curred on the 6th day. A striking fact has been that 8 of the cases have been due to pneumococci of Type III, pneumococcus micosus, a much larger proportion of cases jue to this type than we have previously seen. 13 cases were due to Type IT and 11 cases wore due to Type IV pneumococcus. The fact that so many of the cases were due to pneumococcd of Types II and III explains our high mortality thie year, and suggested that the great severity of the pneumonia now occurring in the city at large may be due to the increased frequency of cases due to this type of pneumococed, One of our cases vas due to a gram negative bacillus. This organism resembled somewhat Friedlander's vacdllus but had no capsules and did not grow in a typical manner on agar We have previously had other cases due to a similar organism and it seems that this bacterium is a etiologic agent in pneumonia not previously described.  The studies concerning the epidemiology of pneumonia have been continued by Dr Ernest Stillman. He is studying the flora of the mouth in a series of persons vithout pneumonia, using the persons coming to the dispensary for this purpose, and is also studyingtthe mouth flora of the members of the families of patients, and also  cf tne nurses caring for pneumonia patients. It is planned to continue this study |  aqhcbae Re arm . ~— Z  dicta Reg —  3 Wray  HT          2 EA  wos  \\\\-  ~Ti.g tee rear. Tuco results so far sontirm those previously obtained. In the mouths  ot oO * !  i ' wi 4 ay ao oO $s a) ec  : S tie preu..csocsl presons aro of Typo IV, except in certain instances  ~O.5 th@ tersens clos3ly associated vith pneumonia patéents, in which case pneumococci  7. i.xe. trros mar se present. In three instances(noimal persons} without history of  ivact, pneumcecoscuc mucosus has been isolated. Dr Avery has now collected about  X strains of this type of pneumococcus, including those fron patients with pneumonia,  a3 912 25 those from normal nouths, and he is raking 2 thorough study to detersine,  2 possible, -sesher any conetant differences(cultural or imsunological) exist between  2 Strains fount in diseaso and those precent in health. It is too soon to report  i. the results obtnined. ' ur Doshes jis coatinuing his study of anti-blastic immunity. At present he  - atteupting to isolate the substances in the serum which aro responsible for the in-  - ition of the negerdal protedl;tic ani glycolytic action.  D> Chiskering returmed on Decerber first from. California where he had been vreang vith Dy Gay on the treatuent of typhoid fever by intravenous injections of -2n8itised tyrkoid vaccines, The work was aided ’y a grant from this Institute made 148 ausuum. The therapeutic results are interesting and suggestive. One observa~ sa0u made oy Dy Chickering seeus tobe of considerable value, that is that the mumber oi typhoid bacil1s in the blood during the course of the disease is of considerable trognostic importance. Apparently the injection of the vaccine is followed by a de --9ase in the number of bacilli in the blood or they entirely disappear. The work a8 also confirmed the observation of Gay that such injevtions are followed by a wave t: leucocytosis. The injection of vaccines is usually followed by a severe general vuactvion aud certain of their observations suggest that such reactions may in some  ture way be of protective value, even when they are non-specific in character.  pas  Ae ol       2 ff ; ~rX- wei E  Tas subject is worthy of further study. Fer the present, Dr Chickering is busily  te ng eae bee:  sngaged in prvparing a supply of bacterial precipitates, as studiod last year by Gay ond Chickering as it ids planned te treat a series of cases duo to pneumosoeccus ef Type II with this preparation.  Dr Chesney has almost completed a study eof tho rato of growth of pncumo- coccd, especially of th: phonemonon which has ben describod as bacterial \"lag\". In broth cult. s there usually first occurs a poriod in which multiplication is wry slow. Fora tine no militplication whatever may occur, indosd, as Gillespio hos shown whon th numbc inoculated is small there may ba no growth at all. Follow ing this letont period a poriod cf vaximum rato of multiplication occurs and then acain a yricd of decline. Chosey has shown that the occurrence or degree of lat-nt poriod,or lag, depends upon tho stzge of growth of tha culturo from which tho subehiture is mado. If tho forrer be in the stago of maximum or logarythmic a growth, the subculture may show no lag whatever, whoreas if the culture be in the  earhy or late stage tho lag may be marked. The curves of the firat fow hours of     growth of the sub-culture parallel almost exactly those of the original culture during the same period)h If during tho period of maximal rate of growth, the cul- ture de placed in the ice box and kept at 0°C, the culture coases to grow, but on rericval from the ice box it will again grow at maximum rate(without lag), even if kopt on dice for as long as three days, and subcultures from this same culture kept on ice will also grow at maximo rate, without lag. |  Pneumococe’ of Type IV, when freshly isolated fron ths human body, show a very marked lag, nuch greater than pneumococci of Type I or Type II frashly isco~ lated from the human body. The minicum generation time of pneumococci (Types I and IZ) has be sn found to be from 25 to 30 minutes.  The idea is entertained that lag is an expression of injury,which view  is sor’ ad by the experiments so far performed. Attempts have been made to deter~       WE Sr ee SRN oe 2  ate  + tera:     sete oo es aay  mina whether cultures showing marked lag are less virulent for mice than those not     ~%-  oe  ke  ohowang Lar, cat it has not been possible to secure clean-cut results, as yet.     “itu. ta Cullen, Dr Chssney hes followed the change in H-ion concentration “hich o¢cur in a calture of pneunccoccus during its grcewth ond the experiments done thus far show «. striking parallelia: betwen tho growth curve and the H-ion con- ccntration curve up to the point where growth coeasos. After this point is reached, tho i-icn cencentration re:ains constant for days, although the pmsumococci dacrease in numder and autélysis occurs as is cvidenced by 2 partial clearing of the culture. Cultures of both Type I end Type II pneumococci attain the sems H-ion concentration, ana growth in cach caso caasas at this particular level of H-ion concentration. It is interesting that at the tim at which this lovel is reachad, the culture of meu- aoececua Type I shows twice as many organisms per unit volum as does the culture of Type II. This would indicate that the metabolic activity as masured by H~ion con contration, is areater in the case of Pnoumococcus Type II than in the case of Pneu- noceoccus Type Ty.  Dr Moore has been civen charge of ‘the preparation of irmame serum and  is also continuing his study of the action of ethylhydrocuprein. Attempts are be- ing made to produce rapid and effactive immunity in horses by the daily injection of small doses of cultures, as was shown last year could ba done in rabbits. ‘The préparation of the immune serum for experimental and therapeutic purposes has now Srown to be a considerable undertaking. A very large number of clinics, boards of health etc, are now determining types of pneumocecci by the methods devised here and almost daily requests ars received for serum for such diagnostic and experi- mental purposes. We are also supplying serum for a few clinics for therapeutic usése It is impossible at present, however, to do this except in a very limited Serux of Type I and Type II is now being prepared, however, by the New York  Way » C.t7 Board of Health, the New York State Board of Health and the Pennsylvania State    7 ee ee . fe ates ast '  -\\\\- djoard of Heabth so that it is hoped that we shall be relieved of the need of com tinuing or extending this work. The New York State Board of Health is making pre= saraczions t¢ determine the type of infecting agent for physicians in the larger cities of tre state. In this vay will undoubtedly ba obtzined observations and statistics of great evideminlosic=~l significance,  At prosont o series of coses due to Pneumococcus of Type II and Typo II!  <6 veins treated by ontochin(ethylhydroduprein). Lost spring Dr Moore made a ctady of the bactericidal effect of ths blood soruni of rabbits following the adnin- istracvion of optecnin. Curves made by plotting the b-ctericidal power at intervals Pollowing the aduilnistretion of the druc wre ods. Similar studiss were also wis On 2 few acrml persons. This study permitted the development of a technique suich may now be employed in the study of patients. The number of patients treated is still too tow for any conclusion to be drawn as to tho therapeutic effect of the  x3, but so dar the results cre promising. In any case, the effect of the drug in. increasing the bactericidal power of the serum can be readily demonstrated and repeated doses have a cumlative effect. We have already had a distressing example of the toxic effect of the drug,khowever. One patient 16 years old, received four doses of the drug 0.5gm. each, over a period of 36 hours. Following this she be- cam? completely blind, and the total loss bf vision lasted six days. She then becane able to distiguish light from darlmess and there has followed a gradual im- provement. It is at present impossible to say whether or not complete recovery will occur. This seems te be one of the most severe cases of amblyopia which has ocourred following the use et this drug. Some cases are now being treated giving very enall doses of the drug every two hours, but administering a total amount of 1.5 gm in 2% hours, as it is not likely that any smaller doses can be of any valus.  It is planned later, in a series of cases to cambine the drug with immune serum.          Last yeer Lister in South Africa, published a short payer setting forth that when pnewmsecoccd are grovm in dirane sera they become more susceptible to won action of nomml ssr2n as shom by the power of normal serum to cause agglu- tin:tier und clse opsonization. This phenomenon he called piantication(to fatten slaughter). He showed also thet such organisms becore less susceptible to the act: cn of tha izmuane serum. A sories of studies have therefore beon made by Miss “or te investigate this phenomenon, studying pnoumoceccd of various tyres. It has tocr found trae tint when pneumococcus of Type I for instance is grown repeated- ly in serum of Type I it may become Ho a slight extent spontaneously egglutinable vith nerm:l ssrua, but more strixing and of grecter significance is the fact that it boctsws much less actlutinadle with the homologous serum, and alse now becomes readily agglutinable with serum of Tyre II. With this change in agglutinability the pneumococci lose their virulence. If such pneumococci were now observed for the first tine it would be difficult to at once determine the type. Passage through eninals, however, readily brings bacx their virulence and elso makes evident the specific cgglutinedility, which is always of tha some character as thet originally pre sont. The change in the orcanism occurs after five to six passages through oroth containing one tenth part of immune serum. Orzanisms have now been passed thooack as many as 95 transfers in serum broth over 2 period of one year, ‘The chanzed character of the organism may persist for as many as 50 transfers on broth, thcugh, as previously stated, it quickly reverts on animal passage. The inter— pretation of this phenomenon is at present difficult, and studies are heing made vy immunization of animals against these changed types etc., in the endeavour to fing underlying factors. At the presentt time the significance in relation to the question of epidemiclogy and origin of tyres does not seem to be great. In any  case it is entirely different from the socalled transformation of organisms as  described by Rosenow,  oo ee  be  i ast  rE  UE NTI EGS IE SO, OEE IT RT! oR Chae BR Sake I ELUTE TI rs re ‘             | Sao 4 i hogan } .  :  i  i  j  a —~- —-_  i  Diabetes: Only children and adults suffering from the most severe type 6: this disease are being admitted for study, These cases are under the immediate core of Dr Stillman. Dr Allen's study of this disease has made two radical inno- wtions in the treatment, first, the valuable effect of prolonged fasting has been onstrated, and second, the important rOle which exercise plays in the treatment wos been shown. Moreover, it has became evident that the treatment of the patients —wr they have becans aglycosuric consist in education « teaching them how to live “itn their restricted functions. This cannot be done by general instructions, but -awh patient(or 4f a child, the parent) mst take a course of instruction in the diet <tcher, and he mist learn te examine his own urine accurately for sugar and diacetic ald,  For the accurate clinical study of diabetes and the proper treatment of the istients it has been necessary to develop a quite elaborate organization of the diet “itchen and clinical laboratory. This has been accomplished largely by the efforts of Dr Stillman and I think it is no more than true and proper to say that this has vecoms a model that is being im jtated throughout the countty. The methods em ployed are constantly being demonstrated to very large mutsbers of visiting physicians. Dr Stillman is getting ready for publication an a monograph a complete deacription of the methods emplayed, with full reports of the caass treated for illustration.  At the present time, Dr Alien is giving his time largely to the preparation  of his experimental materia) for publication, With the aid of Mias Wishart he is continuing and finiehing some necessarily long and tedious experimentse  Dr Fitz has made sam studies on the question of renal function in patients “ita diabetes, especially with reference to the disturbed salt metabolism frequently presente He is now, under Dr Van Slyke's direction, working on methads for the de~ vemulnation of the abnormal organic acids in the blood and urine for the purpose of  cstermining if possible the laws governing their excretion.       Pn ce lle vee ee  ta a cw ae “5 -  -d-  De Palmer has carried on a esries of studies concerning the of sugar in the tissues,  concentration Althouch moh work on the fate of glucose in the anim  orcaniam has been reported, little ettentién has been given to the concentration  of this substanos dn the tissues. ‘the Purpess of this investigation is to deter nine the amount ef glucose in the tissues under varying conditions(nermal and diak betic animals, hyrperclycasnia artificially produced) with the view of throwing  Sidon. It was necessary to develop a suitable mathod of investigation, which gas bean accemplishec, (Up to the present 26 dogs, 18 of which were normal and 8  +4 various ateces of Clabetes, as well as 16 rabbite and 1 monkey have been used)  — Vostained, The regultg thus far cre ag follova, The concentratian of sue  oT in the verioug tissues, though considerably lower, vzries directly with the  -oncentration in the blood, There ssems to be little difference in this ree  S160t between normal and diabatic aninals,  Acidosis + Dr, Van Slyke, lf. Cullen and Dr. Stillman have carried Mm an extensive study concerning acidosis in diabetes, As mentimed in a pre vious report, Dr. Ven Slyke hag devised an accurate but simple method for the study af Cog Capacity of the blood, which seems to be the best clinical method available. The use of some such method is of Breat importance where the method of prolonged fasting is being employed. This and other methods for de termining ecidosis have been used in the study made here. Fran this study Pr. Stillman  1  “as been able to separate the cosas of dissetes inte four types as regards  Cldosisa,  i. Ca368 with only slisht tendency to form abnormal acidg either eer ak  wots Shia or on a adet. Little or “40 @xcretion of acetone bodies. No tendency     ov Les, Dicart onate vo fall. Cases of this type are usually mild,  Re RE ate eee    (2) Cases with 3 tendency to _ acute acidosis on fasting, Patients may be-  jiu tha treatment in apparently fairly good condition, but the plaam bicarbonate Zalle rapidly when the fast is institu’ sd, and may resubt fatally if acidosis is not detacted and checked (by feeding) in time. Since the present methed of plasna bi- sartonate deteruination has been employed for detecting acidosis, patienta of this typo have without exception been detected and successfully treated. A peculiarity  of such cases is that as the second or third attempt to make the urine sugar~frea by  tasting ne acidosis hay occur.  (3) Saseg shoving acute acidosig, curable by fasting, This type shows a  -snaviour exactly Gpposite to that of typs 2, 4 patient entering the hospital ina scnuition of serious acidosis, with plama bicarbonate at half the norma] level and ite physical anc wental syaptons of impending coma, May as the result of fasting and orate carbonate dosage, or even without carbonate, clear up go rapidly that in twe <r three days! time both the blood tests and the clinical signs are normal. Such “tients put on a rational diet then tend te remain free of acidosis,  (4) £209 showing chronic acidosia, These Patients even after long treat~ 2nts show subnermal plagna bicarbonate and , bypernormal ammonia excretion. They form acids in abnormal amounts and eliminate then less efficiently than do normal indivie uals. These cases are in an unstable condition, and are the mest difficult of all to treat satisfactorily. Eventually, however, som of them appear to craduate inte the nildex type described under (1),  -Nerhritig, Dr McLean is Continuing the clinical study of nephritis, em  rloying the nuwerical laws governing the excretion of urea and sodium chloride. In  “e individual cass one of the execretory functions is frequentiy affected while the ‘thers remain normal. Urea excretion function apparently depends on kidney factors dene, but conditions regulating salt excretion function are more complex and apparently  vs lurzely extraerenal., The Gases of nephritis ac far studied have been mainly cases       hi  “th advanced frictional Chanze, @SreCially those liable to uremla, and also cassa saith edera, fn addition, cases of primary hypertension without apparent kidney Sa.cuisnal Chances are vedng studied,  Studies are also being made of the effect of differences in nitrocen intake (very larsa amounts, as well as very gnall amounts) on the index of excretion. Such “15768 in intake apparently cause no chanse whatever on the index of urea excretion, | Heart disease: Dr Cohn and Dr Jamiason,  By Preference patients sufferin~ fron auricular fibrillation and cedena are <@ing admitted. In these patients the effect of cizitalis on heart-rate, kidney funn sion, blood pressure, fever(when this is present) and on the electrocardiogram is being  atidied. Blood pressure in Patients with fitrillation is veins studies by recording     iw percentage of total beats that reach the wrist with different levels of externa)  ¢SSure. From these records Curves are tein> mde which are believed to zive a more suvistactory account of the effect of di-italis on blood pressure than do fisures can r3sed of averaces,  The time when the Clinical effect of digitalis becomes evident ia being “ocpared with the time when alteration in the 2 wave occurs. Studies are also bem ing made te decide whether a larcer dose of digitalis given in a short time has the sare effect on the electrocardiocram aS & small dose given over longer periods.  The action ef dimitalis in 108 cases of pneumonia in respect to chancea ia the T wave and in conduction time has been atudied. 58 cases that took no disita~        | lis served ag Gontrels. 50 oases took digitalis. Of these only 40 were available  ter Study, the others not havine had an anount of dizitalis sufficient to influence the curve. Of the 40 available Cases that took disitalis, 37 were studied. Briefly stated, the observations showed that in the absence of digitalis 90.3 per cent of the 21868 showed no chances in the electrocardio-ran while of those who took the druc,  “8 per cent showed chanze of ene sort or another, These figures show that dicitalis       | ~ Tiv<  “odaces an effect even during the febrile period in pneumonia.  