Copyright 0 I996 by the Society of America Perspectives

Anecdotal, Historical And Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

Recollections of HOWARDTEMIN (1934- 1994)

John W. Drake* and James F. Crowt

*Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 and ‘Genetics Laboratory, University of Wisconsin, Madison, Wisconsin 53706

N 1960 HOWARDTEMIN finished a postdoctoral year ers, MICHAEL(now an attorney) and PETER(now a pro- I at Caltech and moved to the University of Wiscon- fessor of economic history). His father HENRYwas an sin. His brilliance was evident to his Caltech associates, attorney, his mother ANNETTE (nee LEHMANN)was what but as to promise he gotmixed reviews. GEORGEBEADLE we might nowcall a volunteer. Bothwere strong- and RAY OWENboth recognized HOWARD’Sability but minded and voluble. HENRYconducted a diverse one- differed in their assessment of his future. RAY was con- man legal practice; at home he held high expectations fident, BEETSnot so sure. They decided to bet a fifth of those around him. ANNETTE had wanted to be a of whiskey on whether HOWARD woulddo something physicist butfound that worldclosed to women. A important within five years of graduation. After five bright and busy person, she founded a Citizens’ Com- years he had a publication record that was respectable mittee for Public Education in Philadelphia and a free but not unusual, and he had kept plugging away at an summer camp for children who would otherwise be heretical idea. RAY was particularly impressed by one of denied that experience. She was active in both Hadas- HOWARD’Spapers and thought his accumulated work sah and the synagogue, where she chaired the School good enough to win the bet, but BEETSdemurred. A Committee. Thus, whatever may have been the genetic few years later he reluctantly conceded and the betwas contributions to his abilities, HOWARDalso benefited paid off (a bottle of Old Crow, we are told). Of course, from an environment in which both analysis and action if they had waited until the discovery of reverse tran- reigned. scriptase in 1970, there would have been no question. As an undergraduate at Swarthmore, HOWARDstud- HOWARDwas honest, strongly individual, and willing ied . He quickly became a practicing scientist, to state and stand by his convictions. These traits were participating in two summers of research at the Jackson evidenced at his graduation from Swarthmore, where Laboratory. His first paper, dealing with the genetics of he had had a disagreement with a college policy and congenital anomalies (INGALLSet al. 1953), was based refused to dress for the graduation ceremony. (Years on work done there. His final oral examination in 1955 later, after he became famous, Swarthmore awarded for an honors degree at Swarthmore iswell remem- him an honorary degree; this time he donned cap and bered at that institution, because his thoughtful and gown.) His outspokenness was also evident at the 1975 far-seeing responses elicited a vigorous debate among banquet in Stockholm, when he shocked the external examining committee that soon subverted the audience by expressing his outrage that the one the examination into a professorial exchange. known major cause of cancer, cigarette smoking, was There have been numerous tributes to and reviews so blithely overlooked, even at the event itself. At a of HOWARD’Slife in science (e.g., COOPERet al. 1995). gathering of Nobel laureates in 1991, one speaker said Here, instead, are personal accounts by two of us who that if the world were more loving, many social prob- knew him well, but at different times and in different lems would be solved and world tensions lessened. ways. The first deals with his and his Caltech HOWARD,with AIDS in mind, used his turn to shock an years, the second with his life in Wisconsin. international television audience by saying, “If you love, Caltech and virology (J.W.D.): HOWARDinitiated his use a condom.” Perhaps the most characteristic exam- Caltech graduate work in 1955. He settled first into the ple was his steady belief in and defense of theprovirus laboratory of ALBERT TYLER, a kindly, scholarlydevelop- theory during a time when the transfer of information mental biologist who was interested in fertilization and from RNA to DNA was unthinkable. early development. I had been working in the same lab, HOWARDMARTIN TEMINwas born on , both of us having arrived at Caltech with an interest in 1934