As to wrethor the drug is beneficial in pneumonia the observations offer -) ;rccise information, We can, however, draw some deduction fron the following ts: din examining 126 cases(those (108) already mentioned, torether with 18 other -.5S not inoludec in them for certain reasons), 12 cases(9.5%) developed either fib~ ‘lllation er flutter, 9 recovered from the irre ,wlarity; 1 left the hospital with it; 2 died while it was still present. When the irrercularity beczan 4 cases had had .2 di italis; the others bad had very exall doses which could not be rerzarded as the -2.se@ Of tue irregularity. If the irrecularity existed and lasted sufficiently long, | 2italie was able to reduce the heart rate by blocking auricular impulses, just as it -#S under ordinary conditions. In these cases showing these irregularities there~ sore the beneficial action of the drug is very evident.  We are continuing the study of the action of digitalis in fever with the view cz ottaining an effect more rapidly than has been possible by the use of the doses rieviously employed, narely 0.4 cram per day by mouth. Digitalis is now being em ployed in doses of 1.0 cram or more by mouth in 12 to 24 hours. Cpystalline strophan~ thin ds also being given, using very suall doses, on account of the unexpected irregue larity in results encountered some time ago. These studies have ‘been begun too rea cantly te give a report of results. |  Experiments are being conducted with the purpese of studying hypertrophy of the heart and its influence on the form of the action current of the heart. The nature of hypertrophy is being analyzed by studying the weight of the layers of the heart as obtained by dissections made according to the method of MacCallum and Mall. “hen the layers are separated by this methods they are not coextensive with single caanbers. The methed of study is as follows:~ after X-ray plates and electrocar dicgrams are obtained, hypertrpphy is produced in one group of dogs, which are made  to work by running on a tread mill, and also in other groups in which different val~    ~ YK  vulnr lesions have been artificially preducei, Xeray pletes and eleotrocardiograms  are made reyeatedly durin; ths period ef observation. On the conclusion of each ex- viens, the heart is dissected in the way described and the layers weighed and the  Li'LtS tarared with controls.  Chezdcal Laboratory: Dr Van Slyke, with the assistance of lir Cullen and lz .kLecn has continued tre wore on tne fate of protein digestion products in the body, ocmocrith Dr Layer. The work with Dr Leyer had snom that amino acids from digested croteins enter the circulation urnchnnged, and “0. it are absorbed by the tissues, each 3: which possesses its own store of free arino acids. It is mown that the greater mat of tus protein nitrozen absorbed is turned into urea and excreted within a few wee > appeared possible,however, that urea formation might not begin util the vicsusa da general have taken up such an aiount of the avsorbed amino acids as they eijaire, the ureaeformine mechaniagn serving to rid the body of the excess. Consequently lo-s after a fortyeeicht nour fast were fed with meat and the blood urea was determined several times yer hour. Ir some experiments X-ray photocraphs were made in order to correlate the nrogress of tne food throush the alimentary canal with the production of avsae It was Zound that urea -ormation does not wait until an excess of amino acids abéve tne requirerer.ts of the orcaniam have been absorbed, but begins the moment ab- sorrtion starts. Increase in bleed urea occurred within mich less than an hour after reading, as soon as the X-ray showed the first passage of food inte the ducdemm, and 23 S000 as any increases in the amino acid nitrogen of the blood could be detected. The urea-Pormming mechanien is, therefore, immediately atimilated into action by the absorp- tion of amine acids, and does not wait until they have accumilated in excess.  One other point in protein metabolism attacked has been the question as to “tether not onl¥ aming acids, but also peptones or other intermediate protein products are absorbed from the intestine into the circulation. The blood proteins were pre~  cioitated under conditions which leave intermediate products, not only peptones but    ~ tr-  vex, aloumesés, in solution. The amino nitrogen was then determined in the filtrates verors hydrolysia(free amine acids) and after hydrolysis(increase = intermediate prom -.0ts,rentones or albumoses). It was found that, althouzh in the sare experiments .rlé increases in fre amino acids of the blood occurred during digestion, no increase 24 all da the intormediate protein products could be measured. Consequently little cr no eoserption of \"peptone\" as such occurs. All the protein,eo far as determinable <y présvat rethods, is hydrolyzed.  -iss Vinorrad is encaced in a determination of the auino acids yielded by we Mlewdn and casein froa human milk. This problem is undertaken at the suggestion oi Dr Helt. Tra observation of clinical experience 33 that less protein is required -y is2?anta in tha form: of human mili than as cons! Ldlk, It is mow, from work done sist year in co-operation with Dr Osborne of New Haven, that the albumin of cows! milk +S tach richer in hexone bases, especially lysins, than is casein, and lysine has been -vi.d Dy Osberne and Lendel to be ene of the amine acids absolutely essential for growth. “ian udlk contains mach mere of its protein in the form of albumin than does cows! mad Le sai the richness of the albwain in necessary amino acids, such as lysine, may explain ts9 hish nutritive value ef human milk proteins, if the albumin of human milk ig sini- lar in couposition toe that ef cows! milk.  Dr Van Slyke has studied, with the co-cprergtion of Dr Lesee of the Lying-In dospital the present theories cencerning the nature ef the taxic ascent in toxemia of pregnanay’. The two leading theories have been that the texemia is caused by either (1) anine acids,which the decenerated liver fails te turn inte urea, or(2) acidosis, tuo foetus intexicatinz the mother by acids which it forms. Both theories are false. In no case of texemie have abnormal amounts of amino acids in either urine or blood been sound. Nor is acidosis cenerally present, except in isclated cases, and even in ths lnvver it ds not severe, Yurthemmore tho fostal blood obtained from the umbilical cord *¥ Gslivery saows bicarbonate content no lewer than the maternal blood, The real toxic  eat 38 not indicated by any chemical theory which has previously gained prominence,", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-agay.miww-fgxj", "00000000-0000-0000-1EBE-43F6D914A0DF", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "The 'Undiscovered' Discovery", "101584575X11", "101584575X615", "1970", "September 1970", "This article chronicles the work of Thomas J. Francis, Jr., on bacterial transformation at the Rockefeller Institute beginning in the late 1920s, and the ways in which Francis influenced Avery's research.", "Articles", "Transformation, Genetic ; DNA,History of Medicine", null, "7", "pages", "Text", "English", "Copyright 1970, American Medical Association. All rights reserved. Applicable FARS/DFARS restrictions apply to government use.", "Copyright may apply", null, null, "Reprinted from the Archives of Environmental Health  September 1970 Volume 21 “FEE ] Copyright 1970, American Medical Association  256  The “Undiscovered” Discovery  Wendell M. Stanley, PhD, ScD, Berkeley, Calif  2 Tw. \"lez  ce  Seon oy gas Slog  Prologue  Tus STORY deals with the very impor- tant discovery that DNA could possess and transmit genetic information and some as- pects of the scientific climate existing at the time which probably were responsible for the fact that the discovery was really “un- discovered,” generally unappreciated, and failed, for several years, to affect the trend of science. It is written in honor of Thomas Francis, Jr., MD, because, when Dr. Francis came to the hospital of the Rockefeller Insti- tute for Medical Research in the summer of 1928 to do some research under the supervi- sion of Oswald T. Avery, MD, he became interested in bacterial transformation. He tried to obtain the transformation of a type of pneumococcus in vitro but unfortunately did not succeed. The time was not yet ripe, for later when Alloway came to work with Dr. Francis, he took on the problem and did succeed as did Dawson and Sia later on. Dr. Francis never returned to research on bacte- rial transformation, but during the time he was at the Rockefeller Institute he de- veloped a close and lasting friendship with Dr. Avery, the main architect of the DNA discovery. They shared an active interest in respiratory disease, Dr. Francis mainly with influenza, and Dr. Avery, with pneumonia, and they enjoyed getting together for late afternoon social hours during which science was often discussed. In this one respect it is unfortunate that Dr. Francis left the insti- tute to go to New York University in 1938 and then on to the University of Michigan in 1941, for I know he would have greatly enjoyed a continuation of his close associa- tion and almost daily conversations with Dr. Avery during the exciting years immedi- ately preceding the publication about trans- forming DNA by Avery, MacLeod, and McCarty in 1944. Dr. Francis had a great capacity for friendship.  Submitted for publication Dec 29, 1969; accepted Jan 5, 1970.  From the Virus Laboratory and Department of Molecular Biology, University of California, Berke- ley.  Reprint requests to Virus Laboratory, University of California, Berkeley, Calif 94720 (Dr. Stanley).  Arch Environ Health—Vol 21, Sept 1970 THE “UNDISCOVERED”  The Discovery  The first of a class of substances to be called “nucleic acids” by Altman in 1889 was isolated by Miescher in 1869. As so often happens with a discovery, Miescher was somewhat uncertain about the nature of the nonprotein, phosphorus-containing mate- rial he had obtained from pus cells. He called it a “nyclein’ and described its prop- erties in a paper he submitted to Hoppe- Seyler who was SO perplexed he held up publication while he and his students re- peated and extended Miescher’s experl- ments. Hoppe-Seyler became satisfied that Miescher’s results were correct, for in his journal in 1871 appeared not only Miescher’s paper but three from Hoppe-Seyler’s labora- tory plus an additional one from Miescher, all on this new class of substances. Miescher extended his work to include fish sperm, and, in 1879, Kossel, working in Hoppe-Sey- ler’s laboratory, entered the field and gradu- ally gained control. Kossel dominated the field until the entrance of Levene about the turn of the century.  The structural organic chemistry of the nucleic acids was worked out in beautiful detail by Levene and co-workers during the next 35 years. Levene considered DNA to be a tetranucleotide containing repeating units of the bases adenine, guanine, cytosine, and thymine. In the book N ucleic Acids by Lev- ene and Bass published in 1931 they state on p 262:  On the basis of the data discussed in previous chapters, namely, on the basis of the isolation of four bases in equimolecular proportions on complete hydrolysis of ribodesose nucleic acid and on the basis of the isolation of four nucleo- sides on partial hydrolysis, it is warranted to attribute to the substance 4 tetranucleatide structure.  On pp 276 to 277, it is stated:  Thus the tetranucleotide structure of yeast nucleic acid has been re-established by the analytical method. It will be seen later that the formulation of Levene has been confirmed by the physicochemica! method.  Even at that time Levene was uncertain about the molecular weight, for on P 989 the following occurs:  On the other hand, it must be borne in mind that the true molecular weight of nucleic acids ig as yet not known. The tetranucleotide theory is the minimum molecular weight and the nu-  Arch Environ Healt  pISCOVERY—STANLEY 257  cleic acid may as well be a multiple of it.  Probably because of the general accep- tance of the tetranucleotide theory no at- tempt seems to have been made to relate biological activity to the nucleic acids. The early piologists certainly connected the nu- cleus of the cell to piological activity, but because of the diversity displayed by Pro tein, tended to believe that protein played the central role in the transmission of genet- ic properties. One of the first clear state- ments was that of the great American cytol- ogist Edmund B. Wilson who in 1895 stated, Chromatin is known to be closely similar to, if not identical with, a substance known as nu- clein, which analysis shows to be a tolerably definite chemical compound composed of nu- cleic acid and albumin. And thus we reach the remarkable conclusion that inheritance may, perhaps, be effected by the physical transmis- sion of a particular chemical compound from parent to offspring.  The efiect of the Levene school of thought is clearly evident for in 1925 Wilson wrote in his book The Cell the following:  Apart from the characteristic differences be- tween animals and plants, the nucleic acids of the nucleus are on the whole remarlably uniform, showing with present methods of anal- ysis no differences in any degree commensurate with those from the various species of cells from which they are derived. In this respect they show a remarkable contrast to the pro- teins, which, whether simple OT compound, seem to be of inexnaustible variety. It has been suggested accordingly, that the differences be- tween different “Chromatins” depend upon their basic ot protein components and not upon their nucleic acids.  There is little wonder that scientists in the 1930’s considered proteins to be impor- tant in genetic transmission and showed lit- tle interest in the nucleic acids, apart, of course, from their structural organic chemis- try. A further indication of this lack of interest is the fact that in their 900-page volume Practical Physiological Chemistry published in 1931, Hawk and Bergheim de- voted only 12 pages to nucleic acids and nucleoproteins. The discussion indicates that the nucleic acids could be divided into two classes—animal and plant—with the former containing deoxy pentose rather than pentose and thymine rather than uracil. Levene was credited with the structural work supporting the tetranucleotide theory. I am convinced  p—Vol 21. Sept 1970 258 THE “UNDISCOVERED”  that the general acceptance of this theory was largely responsible for the reluctance on the part of scientists to consider that nucleic acids might carry biological and especially genetic information. This was, therefore, the scientific atmosphere surrounding the early work on the transforming principle of the pneumococcus. When Avery, who had been working on and off on transformation since 1928, finally directed the work toward the purification and isolation of the agent re- sponsible for bacterial transformation, he certainly did not expect to end up with nucleic acid. As the experimental work con- ducted with his two younger associates, MacLeod and McCarty, proceeded it be- came obvious that the transforming princi- ple was in fact DNA. Finally a paper was submitted on Nov 1, 1943, and published Feb 1, 1944, in the Journal of Experimental Medicine entitled “Studies on the Chemical Nature of the Substance Inducing Transfor- mation of Pneumococcal Types: Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated From Pneumococcus Type III.”  One has only to read this paper in detail to recognize the turmoil and mental anguish these investigators must have gone through as the work proceeded. Although mentioned in the title, nucleic acid is not mentioned in the first eight pages of the paper. On p 9, a table showing the results of elementary chemical analysis is presented with the statement that while there was close agree- ment with the figures for nucleic acid, the analytical figures by themselves did not es- tablish that the isolated substance was a pure entity. Obviously, although the results indicated otherwise, they were still thinking about the possibility of a trace of highly biologically active protein. They tested their preparations extensively with known en- zymes and tissue extracts since certain of these were found to destroy the biological activity. They found an enzyme that would destroy the activity. This was the enzyme reported from Greenstein’s laboratory in 1940 and called “deoxyribodepolymerase” by Greenstein in 1943.  Finally after extensive enzymatic and ser- ological analysis and some physicochemical and quantitative studies, the tentative con- clusion was reached on the 14th page of the  DISCOVERY—STANLEY  paper that:  The fact that transforrning activity is destroyed only by those preparations containing depoly- merase for desoxyribonucleic acid and the fur- ther fact that in both instances the enzymes concerned are inactivated at the same tempera- ture and inhibited by fluoride provide addi- tional evidence for the belief that the active principle is a nucleic acid of the desoxyribose tvpe....  Thus in both the electrical and centrifugal  fields, the behavior of the purified substance is consistent with the concept that biological ac- tivity is a property of the highly polymerized nucleic acid. Ultraviolet absorption curves showed maxima in the region of 2600 A and minima in the region of 2350 A. These findings are characteristic of nucleic acids. Yet in the immediately following para- graphs the terminology that was used was “transforming principle” or ‘‘active materi- al,” and not “nucleic acid.” The paper con- tains an excellent discussion and the sum- mary is as follows:  1. From Type III pneumococci a biologically active fraction has been isolated in highly purified form which in exceedingly minute amounts is capable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants of Pneumococcus Type II into fully encapsulated cells of the same specific type as that of the heat-killed microorganisms from which the inducing ma- terial was recovered.  2. Methods for the isolation and purification of the active transforming material are de- scribed.  3. Th> data obtained by chemical, enzymatic, and serological analyses together with the re- sults of preliminary studies by electrophoresis, ultracentrifugation, and ultraviolet spectrosco- py indicate that, within the limits of the methods, the active fraction contains no demon- strable protein, unbound lipid, or serologically reactive polysaccharide and consists principally, if not solely, of a highly polymerized, viscous form of desoxyribonucleic acid.  4. Evidence is presented that the chemically induced alterations in cellular structure and function are predictable, type-specific, and transmissible in series. The various hypotheses that have been advanced concerning the nature of these changes are reviewed.  The final conclusion in the paper is sim- ply and clearly stated but in a manner characteristic of a very conservative scientist as follows: “The evidence presented sup- ports the belief that a nucleic acid of the  Arch Environ Health—Vol 21, Sept 1970 THE “UNDISCOVERED” DISCOVERY—STANLEY  desoxyribose type is the fundamental unit of the transforming principle of Pneumococcus Type ITI.”  Clearly the evidence presented was sub- stantial and the investigators recognized that they had made a significant discovery. Why, therefore, was this great discovery not immediately recognized by the scientific world and why did it not influence the direction of biomedical research? Why did not the discovery that nucleic acid could carry and transmit genetic information re- ceive the recognition that it so richly de- served, for this was a major discovery, one contrary to general thought, and hence one that should have immediately affected scien- tific thinking in several fields. I am con- vinced that an unfortunate combination of circumstances was responsible. Perhaps of major importance was the fact that the dis- covery was quite contrary to the dominant thinking of many years and, hence, required not only a vigorous presentation but also a vigorous and continuing promotion for ac- ceptance. This was not forthcoming. In fact, although the authors made the correct con- clusion based on the scientific evidence, they were modest and somewhat cautious in their presentation. In their paper, after the first statement of their “belief that the active principle is a nucleic acid,” they returned in the very next and succeeding paragraphs to terms such as “active material” and “‘trans- forming substance.” In the discussion they stated:  So far as the writers are aware, however, a nucleic acid of the desoxyribose type has not heretofore been recovered from pneumococci nor has specific transformation been experi- mentally induced in vitro by a chemically de- fined substance,  thus expressing surprise not only in finding DNA in pneumococci but in the biological ac- tivity. Later on they said:  If it is ultimately proved beyond a reasonable doubt that the transforming activity of the ma- terial described is actually an inherent property of the nucleic acid, one must still account on a chemical basis for the biological specificity of its action.  Similar hedges about the possibility of im- purities and about the possibility of confirma- tion of the results are in the summary. Thus it would appear that they felt that they had not proved the point “beyond a reasonable  259  doubt,” and thus did not tend to imbue the reader with confidence in their results. Yet today most scientists would regard their evi- dence as impressive.  Another factor was Dr. Avery himself. At the time of the writing of the paper he was 66 years of age and planning his retirement. He was a warm and wonderful man of great intelligence but nevertheless a cautious and timid man. He had spent most of his scien- tific effort on the pneumococcus and, although he had made many important contributions, his dream of conquering the dreaded acute lobar pneumonia by means of specific sera had just been punctured by the discovery and development of the sulfa drugs. He had encouraged work on the transforming prin- ciple over many years. Perhaps the best pic- ture of Dr. Avery at this time can be gained from a letter he wrote to his brother, Roy C. Avery, on May 13, 1943. This letter should be published in full sometime but some excerpts may suffice here. After relating the ups and downs of the early work on the transforming principle, Avery comments, “Some job—full of heartaches and heart- breaks. But at last, perhaps we have it.” After describing some of the properties of the nucleic acid Dr. Avery concluded, “This does not leave much room for impurities— but the evidence is not great enough yet.” Later on he stated, “If we are right—of course that’s not yet proven.” Near the end of the long letter he wrote:  Sounds like a virus—may be a gene. But with the mechanisms I am not now concerned —one step at a time and the first step is, what is the chemical nature of the transforming principle? Someone else can work out the rest. Of course, the problem breathes with implica- tions. It touches the biochemistry of thymus type of nucleic acids which are known to con- tribute the major part of chromosomes but have been thought to be alike regardless of major species. It touches genetics, enzyme chemistry, cell metabolism, and carbohydrate synthesis, etc. But today it takes a lot of well documented evidence to convince anyone that the sodium salt of desoxyribose nucleic acid, protein free, could possibly be endowed with such biologically active specific properties and this evidence we are now trying to get. It’s lots of fun to blow bubbles but it's wiser to prick them yourself before someone else tries to. So there’s the story Roy—right or wrong its been good fun and lots of work. This supplemented  Arch Environ Health—Vol 21, Sept 1970 260 THE “UNDISCOVERED”  by war work and general supervision of other important problems in the lab has kept me busy, as you can well understand. Talk it over with Goodpasture but don’t shout it around— until we’re quite sure or at least as sure as present methods permit. It’s hazardous to go off half-cocked and embarrassing to have to retract later. I’m so tired and sleepy I’m afraid I have not made this very clear—but I want you to know-—and sure you will see that I cannot well leave this problem until we’ve got convincing evidence. Then I look forward and hope we may all be together—God and the war permitting and live out our days in peace.  It would appear that by the time the paper was submitted for publication the fol- lowing November, Dr. Avery, despite the terminology used in the paper, had con- vinced himself that the work and the conclu- sion were correct and he was ready to retire, leave the rest to others and to live in peace. The war was still on, making especial de- mands of the younger men. The interests of his two younger physician associates had changed, for MacLeod had left to accept a position as professor at New York Universi- ty School of Medicine in 1941 and McCarty had accepted additional obligations by join- ing the Rockefeller US Naval Medical Re- search Unit in 1942. Although two addition- al papers on the transforming substance were published in 1946 by McCarty and Avery, one on the effect of deoxyribonu- clease and the other on an improved method for isolation, Avery had no desire to argue the merits of the discovery before the scien- tific world at that time, a world that was fully preoccupied with the war. I am sure that he felt the pride of accomplishment within himself and that sooner or later the world would recognize that accomplishment. But the fact remains that no one undertook the task of describing the discovery and arguing its merits and significance before scientific audiences across the nation; hence, several years passed before there was gen- eral acceptance.  The Rockefeller Institute for Medical Re- search, as an organization, seems to have given scant official attention to the discov- ery. In the 1944-1946 “Descriptive Pam- phlet” of the institute there is the state- ment:  More recently it has been found that desoxy- ribonucleic acid is intimately associated with  DISCOVERY—STANLEY  the structural organization of pneumococci and, indeed, that certain nucleic acid polymers of the desoxyribose type possess the capacity, un- der appropriate conditions, to induce transfor- mation of the various types of pneumococci. Thus, the nature of the capsular polysaccharide appears to be dependent upon a metabolic system which at some point is specifically ori- ented by desoxyribonucleic acid.  The identical words appear in the 1946-1948 “Descriptive Pamphlet,” and there is no evaluation whatsoever of the significance or importance of the discovery. One would nev- er guess that one of the really great discov- eries within the history of the institute was being discussed. Hotchkiss, then at the insti- tute, did take up research on the transform- ing DNA and made important contributions as did Alexander, Leidy, and Zamenhof.  Still another factor that undoubtedly con- tributed to the overlooking of the work on the transforming principle was the series of important scientific accomplishments that took place in the early 1950’s. By this time Chargaff had demonstrated quite convinc- ingly that nucleic acid could not be a simple repeat unit of the four nucleotides by show- ing that the base composition of different DNAs differed considerably, although A al- ways equaled the T content and G the C content. Ideas with respect to the structure of the nucleic acids were beginning to change. In 1951, Lederberg and Zinder dis- covered transduction and Pauling described the a-helix for peptides and proteins. In 1952, Hershey and Chase published their famous experiment showing that the genetic information of bacterial viruses was carried in their DNA, work which shared recogni- tion by the 1969 Nobel Prize in Medicine. Needless to say this work was generally regarded as supportive of the earlier work by Avery and colleagues and did much to se- cure acceptance of DNA as a transmitter of genetic information, but even then the suspi- cion remained strong that protein might be involved. Also in 1952 Schachman, Pardee, and Stanier discovered ribosomes and showed that they contained RNA and not DNA. In 1958, Watson and Crick revolu- tionized scientific thought by their presenta- tion of the double helix for the structure of nucleic acid. And in 1956, Fraenkel-Conrat and Gierer and Schramm made the impor-  Arch Environ Health—Vol 21, Sept 1970 THE “UNDISCOVERED”  tant discovery that the biological activity of the tobacco mosaic virus nucleoprotein was carried solely by the RNA of the virus, thus proving for the first time that RNA could also carry and transmit genetic information. Truly an unbelievably rich harvest of scien- tific discovery occurred during a five-year period, and today Lederberg, Pauling, Her- shey, Watson, and Crick are Nobel Laure- ates as a result of accomplishments made during that period. With so much to occuvy their minds and so much scientific progress to digest and assimilate, it is small wonder that scientists continued to fail to give prop- er recognition to the discovery of transform- ing DNA and simply accepted it as a fact of life. Perhaps full acceptance of the role played by nucleic acids in the storage and transmission of genetic information did not come until the genetic code and the synthe- sis of DNA, RNA, and proteins was under- stood.  These, then, are the factors which I be- lieve to be responsible for the failure of the scientific world to accord proper recognition to the discovery of the DNA nature of the transforming principle of the pneumococcus. It is dificult to assess which, if any, of these factors was more important than the others. Certainly the scientific atmosphere at the time was very important. The general ac- ceptance over many years of the “tetranu- cleotide theory” as the basis for the structure of DNA, with the great organic chemist Le- vene as its proponent, certainly was a major obstacle, for it was argued vigorously and convincingly that DNA could not possibly carry biological information. The work of Brachet and Caspersson in the early 1940s, relating DNA to protein synthesis within cells, had little effect. The theory died a slow and lingering death and the person most re- sponsible for its demise, Chargaff wrote on p 368 in a book, Nucleic Acids, co-authored by Davidson, published in 1955:  The tetranucleotide theory continues, for this reason, to lead a stubborn existence. There never were any but psychological reasons for its formulation, as has been pointed out before; but even in a very recent and massive treatise there will be found the statement that thymus nucleic acid is a large chain consisting of 500 to 1,000 tetramucleotide units and that each tet- ranucleotide is formed by the combination of  DISCOVERY—STANLEY 261 four nucleotides containing adenine, cytosine, guanine, and thymine, respectively.  If the true structure of DNA had only been known and accepted, it is highly prob- able that the discovery by Dr. Avery and his colleagues and its significance would have received ready recognition. Furthermore, in- stead of a timid and unusually cautious presentation the authors might have set forth their conclusions with greater firmness and confidence and this would have fostered ready acceptance. If Dr. Avery or one of his younger associates, all of whom were trained as physicians, had been trained as a profes- sional biochemist there might have been a difference in acceptance of the discovery, as well as a high probability that a person so trained would have wanted to carry on the work. The war was undoubtedly a major factor but one which is difficult to evaluate. Many persons felt tired and worn out by the end of the war in 1945, but had Dr. Avery been ten years younger, instead of looking forward to retirement, things might have been different. Many factors played a part and with the rush of scientific discovery of the early 1950’s it is easy to understand why the discovery of transforming DNA did not receive the public recognition that it so rich- ly deserved. But of one thing we can be sure —-Dr. Avery, at the time, recognized the significance of the discovery and he was inwardly completely happy and at ease with himself. We have all come to realize the greatness of the discovery and I am sure Dr. Avery knew that this would happen, sooner or later. He did not need public acclaim for he was inwardly content because of his knowledge of the discovery and the honor accorded him by his close friends.  An Apology  In 1935, I reported the isolation of a crystallizable protein possessing the proper- ties of tobacco mosaic virus. By 1938, as a result of discussions with Bawden and Pirie in London in July 1936 and subsequent work in their laboratory and in my labora- tory, I knew this crystallizable material was a ribonucleoprotein. I also was familiar with the work that was going on in Dr. Avery’s laboratory on the transforming principle and. over River’s objections, I included a dis- cussion of it in a chapter entitled ‘“Biochem-  Arch Environ Health—Vol 21, Sept 1970 262 THE “UNDISCOVERED”  istry and Biophysics of Viruses,” written for Doerr and Hallauer’s Handbuch der Vi- rusforschung, published in 1938. In this dis- cussion, I commented: “This phenomenon is virus-like, and it is because of this and the fact that it may become important from the standpoint of the chemistry of viruses that a discussion is included here.” After describ- ing the method of preparation and biological properties of the purified material, I ended the discussion with:  No chemical tests were made on these purified preparations, hence nothing is known about the nature of the active agent. It is to be hoped that the study of this agent will be continued be- cause of its virus-like nature.  I was also interested in the RNA of tobac- co mosaic virus. In 1942, Cohen and I re- ported the isolation of this RNA with an unusually large molecular weight and we reached the conclusion “that the nucleic acid exists in thread-like molecules, the length of which is that of the intact virus molecule.” It is obvious that despite my 1938 writings, I was not impressed with the significance of the 1944 discovery by Avery, MacLeod, and McCarty or I would have pre- pared high molecular weight tobacco mosaic virus-RNA once again and tested it for virus  DISCOVERY—STANLEY  activity despite the fact that RNA was not suspected to have genetic properties. It re- mained for Fraenkel-Conrat to do this im- portant experiment in my laboratory 14 years later. I have searched my memory and have failed to find any really extenuating circumstances for my failure to recognize the full significance of the discovery of transforming DNA. Some of the factors mentioned in the body of this paper may have had some influence, and with the out- break of World War II in 1941 my labora- tory effort was converted almost overnight to the development of preventative vaccines for our armed forces and this total effort contin- ued until well past the end of the. war in September 1945. But there should have been time for me to accord proper early recogni- tion to the discovery of transforming DNA in 1944, and for my failure to do this I apologize. I am pleased to be able to record in this paper the fact that in 1947 Lasker Awards in Basic Research were presented to Drs. Avery and Francis.  This communication was developed from notes which were used in connection with a speech I pre- sented on the occasion of the dedication of the Avery Memorial Gateway at the Rockefeller Insti- tute in New York, Sept 29, 1965.  Arch Environ Health-——Vol 21, Sept 1970  Printed and Published in the United States of America", "Stanley, Wendell M. (Wendell Meredith), 1904-1971", null, "Archives of Environmental Health", "Heldref Publications", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-8wsr_xqq4_ikac", "00000000-0000-0000-1F89-5A0136F0760D", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research, April 1916", "101584575X110", null, "1916", "April 1916", "This April 1916 Director's Report not only addressed the need for additional laboratory and patient rooms at the hospital, but also detailed the work on pneumonia conducted in the hospital in the first quarter of 1916.", "Official reports, Excerpts", "Immune Sera,Diabetes Mellitus,Nephritis,Heart Diseases", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "28", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "a Rm een     ah 4  REPORT OF THE DIRFCTOR OF THE HOSPITAL OF  THE ROCKEFELLER INSTITUTE FOR NEDICAL RESEARCH  April, 1916. April, 1916. To the Board of Scientific Directors of the Rockefeller Institute. Gentlemen: -  Tne Director of the Hospital has the honor to submit the following sugzestions in regard to future development of the hospital, and report of scientific work carried on during the past quarter.  NEED FOR INCREASED LABORATORY SPACE : Since its organization, the activities of the hospital have gradually increased, both as regards the number of patients studied and the amount of work carried on by the physi- cians in the laboratories of the hospital. This increase in activity has rade manifest certain limitations in the facilities of the hospital and has inade evident that, for further increase, or even for the most aiven- tageous continuation of the work at its present extent, certain changes are advisable. The most evident requirement which has appeared during the past year has been the need for extension of the space devoted to laboratories, The work carried on in the chemical laboratories has in- creased very materially and as a result, the space originally devoted to this work has been mich overcrowded, four and five persons working in a space originally intended for two or three. The bacteriological laborator- ies have also been much overcrowded. It has been impossible to provide Dr. Allen with proper lsboratory space for carrying on his work on diabetes, and he has had to use several single rooms, widely separated.  The Director is of the opinion that in order that those who are  engaged in making clinical studies and caring for patients may also carry       on expeérinental researches relating to the jiseases being investigated, it is very important that the laboratories in which these men work shall be in as close proximity to the wards as possible. He drew attention to this matter in his first outline of the proposed organization of the hospital, and his subsequent experience has confirmed this view. Moreover, owing to the advisable limitation in the mumber of patients, it is important that t:.ese patients be studied from different standpoints, that is, that fre- quently chenical, bacteriological, or biological and physiological studies be carried out simultaneously on the sane patients. To make this possible it is important that the men working along different lines work in harmony and in cooperation. If the laboratories are widely separated, this is in- possible. For this reason it is better that the men work in somewhat crowl- ed quarters than that they carry on their work in separate buildings, or in laboratories widely separated one from another. It is to be hoped, however, that it may be possible to make some extensions in the space provided for the chemical and bacteriological laboratories. To do this, I have suggested that certain of the rooms now used for bacteriology be equipped for use as an addition to the chemical laboratory, and that temporary changes be made on the west end of the sixth floor to provide for some of the bacteriological work. It is suggested that the changes made on the sixth floor involve as slight structural changes as possible, so that if later it is decided to use this floor for patients, it can be restored to its present condition with comparatively little expense. Sketches of the changes proposed will be presented at the meeting of the Board.  It is expected that Dr. Allen's werk for the first part of next year,  at least, will consist mainly in experimental work on dogs, and it is there-       FOr ta e r a     fore sugzasted that suitable arrangements be made for him on the second floor ef the present laboratory building, which will be made vacant by the removal of the chsnical laboratory to the new building. It is hoped, also, that on  this flecr an oferating room can be provided for Dr. Cohn and also one for           other workers in the hospital, whose work ney require opsrative procedures on dogs.  ADDITIONAL ROOM FOR PATIENTS : The growth of the work on cancer will probably increase Slightly the mumber of patients cared for in the hospital. It is hoped that next year it will be possible to care for this Slight increase by moderate crowding of the space now available on the ord, 4th and 5th floors, and also possibly by using the isolation building for convalescent patients, when necessary.  It is in.portant, however, that at this time the Board consider possible nethods for future increase of tha number of beds available. The Past experience suggests that the need will best be met by providing addi- tional single rooms. In a mixed service as we have, consisting of both men and women, it has been found difficult to separate patients suffering fron. various diseases without overcrowding certain wards and leaving other wards containing comparatively few patients. Such seperation in certain cases seas very important. For instance, certain experiences we have had have indicated that it is very important not to treat Pneumonia patients in ths wards containing patients with heart disease. It has not been wise to Place diatetic patients, who ars not acutely il1, and whose mental state is of considerable importance, in wards containing acutely sick pneumonia  patients.    It has also been found advisable to keep patients on whom very accurate chemical or metabolic studies are being made in single rooms. Moreover, the demand on the part of persons of the better classes for ad- mission to the hospital has grown, and it is found that in many ways these are the best patients for study, as they are more ready to cooperate in the studies being nade. These patients also require separate rooms.  It would seem, then, that the first extension of the hospital facil- ities should proviie for aiditional single rooms. This can probably best be jonas ty using the seconi floor, now occupied by nurses, for this purpose. In ths original planning and construction of the hospital this floor was built with this possibility in mind, and this floor could be used for this Purpose with compsrativaly slight change.  NURSES HOME : Such a use of this floor would, of cours3, necessitate nakins provision fcr the nurses alsewhsre. The marses quarters are already overcrowiei. In very accurate work ani complex studies, such as are méde in the hospital, the numbsr of nurses requirei is large, mich larger than nay be thought necessary by those who have only had experience in the ordinary general hospital. The mrses are neaied, not only in caring for the immediate neais of the patients, but they are employed very largely in assisting in naking observations and in assisting in tha technical pro- cedures «arried out. It seems, therefore, that sooner or later a nurses home should be provided. When this is done, provision should probably be made in this building for providing quarters for the doctors, as wall.  Vhen this is ions, the second floor should ba available for patients.  REORGANIZATION OF THE X-RAY LABORATORY : It has been well recogsized by all those familiar with the work in the hospital that tho  X-ray work has not bean carried out in a manner commensurate with the other       work done, The squigpment ard service have not been even so good as that of i the average general hospital. It is hoped that provision can now be nade  for greatly improving this service. The work on cancer which it is pro-     Fosed to do makes such a development very important. Moreover, it is hoped to make certain studies concerning the changes in the size and shape of the heart that will give information as to output of theoretical and practical importance.  To provide space it is proposed to enclose the open space on the eighth floor, snd to use all the space on this floor not occupied by the operating suite. Plans of the proposed changes will be presented. Addi- tional equipment will ba required, the estimated cost of which will also bo presented. It is also hoped that one man can be obtained who will give all his tima to this work. With tha excaption of this man, the changes in tha hospital arrangements which will be suggested involve very little change in thea size of the staff. The chanzes will make such future increase possibls, but are not for the purpose of providing for any immediate changes. They will relieve the prosent crowding, and provide proper facilities for  carrying on the work as at prossnt planned. Report of the Scientific Tork carried on during the Past Quarter.  ACUTE TLOBAR PNEUMONIA :; During the present winter (up to April let) 70 cases of acute lobar pneumonia have been treated in ths hospital. Nf these, 14 dies, a mortality rate of 20%, which is a distinct lowering of total mortality in a very bad pneumonia year. The cases dua to pheumococcus “zype I were treated with serum, unless they were very slightly  ill or were admittai very late in the disease, when serum treatment did not    seem indicated. Of the cases due to type II pneumococci, a few were treated with the extract of serum precipitate. Many of the other cases were treated with optochkin, Our purpose is to treat cases of type II with a combination : cf optechin and serum, or optochin and extract of serum precipitate. It has oeen inportant, however, to study first the action of each of these agents alone. The following table zives a sumary of the cases and the results  as regards nortality.  Cases of Acute Lobar Pneumonia 1915-16 to April lst.        Treated Cases. Untreated Cases  Tyre No.Cases Mortal- % No. Vortal- No. Mortal- ity ity ity I 22 1 4.5 17 serum 1 5 0 IT 23 5 21.7 8 oReesce” 8 9 3  3 optochin+ 0 extract III 12 6 50, 6 optochin 3 6 3 Iv 13 _2 15.4 ' 2 optochin 0 ll 2 70 14 20 %  The danger of drawing conclusions from mortality statistics in pneumonia must constantly be borne in mind, but as they stend the results seem interesting and instructive,  The results of serum treatment in the type I cases add further evidence for the value ef the inmune serum in this type of case. Our conclusions as to the value of the serum, however, are based on the study of ‘the cases, rather than on the mortality statistics.  The statistics give little infermation as te the value of optochin  in the treatment of prieumonia. However, the studies of the cases have given     us valuable infornation in regard to the use of the drug. Seventeen cases       have been treated with optochin, 9 of type II, 6 of type III, and 2 of type IV. Of these severteen cases, twelve have been very carefully studied. These studies have yielded important results as rezards the size and spacing of the doses. Fron. these stuiies it seens that careful attention should be given tc the size of the body in deciding the size of dose required. It is now troight that at least .024 gm. per Kg. per 24 hours should be given. In the aurlier studies it was thought that in certain cases the drug was not con pletely abscrbed, but it seems now that these apparent jiifferences have been due to insufficient and improper dosage. It is epparently best to give a gcod sizei jose at tha beginning and to follow this by snall doses fre- quently rapeated. These results have been obtainei by naking studies of the bactvricidal power of the blood very frequently during the period of admin- istration. By this biological test tha amount of optochin in the blood at any given tins may b> estimated. \"ith the axcaption of tha casa of am- blyopia which was observed earlier in the ysar, no further bad effects of the drug have been seen. In the case mentioned, recovery was complete.  It is too seen to spesk of clinical results in the cases treated with optochin, but th: inpressions obtained hava been vary decidedly favorable. The clinical results obtainei, together with the experimentel evidence, make it important that the study of this drug be contimed further. It is hoped that ty combining the drug with serum much better results cen ba obtained in cases of type II ani type III than with either serum or optochin alone.  Stugies have been mais by Dr. Moora conesrning the scquirement by bacteria of \" fastness \" to optochin. This is undoubtedly an important question in relation to treatmont. Th: vary interssting observation has bean  made that whan pneumococci are grown in serum to which optochin has been added,             either ty administration during life or addition in vitro, the pneumococci acquire very large capsules, much larger than those seer when the bacteria are grown in serun alone. By repeated transfers in optocnin-containing nedium, the bacteria are able to grow in very much higher concentrstion  of the drug than was originally the csse. After only a few trensfers slight powers of resistance become evident, but several weeks or months  zre reguired before the changes beccne of extreme grade. It is of interest tust when the organisms become very resistant to optochin, they zlso becore bile insoluble.  Tie supply of optochin in this country is very limited, but per- nission has now been obtained fron. the Germen goverment for a sm.a1] amount te be export2d and also from the mnglish government to allow it to caus throu;h, so it is hoped that we shall now soon have enough for our purposes.  Dr. Chickerinz is contiming his study of dissolved precipitate. In his work with Dr. Gay last summer observations were made which suggested that such precipitates acted not only by supplying immune bodies, but also stimulated leucocytosis and caused mobilization of tha normal antibodies. The present studiss ars being made tc determine whether or not the pneu- ‘mococcus precipitates thave any such action. The work has not yet progressed far snough to permit conclusions to be drawn.  Pnaunococcus micosus - Type III. As previously mentioned, pneu- mococci of this type have been very frequently found in normal mouths. On  he other hand, pneumocecci of types I and II are never found in normal mouths except in the case of contacts. Since the pneumonia caused by pneunecoccus micosus is relatively infrequent and is of very great severity, these results with pneumecoccus micosus are very surprising and make aifti~  cult the interpretation which has been placed on the findings of type I and       + om mn seeanneege       II as relates to epidemiology. It is conceivable, however, that the pneu- mococci of type III found in normal mouths differ in some way from the pneumococci of type III found in disease,  To test whether immunological differences could be found, Dr. Avery has collected about 70 strains of this type of organism from normal mouths, and from patients with pneunonia. He has studied with much care, but no erltura. or other differences could be detected. Rabbits were inmunized to various strains and cross reactions with the various strains studied. The results, however, were negative; no differences between the strains could be made out.  It will be recalled that in our earlier studies of this organism it was never fossible to obtsin an immune serum which would agglutinate these organisms unless the latter were first treated with acid to remove tha capsules. In our work rabbits, sheep ani goats were used in the attempt to produce an immune serum, but on sccount of the results obtained, horses were never employed. The immune serum from smaller animals had slight protective power for rats, but it had absolutely no effect when tested with mice.  The State Board of Health under Dr. Wadsworth's direction, however, proceeded to inminize a horse, and after several months inmunization sent us Some serum for testing. To our great surprise it was found that this serum had well narked aggiutinating power for pneunecoces of type III, and also soms protective power for mice, 0.2.cc. serum protecting against doses as higa as 0.001 cc of culture. The agzlutination reazttion is very short and complete in low Jtlutions, but does not occur in high dilutions. The degres  ot protective power 2oes not suggest that the serum can be of much effect in treatment, but the demonstration has been made that 2n inmune serum can be obtained from horses and it is not impossible that methods may be found for increasing its potency. This serum was tested by Dr. Avery against all of the strains which he had collected, and was found to be active against all but 3 cf them. These 3 strains were non-virulent and were from normal nouths. However, it was active against other non-virulent strains from normal mcuths, so that it has net been possible by this serum to distinguish between the parasitic and non-parasitic strains. In general, the strains from normal mouths 2re less virulent for mice than are the strains from cases with pneunonia, but this is not constant. The frequent occurrence of pneumococcus mucosus in normal mouths, therefore, is still obscure. Further studies are being made on this problen. Ths imme serum had no effect on four strains of cocci with large capsules and forming mucous, which, however, were bile insoluble, were haemolytic and did not ferment imlin, end have therefore been called strettoceccus muicosus. The serur, therafore, offers a ready ueans of distinguishing between Pnsunococcus naicosus snd Sterptococcus macosus.  Dr. Avery hes also studied the effect of growing pneumococci in dilute solutions of bile. It has been found that by prolonged growth in this medium, gradually increasing the concentration of bile, the becteria become accustomed to concentrations of bile, which with the untreated strains cause immediate solution. This property, however, is lost efter one or two transfers on non-bile containing medium.  Epidens.c.ozy. Dr. Ernest Stillman has, during the past winter, made a study of tia types of pneunococci present in the meuths of a large  munber cf uvivial parsons, contacts 2nd convalescents. This material, in    ary a  addition te thtt already ottained by Dr. Pochez and Avery, gives considerable information in regard to epidemiology, though there are still many obscure Guestions. Dr. Stillman has studied 270 specimens of sputun. from 246 normal nouths. In 152, or about 50% of the instences, pneumococci were present. There has been no difference found in the percentage of persons harboring pneunococcus és studied in the different mouths. This study, however, has been entirely carried out during the so-called pneumonia nouths. Cont inua- tion of the study through the summer may show differences. In 2 instances the pneumococci found were of type I, 4 type II, 14 atypical II's (a,b,or x), 23 type III and @3 type IV. In all ceses but one in which type I or II were found, the persons harboring then. vere closely 2zssociated with persons suffering from tha disesse. The epidemiological significance of the carriers of atypical tyre II and of type III is more obscure. It seens thst in this respect the organisms more closely resemble the type IV pneumococci. The extrer.s virulence of the orgsnismsa of type III, however, makes this difficult to understand. In this connection a surprising obser- vaticn has been made. Pnewmococci of type III very quickly diseppear from the mouth end sputum after an attack of pneumonia due to this organism. On the othar hand, they may persist for months and even years in normal mouths.  It is hoped and expected that other interesting and valuable informz- tion will ba obtained fron. this statistical data after the work has been complete 2.  Production of Innune Serum. The two most important practical points to be solved in the study of immune serum are improvements in the methods of producing serum se as to produce a more active serum end, second, methods of  determining proper dosage.    A tes eat  Considerable work has already been done on the first of these prob- lems. Last year the study of inmunization of rabbits showed that better results could be obtained by the use of small doses of dead organisms, freqcently repeated, than could be obtained by living cultures. This nethod nas now been epplied in two horses, one being immunized to type I >neumoceccus, one to type II. In both cases, after immunization over a period of 6 to 8 weeks by the method mentioned, sera have been obtained which are as zctive both as regards agglutination and protective power as sny serum. we have previously had, using other methods. Attempts ere now baing male to drive the immunity higher in these horses by enploying living cultures, accoriing to the method used by Dr. Flemer and Dr. AILoss. It is hoped that in this way the effectiveness of the serum can be increased, but in any case a mathod for aconomizing time and horses has been secured.  Dr. Avery has also conducted some experinents to study the haemo- lytic sction of pneumococci in the hope of using this reaction as a delicate test for immune bodies. The experiments bearing on this problem are not yet far enough advanced to report, but he has brought confimuation of the con- clusions drawn from our previous studies, that the haemolytic action is due to substances contained in the bacterial cells, and set free on their solution. Dr. Chesnay's studies enabled him te test the haemolytic effect eof cultures at various stages of growth. The cultures began to have haenolytic power only at a period when Dr. Chesney was able to show that dissolution of part of the organisms in the culture wes occurring. This haenolytic power persists for four or five days, then gredually dissppears and after eight days is entirely absent.  Dr. Chesney has contimed his studies concerning rate of growth  of pneumococci. By means of observetions carried out on the supernatant    3 . Pte EES  fluids ootained by centrifuging portions of a culture at varying intervals during the period ef maximum rate of growth, it has been demonstrated that toward the eni of the period of rapid growth substances or factors are present which bring about the death of a large proportion of the pneumococci, which substances remain in the supernatant fluid after centrifugalization. It has further been shown that the process of destruction or killing of the yneumococci follows or is identical in its rate with that of a so-called honomolecular reaction of physical-chemistry, and in this respect the effect of the fluid constituents of the medis upon the bacteria is analogous to the effect ( observed by other workers ) of bactericidal agents such as phenol end bichloride of mercury upon anthrax spores and upon cultures of bacillus raratyphosus. Furthermore, some evidence has bean eccumul2ted to show that these bactericidal substances in the culture medium disappear in part when the culture is ellowed to stand in the incubator for severe] days, for e filtrate obtained fram a culture at the end of 24 hours incubation shows decidedly more bactericidal activity for sctively growing pneumococci than a filtrate from the same culture 48 hours old, and at the end of 6 days incu- bation, when there are no longer any living pneurecocci present in the culture, the filtrate frem thet culture shows decidedly less bactericidal action for actively growing pneumococci introduced into it than did either the 24 hour filtrate or the 48 hour filtrate.  | It has been possible te cause pnewmocecci to grow less rapidly by exposing them to the action of filtrats or supernatant fluid obtained from 24 hour broth cultures of the same strain, if, after exposure to such an envirorment at ice-bex temperaturss, th pnewuiecccci be renoved and rainocu~ lated inte unused broth. Attempts have been made te fellew the behavior of  pneumococci from actively growing cultures (during the period of maximum    rate or growth) when injected into rabbits intravenously, and to contrast, under these conditions, the behavior of these actively growing pneunococci with the behavior of pneumococci which would show a definite lag in the test tube. The study is not complete, but the preliminary experiments sugiest that ferhaps there may be a differenee in the behavior of \"young\" and \"old\" cultures of the sana strzin of pneunococcus, when they ere injected intreven- ously into rabbits, the individuals from a culture during the period of nheximun rate of growth tending to increase in mmber in the blood stream eof the rzbbit somewhat more rapidly during the period immediztely follow- ing injection than io the individuals from a culture which is no longer growing rapidly. It is proposed to carry out this study at greater length. Dr. Dechez has contimied his studies relating to the enzymotic activity of pneumococci. Ina previous report it was shown that antipneumococcus serum possess the capacity to inhibit the growth of pneu- mococcus in vitro. After this phenomenon had been definitely confirmed, an explanation of its mechanism was sought. In the course of the investi- gation it developed that antipneumococcus serum has the capacity to inhibit more or less completely the peptolytic and glycolytic functions of the Pneumococcus. From this it has been assumed that inhibition of growth is in part at least dependent upon the antienzymotic properties of immune serum. This quality of inhibition of metabolic function is possessed in varying degree by the sera of normal animals and may have some relationship to their natural resistance, Human sera during lobar pneumonia exhibit the capacity of inhibition, and the degree éf inhibition reaches its maximum grade during the period of recovery from the disease. It is thought that for the animal tody te rid itself of infection the growth of the infecting  microorganism mist rirss be errested, and that only after this has occurred    do the more specific substances have an opportunity to exert their full effect,  An attempt has been made by use of the nethods previously described to throw some light upon the phenomenon of parasitism. It is obvious that, in order tor a microorganism to develop a parasitic phase, it must be able to grow in the 2nimal body, which implies the development of a nechanism for resistance to the antigrowth influences of animal sera. A study of the Pneumococcus shows that the metabolic function s of organisms recently isolated from the human body are nore resistant te the inhibiting factors of both normal and inmune sera than are those of organisms which have led & saprophytic existence for considerable periods of time .  A study has also been nade of the nature of the antienzynotic sub- stances of the inmune sera. It has been possible to obtain specific inmuns bedies ina fraction of the serum which does not exhibit antienzymotic activ- ity, but no light has been shed, so far, upon the actual nature of the sub- stances themselves, or upon their source. They are thought to be non-specific in character, and their efficiency is helieved to be markedly enhanced by the presence of specific inmune bodies. Up to the present time, work has been carried on entirely with living organisms, a difficult procedure .in work ef this kind because of the low virility on artificial cultivation of Preumecocci recently removed: from a parasitic state. For example, it takes seme weeks for a strain of pneumococeus recently isolated from the human body to acquire a capacity of uniform growth on artificial media. A consid~ erable simplification, hewever, has recently been made in the fact that a pepteciybtic fesmant ass oeen prepared which is active in the absence of the livin; zsll. hit wethod should render the interpretation of compsarstive  er res waooveee ny em, wi tosh | SRTOLVAMeATS Wash Wars reujiavie.    DIABETES, Dr. Allen : The principal effort is now being directed toward publication of the dats st hand. The work still in progress centers around the acidosis problem. All diabetic patients are now cared for by Dr. Fitz. Practically no patients ere admitted except those with marked acidosis,ani Dr. Fitz is studying especially the concentration of acetone bedies in the blood and urine. There are two current theories of diabetic intoxication; one, that it is a pure acidosis; the other, thst it is a specific poisoning with acetone bodies. Acidosis is being measured by the c2rbon dioxide of the blood and alveolar air, and by the total acidity of the urine. The concentration of acetone bodies is being determined by a nodification of Marriott's micro-rathod. Obviously the typical patient approaching coma shows high acidosis with high concentration of acetone bodies, but in special cases it may perhaps be possible to separate the two factors and learn whether one or both may be chiefly responsible for the entire con-ition or for individual symptoms, or whether there may possibly be another factor, viz., intoxication by yet unknown intermediary products, especially these arising during fat metabolism. As is known, the treatment employed in this hospital aims especially to prevent this intoxication and to remove it when present by means of fasting and suitable diet, rather than to attempt to neutralize it. But in patients bordering on coma, the question of alkali therapy inevitably presents itself, and evidence is accumulating that intelligent use of sodium bicarbonate is beneficial at a certain stage in some cases. The ‘oses used. ere, however, reletively small, and the alkali is exployed as a temporary adjuvant, and not as the principal treatment.  It is believed that the reproduction ef human diabetes in experimental  animals, and the experimental foundation of the fasting treatment, are now       &  fairly complete, with the exception of the feature of acidosis. 4 few animals have been caused to develop what appears to be the same fatal intox- icaticn found in human diabetes. Hitherto no satisfactory reproduction  of diabetic acidosis in animals has been achieved, and the present study  is therefore believed to ba important, to complete the experimentel counter- Fart cf the clinical disease, and also to afford a basis for a more funda- rental study of the condition than is possible in human patients.  Five patients of this Hospital have been studied in the Russel Sage Calorimeter, under Dr, Eugene Du Bois and staff. From the sane standpoint, a few diabetics from other hospitals have also been studied by Dr. Da Bois. A paper (Allen & Du Bois) now in Fress embodied the results with sll the Rockefeller Hospital patients except one. The results are essentially as follows: (1) The existence of the Lusk dextrose-nitrogen ratio of 3.65 : 1 in certain cases is confirmei, but this is not necessarily a \"fatal\" ratio and such patients way recover considerable tolerance. (2) The respiratory quotient under these circumstences is about 0.69, All the severe cases showed very low quotients, indicating that they were actually of greater severity than most cases called \"severe\" in the literature. (3) The metabol- ist. of severe diabetics is increased when compared on an accurate basis with that of similarly emaciated non-diabetics, or when the same patient with active diabetes is compared with himself when free fron active symptoms. These results sre believed to be important in this disputed field. (4) The metabolism is zreatly reduced by the fasting treatment. One patient", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ebnm_8y85.nk55", "00000000-0000-0000-60BB-8753E56A2E91", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research, June 10, 1916", "101584575X111", null, "1916", "10 June 1916", "Several pages of this June 1916 Director's Report addressed the research on pneumonia conducted by Avery and others at the hospital during the second quarter of 1916.", "Official reports, Excerpts", "Drug Evaluation,Pneumococcal Infections,Diabetes Mellitus,Nephritis", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "8", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "he ee  EPORT OF TH  HOSPITAL  DIRECTOR OF THE     e 5 a fx Ww fx a — < oO “4 A & a fe fs) 5 re e 7 a “ & = fa bi 4] eo oO [oa fa oi e&           Juno 10,1916 Since the last report the work in tho hospital has stod chiefly in a continuation and complotion of work statod in my last roport to . Drogrosa, PNEUMONTAS The study of Dr Stillman in regard to opidemiology, some of the .ts of which woro given in our last report, is now about ready for publication, In (1on to the question of opidomiology an interosting study has rocently bcon mado so-called house opidomic at Nowburgh, N.Y. which was brought to our attcntion by -tate Board ef Health. Dr Chickcring wont to Ncwburgh and obtained cultures ffom ‘in of the paticnts and contacts, The family consisted of tho father, mother and childron, enc a young baby. Tho mother was takon ill first and diod. Following _ four children were takon down in succession. At tho timo of Dr Chickoring's visit vf thesc children woro still 111 and two were convalescent. Cultures wero mado from s four childron, from the fathor and {rem the woman caring for them, From tho fathor two sick children, ono of tho cenvalosconts and tho nurso, pneumococci of Typo I cultivted, Tho culturos from tho othcr convaloscent child woro negative. These lings scom vory significant and suggcet strongly the possibility of contagion in umonia,  The study of Drs. Moore and Chickcring in regard to optochin ie also almost ady te be published, This investigation has beon important in determindng tho propor -age in order to confcr upon the blood bactoricidal pewor. So far the rosults of tho idy indicate that bactcricidal power of the bleed gives a truo indication of clinical ‘cet,  Twenty-néno cascs hav- now been studied, and among the eighteen receiving one g6 @ose followed by frequently repeated emall ones only two have died, and one of 30 received a dese which is now know to be too emall, taking into account the patient's y weight. The ether patient was fairly well treated according to present standards . the organies ebtained at death preved te be relatively \"fast\", If the drug does  prove te be efficacious it seems that it will prodably be because of this acquired    J ZZ ee whi hae ald Reta eat ce Abn ee aoe aden beam oe ee       % \" fastness. By the present method it seems that in all cases the blood can be made bactericidal for pneumococci, A very recent case has been very instructive, The r fatient was an old man and there was difficulty in obtaining sputum. Fram the  saliva a Type IV pneumococcus vas cultivated. The patient was therefore given  optechin and the dosage was correct according to our present conception. The  patient, however, died. When the blood cultures were carefully studied, however,  and also cultures made directly from the lung at the time of death, it was found  that the infecting organism was not a pneumococeus but a streptococcus. The stu- 4 dies of the blood showed that it had acquired very prompt bactericidal power for ; 1 i  pneumococcus, but, as is known, optochin has no effect on streptococci.  Studies concerning proper dosage of serum; At various times during the  course of our work efforts have been made to devise methods which would enable us it     to determine the proper size and spacing of the doses of serum administered, Here- bo F           tofore estimations of agglutinative power have not seemed to give mich information, but lately the matter has been taken up again and very thorough studies are being made of the agglutination titre of the blood. The blood is being obtained very frequently both before and after the administration of serum, Those samples ere all kept and at the end of the disease these samples are all studied under identi- cal conditions and curves are made’ of the agglutination titree In some cases it seems that there is too long a delay between the first and second dose. By study- ing a series of the treated cases in this way it is believed that the present methods of administration can be improved and the amount of serum used probably be reduced, One of our last cases received altogether 875 cc. of serum, The patient wes an al- coholic, had very violent delirium, probably delirium tremens. We felt it inad- visable to stop the serum until we were quite sure that the pneumonic process was at anend, The patient has had severe serum sickness but is now quite well and, con~ sidering the severity of the disease, it is probbble that the serum was life saving. During the present season we have already had over 100 cases of pneumonia.  Up to the first of June from the opening of the hospital 382 cases of pneumonia have    teen treated. Among the cases in which type has been determined, 112 were of ype I, Of these, 70 have been treated with serum, and of these 6 have died, a tortality rate of less than 9%, Of the six who died, two died of complications  rot of pneumococcal origin. Three were treated late in the disease, only on the     day of death, and only one received serum treatment in a manner we now consider satisfactory, So far as serum treatment in pneumonia of Type I is concerned, therefore, we feel fairly safe in considering that it has a very definite ther- apeutic value,  Capsular substances Dr. Dechez has been making a study of this ques- tion and his report is as follows:  Fran the study of the different groups of pneumococcus it has become  apparent that there are differences in the amount of capsular development by the     organisms of the four groups, The virulence of the group as measured in human beings is directly proportional to the degree of capsule formation, Group IV shows some irregularity in the capsule formation. Group I has a well~defined capsule; Group II forms a heavy capsule and Group III, in addition to a very vol-  uminous capsule, gives rise toa large amount of mucoid material in the exudates     Which it causes in animals, The fact has been noted that Berkfeld filtrates of Pneumecoccus cultures which contain no bacterial bodies contain in solution con~ siderable amounts of bacterial eubstance which is precipitable by specific inmune Serum, One would at first think that this specifically precipitable substance arises from the disintegration or autolpsis of the bacteria themselves. This,  however, can be shown not to be the case inasmich as it is present in considerable     amounts before measurable bacterial disintegration has occurred as determined by the presence of soluble heemolysin and from the character of the curve representing the rate of growth, The substance is present in the carly hours of growth (at tho ond of four hours in cultures of type IIT) and is of undoubted bacterial origin because of its specific precipitability, and cannot be entirely due to bacterial  autolysis. The amount formed and the rate of formation corresponds exactly to          mae  “28 degree of capsular development of the different groups. Type III gives rise ‘o the largest amount and Type I to the least. In caltures of type III there is jresent at the end of nine hours and amount of soluble bacterial substance which 13 roughly comparable to the total aeczint of living bacteria in the same amount of cul tures The greatest gormation of the soluble bacterial substance seems to oc- sur during the period of active growth of the bacteria. The substance can bs do- «onstrated in both broth and serum broth cultures and also in the filtered blood of heavily infected animals, From the facts stated it seems unlikely that the soluble bacterial protein present in the carly stages of pneumococcus cultures is due to disintegration of the organisms, but more probable that it is a produco of growth, The close relationship in amount formed to the degree of capsular dovel- oiment suggests that the capsular function is a contimous one, and that capsular caterjal is contimally formed and thrown off into the medium of environment.  DUBETES ~ Dr, Allen, Dr, Stillman and Dr. Fitz : - Rapid progress is sing made in the preparation of the monograph for publication. The preparation of graphic charts of the cases treated is not far fram completion and the data of tho animal experiments are also being collected and tabblated.  About a dozen diabetic patients are kept in the hospital, under care of Dr. Fitz. In addition to observations of the relation of acetone bodies in bload and urine to the clinical condition, Dr. Fitz is now paying special attention to the renal permeability, finding marked variations from the normal to be frequent in severe diabetes. One patient lately showed entirely normal urine, while sugar, acetone bodies and fat were remarkably high in the blood, and the alkalinity of the blood was low. Sodium bicarbonate caused a sweeping out of both sugar and acetone bodies abundantly in the urine. |  Animal experiments, which still continue, show that diabetic lipemia and c‘abetic acidosis can bo regularly and positively reproduced in dogs. By varying the conditions, it is possible ts causo animals to go into cama on mixed diet and  alsc animals to go into come on festing. There are also indications that the var-    ai = BR a ae ne aa ene eae ake eae teams cea somone cn eee -  * thus <0 yvenal permeability can be reproduced in diabetic animals under controll- 1S sonditivas.  CENTER -- Dr, _stirphy, Dr, Montgomery: Nine cases have been treated end  csorved jn the hospital uv te data, shesa, with two exceptions, aro breasi can- rs which have been incumpletely operated on. Treatmont has been started shortly itor oporation, From the foint of vicw of the lymphocytic increase following  11 diffuse dosos of X-rays the results brave been satisfactory, Clinically,  -“ cases, with ono exception, have improved markedly in general health and none  --2 80 far shown recurronces. But as the oldest cases have Zone only four or  vo months after operation we can draw no conclusions as yot. Tho oxcoption to “15 general statement is a case complicated by heart and kidney disease with ex- noive metastasis to the liver and lungs. This woman died shortly after adnis- .t to the hospital. In order to determine what offect this non-penetrating  aituse dose of X-ray has on the spleen in man we have treated by this method a  tical sata gympnatic leukaemia having a very large spleen, In this case the  “S58 were repeeted at frequent intervals. The blood count on admission ves  -0,000, and after a mumber of treatments rapidly febl to about 18,000, The spleen,  cesidss a slight softening and the disappearance of tenderness and pein, has not  teen affected perceptibly in size. Treatment with pene trating doses over the  spleen is now being employed.  CARDIAC NISEASE ~ Dr. Cohn 3 Since writing the last report, no addi- tional problems have been undertaken. The experiments already reported concerning experimental cardiac hypertrophy are being continued,  The study dealing with the action of digitalis in pneumonia is about finished and was reported in brief in Washington hefore the Society for Experimental ‘edicine, It deals with the changes in the A wave and in the P«R time in the elec~ ‘rocardiogram in pneumonia, A second paper, dealing with the dardiac irregulari- ‘ies in pneumonia is being prepared; it deals with the frequency (9.7%) of auricular  sitter and fibrillation, the ireacenca and significance of the occurrence of ex-    % trasystolic irregularities, the occurrence of heart block, and certain alterations in the R wave in the curve, protably due to respiration and probably an exaggera~ tion ef usual alterations,  The collection of material for a third paper on digitalis dosage is al- most concluded. As already reported, the problem is concerned with whether a large dose given ina small period of time (calculated on the basis of physiolog- ical (cat) units) is a satisfactory method af administration, From our observa- tions, we believe that under these circumstances the digitalis is probably excreted in part, before a sufficient amomt can be absorbed by the heart miscle.  The necessarily slow anatomical studies of hearts obtained in experiments and of others obtained from human autopsies are being contimed, The latter in- clude cases of bundle branch lesions, heart block} auricular fibrillation, and one of paroxysmal tachycardia,  NEPRRITIS, Dr. McLean : The study of the mechakiam of urea retantion in nephritis is being continued, In one patient the blood urea content has been varied fram .200 to 2,500 grams per litor by changes in the nitrogen intake, with- out any symptoms, and without any significant change in the mode of excretion, the rate of excretion being parallel with the increased concentration in the blood.  Attempts to influence bloali pressure in cases of hypertension, by varying the diet, have so far yielded negative results.  The study of chlorid excretion and the mechanism of its retaation is being contimed, In one patient with nephritic edema, not due to heart failure, salt feeding produced edema and salt retention without any change in the relation of the plasma chloride to the rate of excretion, It seems probable that salt retantion and edema are rarely or never due to a primary inability of the kidneys to oxcrete salt, but to some cause which resul$s in the retention of salt by the tissues.  CHEMICAL LABORATORY - Dr. Van Slyke : For the most part, the problems discussed in the April report are being contimod along the same lines. ‘The fol-  lowing advances may, however, be noteds    ye  Cullen and McLoan are contiguing work on the fate of protein digestion oducts, utilizing our recently perfected methods for determination of plasma pro- “INS. They have as yot obtained negative results in looking for effects of pro- -in digestion on fibrin, albumin, or globulin of the plasma. There is no evi-~-  nco of the formation of any of the plasma proteins direct from absorbed digestion soducts. On the other hand, during the first two hours of digestion peptides as sll as amino acids do appoar to increase in the circulation, although after six urs only amino acids can be detectod, It appears, thorefore, that during the -rlier stagos of digestion it may be possible for some of the intermediate products + protein digestion to enter the circulation, although during the later stages  ‘uly the amino acids do so.  Tho comparison of the proteins of human with those of cows’ milk, begun / tho chemical work of Dr. Vinograd-Villchur, has been broadened by the cooperation : Dr. Auer and Dr, Avery, who are camparing the respective caseins and albumins iologically. Dr. Auer is testing the identity of the cow and human proteins by .2ans of the anaphylactic reaction, Dr. Avery by complement fixation.  For the further study of acidosis, especially in diabetes, we havo been serfecting for use in further work a new method for the determination of B-oxybutyric acid in urine, former analyses being either time-consuming or too liable to subjec~ tive influence to trust for even approximate results in the hands of ordinary &na- lysts. In the present method 2 to 10 cc. of urine are boiled for an hour in an Erlenmeyer flask under reflex condenser with a solution containing definite concen- trations of sulfuric acid, mercuric sulfate, and potassium dichromate. The oxybutyric acid is oxidezed by the dichromate to acetone, and simultaneously the acetone is precipirated as an insoluble mercuric sulfate compound, which at the ond of the hourts boiling is filtered and weighed. The results are accurate, and the :cnipulations so few and simple that tho danger of subjective error appears to be  ‘liminated.", "Cole, Rufus, 1872-1966", "Rockefeller Archive Center", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-ypjy_8rt9.chqv", "00000000-0000-0000-8D35-B5AC00693555", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Excerpt from Report of the Director of the Hospital to the Corporation of the Rockefeller Institute for Medical Research, October 1916", "101584575X112", null, "1916", "October 1916", "Several pages of this October 1916 Director's Report addressed the research on pneumonia conducted by Avery and others at the hospital since January of 1916.", "Official reports, Excerpts", "Immune Sera,Therapeutics,Diabetes Mellitus,Heart Diseases,Nephritis", "From Physician to Researcher: Early Laboratory Career and World War I, 1904-1919", "19", "pages", "Text", "English", "Reproduced with permission of the Rockefeller Archive Center.,http://www.rockarch.org/", "Copyright may apply", null, null, "DIRECTOR OF THE HOSPITAL  TO THE CORPORATION OF THE  ROCKEFEFELLER INSTITUTE FOR MEDICAL RESEARCH  -OGTOSsER,--1916 .:-  Volume IV January, 1915 - October, 1916                OF  THE DIRECTOR OF THE HOSPITAL  Octover 1916.  Changes _in the Stafé. It is a source of gratification that the number of men desiring to join the staff of the Hospital is constantly increasing and that the training of the men desiring even temporary appointments is of high level, There is now a demandpn the part of well trained men for voluntary assist- antsaips, While it has not been the policy of the Institute to admit voluntary wor sers, the training of certain men applying has been so good that in certain in- stances it has been thouchs advisable by the Board of Scientific Directors to grant such privileges, especially in those cases where men have agreed to remain the entire year, Last year such a privilege was granted to Dr Ernest Stillman, previously Assistant Resident Pathologist at the Presbyterian Hospital. He has worked on the Epidemiology of Pneumonia, During tne summer of 1915 and 1916 privilege to work in the hospital was granted to Dr J.T. Halsey, Professor of Pharmacology in Tulane Uni- versity, He has worked on subjects related to heart-block. During the coming win- ter privilege to work in the hospital has been granted to Dr Francis G. Black of Har~ yard University and Peter Bent Brigham Hospital, Boston, to work on problems con= nucted with infectious diseases a.d to Dr Samel A. Levimesof Harvard University and the Peter Bent Brigham Hospital, Boston, to work on problems in connection with heart disease, It is believed that in the future this training of certain men in the special new methods employed in the hospital may be a highly efficient means for Uringing the methods into more widespread and effective use.  During the year the Hospital has lost the  s:svices of one of the most promising of its younger men, Dr Franklin C, McLean, who                ~ 2 = has been appointed Professor of Internal Medicine and Head of Union Medical College, the new medical school to be organised by the China Medical Board in Pekin, Dr AJR, Dochez, who for two years has very capably served as Resident  Bhysician of the Hospital, was at the June meeting of the Poard of Scientific Di~  rectors made an Associate Member of the Institute. He no longer will live in the  hospital and he will be succeeded as Resident Physician by Dr H.T, Chickering, who for three years has acted as Assistant Resident Physician, Other changes in the  stoff siil te found in the Manager's Ruport,  Cugeus in the Fosrital owing to meed for increased laboratory space, and Lropos¢i changes to accorodate patients,  At the April meeting of the Board of Scientific Directors the Director of the Hospitai made certain suggestions and recommendations bearing on immediate and future hospital neods, Changes to satisfy a number of these needs have al-  ready been made, either in the way propogsad or by ways which, after further con~  sideration, have seened more advantareous,  At the meeting the Director pointed cut that since its orsanisation the activities of the hospital had increased to such an extent that there was an im- mediate need for increased laooratory space, To meet this need slight changes have been made on the Nest end of the sixth floor of the hospital which was origi-  nally intended as a ward floor. Also by building a bridge over the Isolation Build  ing the third floor of the original laboratory building has been brought into im- awadiate connection with the hospital and has permitted the use of this floor by nenvers of the hospital etaff whe, in order to carry on advantageously laboratory  work in connection with the care of patients should have their laboratories very  accessible from the wards,  Improvements in facilities for X-ray work in the hospital were much needed,  vsrecially for the stud: of the action of X-rays on cancer, There is therefore             3-  teing installed on this fleor a new X-ray department which will now be first-class in all respects. On this floor also are rooms in which experimental work on dogs ra; be carried on by Dr Allen and Dr Cohn, Huch of the work of these physicians requires experiments on dogs and it is not thought advisable that these animals be trought into the hospital proper. Another room for operating on dogs is also pro- vided which can be used by other members of the staff who only occasionally need such facilities,  Aiditional Room for Patients, The addition of the work on treatment of cancer will probably slightly increase the number of patients in the hospital, Also with the increased development of other activities in the hospital, some thought is being given to possible expension, If, later, this should be thought adviaable by the members of the Corporation and if resources be available, the experience of the hospital indicates that any future need will best be met by providing additional single room3, In a very complex mixed service such as we have, consisting of both mer and women, it has been found difficult to separate patients suffering from variogs diseases without overcrowding certain wards and leaving other wards containing few patients. Such separation is certain cases seems very important. For instance, it is very important not to treat pneumonia patients in the wards containing patients with heart diseases, It has not seemed wise to place diabetic patients, who are not acutely ill, and whose mental state is of considerable importance, in wards con- taining acutely sick pneumonia patients,  It has also been found advisable to keep patients on whom very accurate chemical or metabolic stydies are being made in single rooms, Moreover, the demand on the part of persons of the better classes for admission to the hospital has grow,  and it is found that in many ways these are the best patients for study, as they are     more ready to cooperate in the studies being made. These patients also require        separate rooms,    =~ 4 =» soar §  yey  It would seam, then, that the first extension of hospital facilities should provide for additional Single rooms, This can probably best be done by using the second fleory hor occupied by nurses, for this purpose, In the original planning end construction of the hospital this floor was built with this possibility in mind, and this floor could be used for this purpose with comparatively slight change,  Nurses Home, Such a use of this floor would, of course, necessitate waking provision for the nurses elsewhere, The nurses quarters are already over- crowded, In very accurate work and complex studies, such as are wase in the hos- pital, the number of nurses required is large, much larger than may be thought nec- cessary by those who have only had experience in the ordinary general hospital.  The nurses are needed, not only in caring for the immediate needs of the patients, but they are exployed very largely in assisting in making observations and in as~ sisting in the technical Procedures carried out. The provision for a Nurses? Home, therefore, would release from the hospital buildings a number of rooms which appar~  ently can very soon be profitably used for hospital purposes,  Report of Scientific Work  The work has largely been a contimation and extension of that done in the  Preceding year, The only new class of patients admitted has consisted of those  suffering from cancer,  Acute Lebar Pneumonia The work on this disease has gradually resolved of itself into investigating modes of specific therapy, first thal making use of mom ' of  immunity reaction, and second that/ employing drugs which have a specific action on the infecting organisms, In attempting to apply the known principles of immunity progress is theoretically possible in two ways. First, it is possible that the  patient himself may be stimulated to himself produce the immune substances, upon                -5e ete  which natural recovery in pneumonia undoubtedly depends, faster and in larger amount thar. he would do unaided, While numerous experiments have been made along these lines up to the present it has been impossible to obtain promising results, The other mechod has been to suplQy him with those immune substances which are contained in the serum of animals artificially immnized. This latter method has been the cne wnich has given the most promising results, As previously stated, this form  cf therasy is only possible if, in every case, the type of infecting organism can be rayialy ard accurately determined, This the previous work in the hospital has made possicia, The results of serum treatment have so far been successful only in the infections due to pneumococci of Type I. Here, however, further experience has con- firmed our previous conclusions, and made it quite evident that serum is of very great value in this type of infection, ;  While the extent of our statistical data is as yet not large, we now feel that we have sufficient to urge that all cases of Type I ih®ection be treated with the appropriate serum, Up to July 1,1916, 396 cases of pneumonia have been treated in this hospital, of which 106 were treated during the year 1915-16. Of these 396 cases, 113 were due to Pneumococeus Type I, 104 were due to Pneumococcus Type II,  37 were due to Pneumococcus(mucosus) Type III, 74 were due to Pneumococci of Type IV; in 48 cases the type of pneumococcus causing the infection was not determinea( occurs ing mainly before methods for determining types had been devised) and 20 cases were diagnosed as acute lobar pneumonia in which the infecting organism was Friedlander's bacillus, Influenza bacillus, streptococcus or staphylococcus, or combinations of these, It is evident that about one third of all cases were due to Pneumococcus Type I, Of these cases due to this type of pneumococcus 72 were treated with in- mune serum, Of these 72, six died, a mortality rate of a little over 8%, Of the six that died, moreover, one died on the 16th day, after recovery from the pneumonia  from pulmonary embolism, one died on the 54th day following the pseumonia from a          ~ § = on  -oneral streptococcus infection with empyema and multiple abscesses, three wore  tae at wR OS  i only on the day of death, and only one was treated for two days, the fifth  sixth, It is evident that if at least two of the fatal cases(those first men~ ‘ioned) no specific treatment could have been of service,  When it is considered that the mortality in the untreated cases due to  Type I infection, judging from the cases here before serum treatment was undertaken and also from the cases observed in other hospitals, is from 25 to 30 per cent, it is evident that the low mortality observed, 8 per cent, is decidedly encouraging, even quite convincing. The importance of this form of treatment is evident when we consider the great frequency of this form of infection, In 1915 prooably 1800 deaths in New York City were due to Acute Lobar Pneumonia of Type I alone. This ie as many as all the deaths from typhoid fever, measles, scarlet fever, whooping  cough and cerebro-spinal meningitis combined, and almost as uany as all the deaths  occurring in the present epidemic of poliomyslitis.  In the previous annual report it has been stated that it Has been impos= sible to produce an irzmne serun active against Type III pneumococci, Our experi- ments were only conducted with small animals, however, Since these were negative, no attempts were made with horses, During the past winter, however, tha.State Board of Health, under Dr Wadeworth's direction, notwithstanding our negative results, pro~ ceeded to deamnise a horee to Type III pneumococci and sent us some of the seruz, To our surprise the serum was found to have some agglutinating power and also slight protective power for mice, While this serum is, as compared with Type I serum, 6X~ . tremely weak and therefore not suited for therapeutic purposes, nevertheless its production dees have wuch theoretical importance, We have repeated this work with horses and have produced a similar serum, This serum has been tested by Dr Avery  on all the Type tIZ strains available, and the results indicate that all Type III pnevmocei are immnologically identical.          It is not yat certain why it should be pessidle to produce an ef-  “eovivs seruw2 against pneumococci of Dype I and only one of much less value Agningt Preumocecci of Tree II, and one of veryslight value against pneumo-  ! fypa Irr/ These difiercnzes aprarantly Sear sone relaticrsnip to thse 2638e6819n of Capsules by organisms of the varioua types, those of Type III avin very large capsules, those of Trpe II moderate sized capsules, and hese o: sype I smaller capsules. CEtudies are veing condusted te deternuine v8 nature of the capsular substance, and its relative antigenic activity, in t.8 hose that light may be shed on this importan® and perplexing problem.  The second method of specific therapy, namely, the use of drugs having specific action on the preuxecoccus, has also veer investigated, em- ploying the drug ethylhydrocuprein, or \"optechin\" which was prepared by Pro- fessor Morgenroth in Germany. In our last report, the results obtained in the preliminary investigations of this drug by Dr. Moore were given. During the past winter studies concerning the action of this drug on patients suffering :rom pneumonia were made. Previous experiments had showed that this drug is vactericidal for prneumoceoced in the test tube, even in dilutions as gfeat as ~ + 5,000,09C and Neore showed that this action was exerted equally on all tyees of pneumococci, It has aise been shown that when thie drug is adminis- tered to animals or patients the serum also acquires bactericidal properties.  The drug has been employed in the treatment of a censiderable nun-  ver of patients in Germany and England but the reports concerning its efficacy     ara conflicting. No standard dosage, however, has been employed, and it weuld        seem that desage is very importent, since in animals considerable amounts must fe ce given to cure, 2ut if very littie more than thess amounts be administered, i}  toisoning results, In other words, the curative dose is very close tc the           | I}. t  -8~  “oxic dose, In uan the toxie effects are mainly seen in the effects on the  eve aud gar; nartial or total clindrness has occurred in a considerable number 32 Une treated cases, While this so far has apparently not been permanent, vsinaness lasting for a -veek to ten days, as in one of our cases, isa very ~erious drawback to the use of the drug, It has seemed that by studying the cactericidal power of the blood of ths patients to whor the drug was adminis- tered much could be learned concerning proper dosage, the probvatle ideal cordi- vion toirg obtained wher 2 vactericii-- To..sr is cttained in the shortest time wid cher this is raintained constant -vithous fluctuation, The latter is os- pecially inporta.t since it Las teer srour shat if the vacteria be exposed to a carncentration cf ths Srug net suffictent te kill, they quickly become ra- Sistant to tue drug and very cucth nigher concentrations are required than was originaiiy ths case. Drs. Moore and Cheay y have therefore made an exhaustive study of the vasterisddal rover of the blood of patients after the administra- vion of optochin in varying doses and administered in various ways, Without going inte the datetle ef their study, it may be stated that the optimum re- te ate obtained when an initial large dose of the drug is given, and this 3 followed by repeated sriall doses given every two hours, day and night. In regulating the size of the dose, the weight of the patient is of very gregt izportance, aud they have found that the patients should receive 0,024 gm.  per xilogram of patient's weight, ner 24 hours, They have thus made it possi- cle to properly test the action of the drug in patients and it is planned to do tais fairly extensively during the coming winter in case a supply of the drug can te obtained, At present it is made only in Cermanv and we are having great difficulty in obtaining a sufficient supply, Last winter the mumber of  Cases properly treated was not large, only 25, These were mostly cases of                ~ Je  -) Ti and III in which the mortality is high, In these few treated cases the mor= ity was 16 per cent, which, «hile not a brilliant result, ie not discouraging. Epidemiology. - The study of thie subject has beon continued by Dr Ernest :ilman, whose results mainly confirm those given in our last annual report. Dr. -(1iman has examined the saliva of 398 normal individuals; in 172 instances pneumo- coi were present. In 4 of these positive cases, pneumococci of Type I were ob= aned, and 3 of these gave a history of close association with a case of lobar pneu~ ia, two of them knowm to be due to Type I organisms, In 4 instances organisms of .; IY were obtained and 3 of these also gave A history of close association with cy \\\\wonia patient; in all 3 of them the patient was known to be infected with Type .4 organiane, Pneumococco of Type IIf were found in normal mouths 44 times and in _st cases no close association with pneumonia cases could be traced, So far as could , determined those Type IlI organisms differed 4n po way (immunologically or other- ise) from those found in disease. This anomalous fact, that pnevmococd of the most hirulent type may occur in healthy youths without giving rise to disease, cannot be explained, Atypical Type II pneumococci were found 26 times and Type IV organisms (110 times, The Length of time during wrich a convalescent may harbor the infecting - organiam was aleo studied, and it was found that organieme of the infecting type may persis$ for a long time, 4n one case even 85 days, These facts all hava an impor~ tant bearing on probéems connected with prevention and it is believed that later prace tical use may be made of them, Preparation of Antipneumocotous Serum, The work entadied in the preparbe tion of antipuewumococtus serum has grown to a considerable extent and now requires 4 consideratle part of the time of one man. Tith better facilities however, offered tv a vew animal house, and with the imprevements devised in the preparation of the sexum to be reutioned betow, It is theught that the Sabor and expenses involved may  bo materially reduced, Ouroag tha present summer De Mcora has been actively engaged          ~ 10 = ome  he bas  in the preparation of serum so that we now have a considerable supply on hand, In aliition, several of the state Boards of Health are praparing the serum so that it is believed that during the comirg season a large ruuber of patients suffering from Typo I infection, both in this Hospital and e]sewhers, can te treatod by this method The method of producing immune serum by using déad instead of living culture aud injecting every day on alternate weeks, instead of only one day a week as for- tury done, has now been tried in horses and found to be very successful, just as cur previous experiments had shovm it to be in emall animals. This method is now teing used, and by it horses can be made immine in six to eight weeks instead of in + to & months as was formerly the case, Sp far it has been impossible to produce any more active serum than was previously done, though efforts along this line are still being persisted in. It has not yet been possible to employ the concentrated serum as prepared by the method of Gay and Chickering on any considerable number of cases. It has been tried in 9 cases of Type {I with apparent good resulte(all the patients recovered), but the number of cases is still too small fer any conclusions to be drawn, It is planned during the coming witter to continue its use, combing it with optochin, A number of other studies concerning pneumonia and pneumococcus infections have been carried on, which however at present have theoretical rather than practical interest, Such studies are those reported by Miss Stryker on Variations in the Pneumo~ coccus induced by Growth in Immune Serum, The Batent Period on the Growth of Bacteria, | by Dr Chesney, Antiblastic Immunity by Dr Doches, Studies by Dr Avery on the Growth of Pnewmotoced in Bile, Experimental and clinical studies on the agglutination curves of sera after the adminstration of immune serum are being made in the hope of obtain ing better indications for proper dosage. Diabetes: Over seventy cases of diabetes have now been under treatment at the Hos-  pital of tha Rockefeller Institute, The methods of treatment that have been recom=                      ended by Dr Allen have now also received wide employment elsewhere, The experience re and the general opinion of those who have had experience else where is that a vy definite and marked improvement has been made in the method of treating this di-~ ‘30, The Journal of \"American Medicine\" has this year presented to Dr Allen a gold jal which 4s given annually to \"the physician who has made the most notable contri~ tion to medical science\", During the past year Dr Allen has devoted a large part of his time to experi= .tal studies on dogs. The patients have been under the immediate care of Dr Still~ ry and Dr Fits, During the early part of the year Dr Stillman gave much time to ..0 organization of the diet kitchen and clinical laboratory, and to so organizing and ‘uveloping the method of treatment that it can be Carried out in a routine manner. -r Stillman was much aided in the work by Miss Cleland who has had charge of the diet -itchen.. She has shown a very spectal adaptibility to thie kind of work and hae made thie department a model that is being very largely 4mitated throughout the country. The rethode used in the dietetic treatment of diabetes have been demonstrated during the past year to a very large number of physicians who have come here for the purpose of learning.  ; Dr Fits, in addition to the care and treatment of patients, has been engaged in studying the kidney functions in cases of. diabetes and in an investigation of the. nestion of oedema of salt metaboliem in this disease, The results obtained are now ready for publication, Much time has been devoted by Dre, Allen, Stillman and Fits to tne preparation of a monograph dealing with the treatment of aiabetes and giving the complete clinical experience ehtained here. This monograph is nearing completion and it 4e hoped will be a most valuable and authoritative contritution to the subjects  Dr Palmer -has carried on a series of studies concerning the concentration of sugar in the tissues of normal and diabetic animale, This study is now completed. Dr Allen has also co-operated with Dr BauBois of the Russell Sage Foundation  in a calorimetric etudy of metabolism in fasting and non-fasting diabetic patients.              ihe results have shown an increased metabolism in diabetic persons as contrasted vith non=diabetics, and also that the metabolism of diabetic persons is considerably ja¢reased during tho fastingtreatment, thus establishing one of the hypothesds on. nich the treatment was based, Turin> the entire vear Dr Allen has continued his study of diabetes in the -rtially Jopancreat ized dogs and during the past half year has investigated especially . production of pipemia, acidosis and coma in dogs, He has succeeded in initating .colutely all the features of the disease as it ‘isssen in man, The nature of dia~ .tic lipenia in man has been unknown, It has long been observed that the blood of ~-9 diabetic patients may be creamy in character and contain as high as 20 per cent rat, This condition is never found in human pathology outside of diabetes, There .2v6 been two theories, one that this fat is derived from the fat of the food, and tue other that it ie derived from broken-down cells, but neither of these theories nas had much evidence to support it, and there has been no explanation as to why such an enormous excees of circulating fat should be heaped yp, Dr Allen has shown that this conditions may regularly be reproduced in diabetic doge. The necessary conditions are that the dog have good digestive power,a severe diabetes, and receive 2 fat rich diet. It has not been seen in Minyiwskits and Sandmeyer's forms of dia~ betes because euch dogs have poor digestion fer fat. Dr Allen's experiments show that the fat of the d]dod is dervied from the food fat. \"Distubbances in fat fundtion™ | stand in direct relation to other diabetic phenomena, Dogs with deidosis do not necessrily have lipemia; this is like the rule with human patients; but all dogs. with lipemia have acidosis, . . Acidosis can regularly be produced in adiabatic dogs by fat feeding. It also occurs when fat dogs, by properly conducted experiment, are rendered glycosuric. These dogs show low CO» capacity of the blood and exceas of ketone bodies in the urine. In proportion to weight gogs excrete less acetone bodies than man, but the  concentration is similar. When the acidosis is increased markedly, coma supervencs.  ~             -13- | Oe th the same program of symptoms as seen in man, namely, malaise and prostration ith decreasing a in the blood, terminating in coma with very low COg. It is casy _ keep the CO2 up to the normal height by means of bicarbonate and other alkalies t the benefit is very transitory. No dogs have been saved by the use of alkalies. It has been possible to produce acidosis which clears up on fasting and aci- _sis which progresses and proves fatal on fasting, just as occur in human patients. Heart Disease, fhe studies concerning heart disease have been carried on ty Dr Cohn with the sistance of Dr Jamieson until February, wher Dr Jamieson left tp join the Canadian ilitary Medical Force, A large part of the work of this and preceding years has consisted in a clini~ :al and experimental study of digitalis, the most important drug we possess in the treatnent of heart disease, The plan has been to study each year the effect of this irug in a special type of selected cases. During the past year the patients studied vere mainly those suffering from auricular fibrillation and oedema, The study of the action of digitalis in acute Anfections with fever(especially pneumonia) has been continued and extended, As a resuit of the clinical and experimental studies the use of this drug in penumonia has been made mach more rational and therefore effective. From the careful etiidy of the heart during pneumonia it was found that al- most ten per cent of the cases showed auricular fibrillation or flutter at some tine during the course of the disease. It is in these patients especially that the use of digitalis may be helpful. It is of great importance, however, that the digitalis oifecta be obtained at the proper time. To obtain digitalis effect time is necessary ang roreover the amount of drug required in the individual case is more or less an inliviiual matter, Bpt the detection of changes in the electrocardiograph curves previously descrived it is now possible to know when digitalis effects are being  obtained before the toxic (poisonous) effects appeare                   ~ 14 =< “ee gt  crom the careful study of 126 cases of pneumonia it has been possible to accurately :amonstrate the beneficial effects of this drug,  Experimental studies concerning cardiac hypertrophy in dogs are being conducted, “ypertrophy is being produced by means of muscular excercise and by producing arti~ :ical lesions of the valves,  Nephritis.  Up to the tine Dr UcLean left for China he was wegaged in a study of the a banisir o! urea excretion and urea retention, Study of the numer ica laws governing -.:g fuuetion las been made in patients with nephritis and taose/ disturbed circulation ale to peart failure, In over 1000 ebservations these laws were found to be valid, In certain cases it was possible to follow the changee in function occurring during recovery from acute nephritis and also during the period preceding uremia, A similar study of the chloride metabolism was carried on, In patients shortly before death {rom uremia a sudden fall in the concentration of the sodium chloride in tho blood plasma was observed, fhis fall is associated with increase of H ion concentration of the blood and Hambweger has shown experimentally that such an increase of H ion concentration causes the sodium chloride to increase in the cells and to diminish < in the plasma,  | These observations led to experiments which were made by Dr McLean and Dr. Var Slyke to test the effect of the introduction of acids bases and salts and non- electrolytes into the circulation on the chloride content of the plasma, It is ox« petted that these studies which were well under way will be completed by Dr McLean aiter his return to this country. Chovical Laboratory,  During the year there has been much activity in the chemical laboratory Dr  ‘far, Cleke and his assistants taking part in many of the studies previously mentioned,  as 7611 as carrying on independent investigations. Dr Van Slyke,with Mr Cullen          ~ 16 < ron  ni Dr lMebean has continued stuiies on the fate of protein digestion products, To ‘arty out these etudies it became necessary to improve the methods for the quanti~ _.tive determinations ef the proteins of the plasma, Marked 4mprovenents in these _othods have been made by Mr Cullen. Nisa Vinograd has continued the study of pro~- cing of human and cow's milk. The results indicate that the albumins from the two cources ‘are quite different, but that the caseins, 60 far as could be determined, ite identical.  Acidosis, An important part of the work carried on in the cnemical labo~ vatory has had to do witn the question of acidosis occurring in diabetes and other conditions, The method devised by Dr Van Slyke for determining COg capacity of tha clood has proved of great value and its simplicity makes it one of the most important, af not the most valuavle of the clinical methods for determining the presence and degree of acidosis. Such a method is of very gteat importance as a control when fasting ie being carried out in diabetic patients, From the atudy of diabetic patiente ty Dr Stillman with these methode it has been possible to group the cases into four classes,  1. Cases with little tendency to show acidosis either when fasting or on  a diet, 7 2, Cases with a tendency to acute acidosis on fasting, These cases are  few but their occurrence makes it extremely important that patients undergoing fasting bacarefully tested irom tine to time) These eases may seem t6 be quite “mild Frew guently on the second and third attempt to make the urine sugar-free by fasting nc acidosis may occur. | ,  3, (Cases showing acute acidosis, curable by fasting. Many severe cases with marked acidosis may show this phenomenon, The occurence of these cases is one of the reasons why fasting is so important in treatment.  4. Gases with chronic acidosis, These are the most difficult cases to treat  in the effort", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-bpv3~p6ax~tw59", "00000000-0000-0000-EDC9-D4FEEFAE3B5C", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Avery, Oswald Theodore, 1877-1955: Papers, 1867-1970", "101584575X117", null, "1970", "30 November 1970", "This is the finding aid for Avery's papers at the Tennessee State Library and Archives.", "Finding aids", "Archives", "After the Discovery: The Transforming Principle's Reception by the Scientific Community", "10", "pages", "Text", "English", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, "te  ’ MANUSCRIPT UNIT ARCHIVES AND RECORDS SERVICES SECTION TENNESSEE STATE LIBRARY AND ARCHIVES  AVERY, OSWALD THEODORE, 1877-1955 PAPERS, 1867-1970  Accession number 70-128  Processed by MWF Date completed 11/13/70  Location V-K-5  The papers of Oswald Theodore Avery, research physician and bacteriologist with the Rockefeller Institute for Medical Research, foremost researcher on pneumonia, one of the founders of the science of immunochemistry, and discoverer of the transforming nature of DNA (desoxyribonucleic acid), were given to the Manuscript Unit by his brother and sister-in-law Dr. and Mrs. Roy C. Avery, Nashville, Tennessee.  Linear feet of shelf space occupied: 1.68 Approximate number of items: 600 items and 2 volumes  Single photocopies of unpublished writings in the Oswald Theodore Avery papers may be made for purposes of scholarly research.  Library of Congress Number MS 71-432    SCOPE AND CONTENT NOTE  The Oswald Theodore Avery papers, consisting of about 600 items and two volumes covering the years 1867-1970, are composed of accounts, biographical sketches, citations (awards, diplomas, honorary degrees), citizenship papers, correspondence, documents, military papers, obituaries, a patent, photographs, speeches, a will, and scientific writings.  The earliest papers in the collection concern the parents of Dr. O. T. Avery. His father Joseph Francis Avery (1847-1892) was a Baptist minister. Born in England, he moved to Halifax, Nova Scotia, in 1870, where he did outstanding service as a Baptist minister. In 1887 he was called to be the pastor of the Mariner's Temple in New York City. Newspaper clippings include several describing his work there and his successful missionary efforts in the city.  Mrs. Avery's papers (Elizabeth Crowdy Avery, 1843-1910) include her pension payments from the Baptist Western Widows and Orphans Society; interest on money she had loaned Acadia University, Wolfville, Nova Scotia; funeral bills; a patent, 1879, for \"Avery's Auraline,\" a preparation for the relief and cure of earache, noises in the head and deafness; papers concerning her partnership with Jane Caroline Irish for the sale of this preparation; and her will.  A scrapbook made by Mrs. Avery contains articles written by her husband Joseph Francis Avery (1847-1892), a Baptist minister. The Reverend Avery's articles appeared in newspapers and church papers in Halifax, Nova Scotia, from about 1873 to about 1882. Included is a booklet he wrote in 1876 entitled \"The Voyage of Life.\"  There is biographical data for Dr. 0. T. Avery including a reprint from GENETICS, Vol. 51, No. 1, January, 1965. Written by Rollin D. Hotchkiss, the reprint contains a selected bibliography illustrative of Dr. Avery's work.  On August 1, 1918, Dr. Avery became a citizen of the United States. Included are his citizenship papers.  There is a clipping from the NASHVILLE BANNER, December 11,  1965, in regard to the memorial gate at Rockefeller Institute erected by \"grateful friends\" honoring Dr. Avery. Another clipping from the NEW YORK TIMES, Sunday, November 5, 1967, is about the concept of the Nobel prize, explaining why Dr. Avery had not received the prize even though he had made the historic discovery that hereditary information is coded, for transmission to future generations, in the structure of DNA (desoxyribonucleic acid).       Forty-seven letters, 1909-1944, written by Dr. Avery, the majority to his brother and sister-in-law Dr. and Mrs. Roy C. Avery, discuss family affairs, and reveal a most kindly, thoughtful and generous man.  On May 13, 1943, Avery wrote his brother describing his research dealing with the transformation of pneumococcal types. This letter was written on the eve of his momentous discovery that DNA is a  ‘functionally active substance in determining the biochemical activities  and specific characteristics of cells, and that by means of a known chemical substance it is possible to induce predictable and hereditary changes in cells.  Correspondence includes one letter, ca. 1892, written by Joseph Francis Avery to his wife from an infirmary where he had undergone an operation; a letter of December 13, 1897, to Mrs. Avery from Jane Caroline Irish in regard to her husband's unsuccessful attempts to get a capitalist interested in marketing \"Avery's Auraline\" and their consequent inability to repay Mrs. Avery any part of the $1,000 she had loaned them; and nine letters from Mrs. Avery's sisters-in-law in England, 1897-1910, which deal with family matters. W. A. Cauldwell in New York City wrote the Reverend Avery on February 3, 1890, that it was the desire of their mutual friend Mr. Rockefeller that the Averys should dine at Delmonico's to meet some of the Baptist people in a social way. There is a note from Emily Vanderbilt Sloan, January 22, 1893, to Mrs. Avery expressing her friendship and interest in Mrs. Avery's work.  Roy Crowdy Avery, brother of Dr. Avery, served in the United States Army Sanitary Corps during World War I. Included are 35 letters which describe his situation in France in 1918.  A letter written on October 21, 1947, by colleagues William S. Tillett and Colin M. Macleod, and Dr. Avery's cousin Minnie Wandeli, whom he and Roy had supported and educated after the death of her mother, expresses appreciation to and gratitude for knowing \"Fess.\"  Included is a copy of President Truman's letter, October 1, 1947, to Dr. Harry S. Mustard congratulating the winners of the second annual Lasker Awards for Medical Research and Public Health Administration.  There is a rough draft of a letter written by Dr. Roy C. Avery on February 16, 1965, to the Director of the National Library of Medicine, Bethesda, Maryland, in regard to the possibility of publishing an American edition of an excellent German text which gives Dr. 0. T. Avery credit for his work with DNA.  The most recent letter in the collection is from A. R. Massar, Luanshya, Zambia, dated July 28, 1970, thanking Dr. Roy Avery for permitting him to read 0. T. Avery's letter written in 1943, which describes his work on the eve of his discovery of the transforming properties of DNA.       Other correspondence includes many letters of congratulations to Dr. Avery when granted various prizes, awards and honorary degrees.  Included is an oral history memoir of Tom Rivers, titled \"Reflections on a Life in Medicine and Science,\" in which Rivers speaks of Dr. Avery's manner of working and writing, his shy and retiring nature and his generosity in sharing his knowledge.  Dr. Avery's military papers include his appointment as Captain in the Medical Corps, United States Army, September 26, 1918, and his honorable discharge, January 30, 1919.  There are two photographs of Dr. Avery taken in the laboratory when a young man; a snapshot of him taken many years later; a snapshot  _of the Avery Memorial Gate at the Rockefeller Institute; a group picture  of members at the annual meeting of the Army Epidemiological Board, Washington, April, 1945, and April, 1946; and a group picture of members of the Board for the Investigation and Control of Influenza and Other Epidemic Diseases in the Army. Dr. Avery served on both of these boards.  Speeches include one given by Dr. Avery on his acceptance of the Kober Medal Award, reprinted from the TRANSACTIONS OF THE ASSOCIATION OF AMERICAN PHYSICIANS, Vol. LIX, p. 43, 1946; and another upon his acceptance of the Passano Award in 1949. Included also is the speech that Colin M. MacLeod gave at the dedication of the Avery Memorial Gate, Rockefeller Institute, September 29, 1965.  Writings include Dr. Avery's annual report, April, 1947, \"Studies on the transformation of pneumococcus.\" A reprint from ARCH ENVIRON HEALTH, Vol. 21, September, 1970, titled \"The \"Undiscovered’ Discovery\" by Wendell M. Stanley, deals with Dr. Avery's discovery of the transforming principle of DNA.  got 20 1972             1877,  1887 1893 1900  1904  1904-1907  1907  1913  1923  1943  1943-1948  1949  1955  BIOGRAPHICAL NOTE  Oswald Theodore Avery     Born October 21, in Halifax, Nova Scotia, son of Joseph Francis Avery and Elizabeth Crowdy Avery  Moved to New York City with his family Diploma from New York City Male Grammar School Awarded B.A. degree from Colgate University  Awarded M.D. degree from Columbia University College of Surgeons  Practiced general surgery  Bacteriologist with Hoagland Laboratories in Brooklyn, New York; became associate director  Joined the staff of the Rockefeller Institute  Made a member of the faculty of the Rockefeller Institute Retired as emeritus member of the Rockefeller Institute Continued bacteriological research as emeritus member Retired to Nashville, Tennessee  Died in Nashville; buried in Mt. Olivet Cemetery ®WN *- ee @  5. 6.  8. 9.  13. 14. 15.  CONTAINER LIST  Box 1  Accounts--Avery, Elizabeth (Crowdy) and Joseph Francis Avery Accounts--Avery, Oswald Theodore Biographical sketches  Citations  Citizenship papers  Clippings  Correspondence Correspondence Correspondence  Correspondence Correspondence Correspondence Correspondence Correspondence Correspondence Correspondence Correspondence Correspondence Correspondence Correspondence  Correspondence -  “A\" = Avery, Mary Avery, Oswald Theodore Avery, Oswald Theodore (May 13, 1943)  Box 2  Avery, Roy Crowdy  wan \"or  \"D - FU \"Ge g\" \"Ke = 7,\" ™ -— Oo\" \"D> Q\" wR  tow  wm. yn wie zt  Diaries, memoirs, etc. - Rivers, Tom  Documents Estate papers  Military papers  Miscellaneous Obituaries Patent--1879 Photographs  , Programs  Scrapbook Speeches  Box 3  Wandell, Minnie Belle--papers  Writings Box 4  Awards--Diplomas--Honorary degrees  ” ote ce - .  |  |  |  INDEX  This is a name index of the correspondence in the Oswald Theodore Avery Papers together with the dates of the letters and information regarding their contents. The figures in parentheses immediately following the name denote the number of letters if more than one. The last numbers refer to the box and folder number in which the material is to be found.  Aggie, 1936, re: visit to Halifax, 1-7  Alan, 1953, re: general news, 1-7  Anderson, Harold A., 1950, re: convocation exercises, University of Chicago, 1-7  Armstrong, George E., 1954, re: appreciation for Dr. Avery's service on Commission on Streptococcal Diseases, 1-7  Aunt Sade, n.d., re: family news, 1-7  Avery, A. (2), 1902, re: family news from England, 1~-7  Avery, Ernest (4), 1891, re: his stay in the country, 1-7  Avery, Lena (5), 1892-1909, re: family news from England, 1~7  Avery, Mary E. (Mrs. Ernest Avery) (2), n.d., re: family news from England, 1-7  Avery, Oswald Theodore (47), 1909-1944, re: family affairs, 1-8  Avery, Oswald Theodore, 1943, re: his research with DNA, 1-9  Avery, Roy Crowdy (35), 1918-1965, re: his World War I service in France, 2-1  Avery, W. I. (2), 1887-1892, re: Mr. Spurgeon, an “accuser of brethren\" in the church; Joseph F. Avery's death, 1-7 7  Baldwin, Edward R., 1932, re: congratulations for Phillips prize, 2-2  Bible Class, 1870, re: wedding of Joseph F. Avery, 2-2  Brigham, F. Gorham, 1932, re: congratulations for Phillips prize, 2-2  Bradley, Ernest B., 1932, re: congratulations for Phillips prize, 2-2  Butterworth, W. Walton, 1950, re: gold Pasteur medal sent to Dr. Avery, 2-2  C ole , Rufus, 1932, re: general news, 2-3  Cambridge University (4), 1949, re: honorary degree of Dr. of Science to be awarded to Dr. Avery, 2-3  Carrel, Alexis, 1932, re: congratulations for Dr. Avery's award, 2-3  Cauldwell, W. A. (2), 1890-1891, re: invitation to the Reverend Mr. Avery from Mr. Rockefeller, 2-3  Chipman, X. Z. (10), 1891-1892, re: business matters with the Reverend Mr. Avery, 2-3  Churchman, John W., 1932, re: congratulations for Phillips prize, 2-3  Clark, Robert D., 1954, re: a thank-you for information on Dr. Fosdick, 2-3  Coburn, Al (2), 1943-1949, re: work with Dr. Avery, 2-3  Coburn, Anne, 1949, re: personal matters, 2-3    DeMuth, J. Smith, 1897, re: certifying that William Gravenhaus died September 19, 1897, 2-4  Dible, J. H., 1948, re: honorary membership in the Pathological Society of Great Britain and Ireland, 2-4  du Nouy, P. Lecomte, 1933, re: congratulations on gold medal award from Ehrlich Foundation, 2-4  Edsall, Geoffrey, 1950, re: appreciation to Dr. Avery for work on Journal of Immunology, 2-4 Ehrlich, Paul, n.d., re: congratulatory cablegram in German, 2-4  Finance Department, Ottawa, Canada (2), 1893, 1903, re: business matters, 2-4  Flexner, Abraham (2), 1933, re: congratulations, 2-4  Flexner, Simon (3), 1927-1933, re: congratulations, 2-4  Gasser, Herbert S., 1942, re: Dr. Avery's imminent retirement from Rockefeller Institute, 2-5  Gay, Frederick P., 1932, re: congratulations, 2-5  Gill, Robert S., 1949, re: Passano Foundation award, 2-5  Heidelberger, Michael, 1926, re: his summer vacation, 2-5 Irish, Jane Carolina, 1897, re: inability to repay loan, 2-5  Jim, n.d., re: congratulations, 2-5  Johnston, J. A., 1887, re: advice to the Reverend Avery about the Mariner's Temple, New York City, 2-5  Jones, Lewis Webster, 1954, re: honorary degree from Rutgers University, 2-5  ‘Katz, Ernst Fritz, 1933, re: a request to Dr. Avery written in German,  2-6  Lechtrecker, Henry M., 1932, re: congratulations, 2-6  Littell, Emlen T., 1949, re: hotel reservations, 2-6  Longcope, Norfield T., 1944, re: congratulations, 2-6  Loveland, E. R., 1935, re: Phillips prize, 2-6  Lundquist, John, 1950, re: awarding of the Pasteur medal from the Swedish Medical Society, 2-6  Maggie, 1909, re: news from Halifax, 2- 7  Massar, A. R., 1970, re: thanks for permission to read Dr. Avery's letter of May 13, 1943; request for a photograph of him, 2-7  Mathers, Isaac H., 1909, re: bank business, Halifax, 2- 7  Morgan, Hugh J. (2), 1932, re: congratulations, 2-7       .g*  Northrup, Jack, 1932, re: congratulations, 2-7  Ormston, Katherine, 1947, re: enclosing clippings from Mrs. Albert D. Lasker, 2-7  Parke, William M. (10), 1906-1910, re: mortgages, etc., 2-8  Parr, Leland W., 1948, re: Society of American Bacteriologists, 2-8 Phil, n.d., re: invitation for dinner, 2-8  Power & Foley, Solicitors, 1906, re: a mortgage, 2-8  Renaux, E., 1950, re: the jubilee for Jules Bordet, 2-9  Resnik, William H., 1932, re: congratulations, 2-9  Rockefeller Institute for Medical Research (8), 1925-1938, re: salary, retirement policy, etc., 2-9  Sawyer, A. W., 1887, re: his regret at the Reverend Avery leaving Halifax, 2-10  Sherman, J. M., 1948, re: honor conferred by the Society of American Bacteriologists, 2-10  Sherring, W., 1892, re: widow's claim, 2-10  Sloan, Emily Vanderbilt, 1893, re: Church work, 2-10  Smith, Fred S., 1890, re: life in Vincent, Louisiana, 2-10  Sprofkin, Bertram E., 1949, re: his coming work with Dr. Avery, 2-10  Spurgeon, C. H. (2), 1886-1887, re: thanks for donation for his orphanage and advice on the Reverend Avery's moving to New York, 2-10  Stacy, John H. (4), 1867-1870, re: Joseph F. Avery's progress in his studies, and his brother, Phillip, 2-10  Streeter, Edward (2), 1936, 1950, re: securities, 2-10  The Tabernacle Baptist Church, Halifax, 1887, re: resignation of the Reverend Avery, 2-11  Thompson, Thomas D., 1887, re: selling property, 2-11  Tillett, William S., and others, 1947, re: esteem and friendship for Dr. Avery, 2-11  Trask, Lillia M. D., n.d., re: congratulations, 2-11  Trask, Thomas (7?) D., 1932, re: congratulations, 2-11  Truman, Harry S., 1947, re: congratulations to the winners of the Lasker awards, 2-11  Vanderbilt, F. P., 1936, re: dividends, 2-11 Vanderbilt, (Mrs.) William H., n.d., re: Christmas greetings and donation, 2-11  Wallace, J. R., 1891, re: bank business in Halifax, 2-12  Weinberg, Arthur von (2), 1932-1933, re: Ehrlich prize (in German), 2-12 White, Benjamin, n.d., re: congratulations, 2-12  White, William Charles, 1932, re: congratulations, 2-12  Willstatter, Richard, 1932, re: congratulations (in German), 2-12", "Tennessee State Library and Archives", "Tennessee State Library and Archives. Oswald T. Avery Papers", null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null, null ]
, [ "row-v3uu_yv77.fyqb", "00000000-0000-0000-234A-25AA05A33614", 0, 1750962675, null, 1750962675, null, "{ }", "The Oswald T. Avery Collection", "101584575", "Transformation of Pneumococcal Types", "101584575X118", "101584575X348,101584575X499", "1943", "[10 December 1943]", "These typewritten notes were probably for a presentation which Avery gave at the Rockefeller Institute between the submission of his article with MacLeod and McCarty and its publication.  See Maclyn McCarty's \"The Transforming Principle: Discovering that Genes Are Made of DNA\" (W. W. Norton, 1985), pages 169-171.", "Speeches", "Transformation, Genetic ; Research Design", null, "16", "pages", "Text", "English", "Courtesy of the Tennessee State Library and Archives.,http://www.tennessee.gov/tsla/", "Copyright may apply", null, null, "tN,  DEC1 1972 Dem, [Teun & | BONGPORATIUN OF PNEUMOCOCCAL TYPEU  wate fOPUGLS tae e  I ruc ay Lies 7\" 5. {nwrsaucvion wiS. ‘ 7 2 ac, NO.  I. Brief historical review  A. Griffith - 1928, unencapsulatec, avirulent R-varients cf cne specific type transformed into fully ence psulated and virulent cells of a different specific type  mxample and technique: Type II R ~»Type III 5 R strain incapable of causing fatel bacteremia Heated Type III suspension contained no vieble organisms. Thus, R cells acquired capsular structure anc serological specificity of Type Iil. Siice 1 illustrates phenomenon  1. Numerous tyses of pneumococcus  2. Tyse specizici % on presence in capsule of @ cae chemical by Gistinct and serologically  polys ari specific for each type.  3. Capsule is imaunclogical unit of type specif ficity anc accessory structure essential in cetermining invasive caracity or organism.  4. Under certain cultural conditions enzymes concernec in synthesis of capsular material may be suppressed or lost. With loss of capsule cell loses its type specificity end invasive properties. ~»K form. Reaction often reversible - animel passage; antvi-R serum. Reversion always back to type from which it came. Reversion SSR different from transformation.  m * 5. In letter instance, R acquired the capsule and type ‘*. specificity of the encapsulated cells used to induce the reaction.  B. Confirnation of phenomenon = yo 1. Newfeid ana Levinthel - 1928 abroad 2. Eaurhenn ~ 1932  *  3. Dawson - 1930 - in isccratory    C. Incuction in vitro  1. Dawson and Size - (intact and heated celis, g ani  1921 Grew R celis in media containing an 2. Al oway 1932 - (cell-free extracts; transforming prince de in soluble forn; Berkefeid filtered Hence in test tube es in mice, showec trensrorna  tion induc and selectively determined by type specificity of  ed $ cells usec. D. Virus - fibroma-+ myxoma  itl  1. Berry and Dedrick - 1936 aS Oerr——eee  Living fibroma virus + heat-killed myxoma >rabdits induced myxomatosis.  2. Berry 1937  Induction by suspensions of washea elementary bodies of myxoma.  3. Confirmation by number cf investigators.  TS  Presen> Study - more cetalied analysis of phenomenon; a     tiempt to determine chemical     nature of transforming principle. Les  Ca . ——e .  . . - fo) ee Y a 1. 7 —> FF “Ts ae Hf ee Model: e > e . oP gt Mee Type II type III Reeeg inten Ue tt .  + 4 2e ae tes =e a8 s 2 LA, . Sie ve: & i. z f a-2es, a nee II. Gulturai Conditions - certain conditions mequisite \\\\ Sees Wes Set ote i . #  A. Broth - optimal for growth. Indiviauai and uwnpredicteole variations in ——tapacity to support transformation. These largely eliminated by charcoal adsorption (MacLeod and Mirick)     B. Serum or serous fluid ~ nce ree rntie”  l.     —_     Anti-R serum first used beceuse of capacity to promote reversion of R-»homologous S.  2. Alloway used ascitic and chest fluid and normal swine serum xz 7 o eS A aE aR contain anti-R)  3.  Present study - human pleural and ascitic fiuics used. t     eA REN cee arcs -  f Effectiveness of a     v ifverent Lots varies. Differences not dependent . RY on content of R antibodté es, suggesting other factors tavolved.s fo trita it  ' a omen xe la ‘ ~  “ 2 > - . . ~ “ Tey  4. Enzyme - various animal sera irrespective of immune properties contad  1  enzyme that destroys transforming principle.  Enzyme inactivetec at 60-65°C, Heating may render ineffective sere effective. 3- Unknown fuctsr - suggested by Bnii-z and enzyme neat 20al t formation. Nature cf unknown fector uncevermined.  6. Properties of serum — stabi  le; may ce storea in refrigerator muny months anc retain original effecti  iveness. .  Recognition of serum factors and prorerties facili Stancardization of cultural conditions required Zc and reproduciole results.  “  C. R strain (R364) ~ derived from Pneunococcus Type II  1. Characteristics - “resatively fixed_in R phase; never spontaneously reverts. Hepeated attempts to cause reversion unsuccessful. Strzin susceptible to transformation | to variety of ait ferent & S tyzes.  (ry tir; VIS aie MTV ‘ere  —_—_—  2. Dissociation - on serial transfer in dlood broth & strain undergoes Spontaneous dissociation number of verients distinguishable by colony form. Only 2364 Suscestibdie; other variants all inactive. “Differaness In hespensivensss g tectforanr Rravanls Ty tame stimebas.  Enshasizes care recuisite ‘Tn selection of suitable R-veriant.  3. Intraceiluler Wehayme — pneumococcal celis release ugon autolysis an enzyne that cestroys activity of transforming extracts. Important in the cultural conditions of induci ing trensrormeticn and in the  extraction of transforming principle from pneumococcal celis. To obtain consistent reproducible results, bear in mind:  1. R strain may uncergo spontaneous Cissociation giving rise to other variants 3 incapabie of responcing to transforming stimulus.  2. Pneumocaccal celis contain intraceilular enzyme which when released  destroys activity of transforming principle.  Hence, important to Select reactive strain and to prevent  }) “destructive changes ai¢ociated with autolysis...  Ae ae ees vas        D. Transforming principle ee ‘Quantitative titration of activity of trensforming material. eee  Sterile material serially ciluted in saline at neutrel pH.  0.2 6. “oF each Gilution edded to three or more tubes containing 2 CC. of \" broth to which 10 J per cent SELOus fiuid has been added. Tubes seeded with 0. O05, cc, or a 1074 Gilution of a 5-8 hour biood broth culture of the R strain’ (R364).  Cultures incubated 37° for 7 18-24 hours. Anti-z% in serum medium causes the R celis to agglutinate Guring growth. The : agglutinated clumss settle to “potton “of tube leaving clear sunernatent. “hen transformation ocew's, eaca osulated § cells unaffected by the R antibodies grow éifrusely; supernatant becoming uniformly turpdid.  inspection wlicne cen distinguish tent watively between positive  anc negative resulis.       wee oe NS Mie wt fe  ®, te eC. NG.     Til. Preparation of trensforming principle  a. Source material Nan erent 1. 75 liter dots of culture of Type III pneumococcus.  “Young, a actively growing (16 hr.)  ens ee Eee  2. Collected on Sharples _centriruge.     This temperature inactivates enzyme known. _to Gestroy t. p.  3. Celis resuspended _in saline - heat-killed 65°C... 20!  B. Extraction  i. Heated cells washed 3 times with saline, removing large excess capsular polysaccheride, much protein, ribonucleic acid and C polysaccheride ~ 10-15 per cent ‘loss or transforming material.  «. Extrected with saline conteining 0. 5 per cent sccium cesoxycholate, by m mechanical shaking. Repeated 3 times.  3. Extracts combined, precipitated by excess ethyl elcchol. Ppt - floetivig fibrous mass. Redissolved in saline.  C. Deproteinization and Removal of S ) polysaccharide a neat AIRDRIE rte pte 8 motte etn aR Noe tT el eta  1. Preliminary deprot inization by Sevag chlorcrorm method. About 3 times tS :  2. SIII Enzyme hydrolyzes. Type III capsular polyseccharide. Enzymatic  dreakdown usually complete 4-6 hours - evicenced by loss of Serological activity. ——- we  3. Reprecivitated by elicohol. Deproteinization repeated until no further protein-chioroform get at interface. ae  ‘Di. Alcohol fractionation TT eo a or te TT en  1. Dropwise addition of ebsolute ethyl alcohol with constant stirring  £. at critical concentration - 0.8 ~ 1.0 volume -— fibrous strands Separate out and coilect on rod.  3. Repeated 4-5 times  4. Yield of fibrous material = 10-2 major portion of active mater             IV.     R  od  ‘Analysis of Purified Material  A.  B.  C.     Effect of temperature  1. . Ext *  xtraction less efficient but activity best preservea when ‘procedures are carried out at 0°-4°C.  General properties  1. Saline solutions (1 mg./cc) - colorless, viscous, clear in diffuse light. in strong transmitted Light, silky sheen on stirring.  2. Preservation -— sxline soiutions retain activity 2-4°C. at least 3 months. Longer periods in frozen state in CO9 cabinet & ~ 4yeF In aqueous solution rapid decrease in activity;. completely inert in few days.  ere ad  Material precipitated from saline solution by eicohol™ and stored uncer aicohol remain active for long~periods.  ote era NT etme     3. Effect of temperature - withstands 30- 60 1 min. 65°C. ‘Higher temperatures not tested.  4. Effect of pH - activity rapidly lost at acidities greater than pH 5. Best preserved at neutral or slightly alkaline reaction.  Qualitative Chemical Tests  lL. Biuret and Millon tests negetive - even on dried material >  eed           2. Dische diphenylemine reaction for desoxyribose strong:y positive        3. Orcinol test (Biai) for ribose weakly positive. However, in similar “Goncentrations pure preparations of desoxyribonucieic acid-of animal origin prepared by different methods give Bial reaction of corresponding intensity.  4. Lipids - no specific tests. Crude material repeated extracted with alcohol and other -12°C. without loss of activity. . Repeated alcohol precipitation and treatment with chloroform results in no decreese in activity.  -Blenentary Chemical Analysis (Dr. Eek) Slide 3  N/P ratio varies from 1.58 - 1.75. Average value 1.67.  Close agreement with that calculated on the ‘basis of the theoretical - structure of sodiun aesoxyriponucleate.  N/P ratio indicates little protein or other substence containing _ N or P present, otherwise Faiion, would be considerably different.  ren oO          ays  Ms, Diy, / AC.NG, f2- 3  Ww- a ‘  D. nzymetic Analysis  1. Crystalline enzymes (Northrop and Kunitz)  Trypsin, chymotripsin ana ribonuciease - no effect on activity. Wo Thong or Broloin Sy \\\\tanlidle If Gatien Tryp licr ony ivates.     Pepsin not tested because pH requirea for action itself ina Liv:        2. Crude enzymes Dog inteytinal micose - Levene and Dillon - polynucleotidase-preparaticn. Rebbit bone phosphatase - Hartland and Robison BO Swine kidney phosphatase - albers, A. and E. Le . Pneumococeus autolysutes mo, Normal dog and rabbit serun.  a bf fh ee Slide 4 [iwtrele y be, as be Ags bobby re “ober “ big 7 \\\\ footers trey i if AG  (Dr. Mirsky ~ desoxyribonucleste) ’ re . 4  ne  Parallelism between enzyme thet causes deoolymerization of known Samples of desoxyribonucieic acid ang that which destroys activit: of transforming principle. Irrespective of phosphatase or esterese activity, only those preparations shown to contain an enzyme cavable  £ depolymerizing authentic samples of DRNA were found to inactivate  _transforming principle. 3. Differential Heat Inactivation of Dog and Rabbit Serum |, Vier     21 different mammelian  at, . N 7'| Greenstein: and Jenrette showm that sera of severel species contain enzyme which causes depolymerization of D.R.N.A.                                “Greenstein has termed this desoxyribonucleodepolymerese. ‘heta z wed cveese—of-viseostty—ol-mintures of nzyme and nucleate in yiscosimeters.  .  Dog and rabbit Serum, tested on partially purified preparation of 7 ? a prer  ‘transforming material.  ata show thet both dog ana rabbit serum unheated is capable of _— ompletely destroying transforming activity. However, when dog | ‘Serum iS heated’ at’ 60° or higher for 30 minutes there is no loss  TPO me, ae, aoe s . . . renee  of transforming activity. That is, the serum enzyme responsible  for destruction is completely inactivated at 60°C. In contrast exposure to 65°C. is required for complete inactivetion of the “corresponding enzyme in normal rabbit serun.     polymerase activity on  _» Same semples of serum also tested for de -., Sodium desoxyribonucleate.  Shawn Oi bastent ; te gas bs el ya - 4 ¥ -  i | 'b “ v % Aye Ry h fice              tion in viscosity end progressive decrease in acid precipitability of the nucleate. a : .. Data in Slide 6 show the differentinl heat inactivation of the  —_—— oN ane eee ree oT ee  cepoiymerase in dog end rabbit serum. ith unheated serua of both species, “viscosity feil to” that of water in 5-7 hours.  ( Depolyne-ization followed redi     |  ee ee ee  Dog serum heated at 60 and 65°C. - no significant reduction in Viscosity after 22 hours. 7. a me  On the other hand, heating rabbit serum at 60°C. merely reduced the rate of reaction.” The depolymerase wes completely destrcyed at 65°C, avy h aba FZ. -  j .  fi cd —7> Thus, striking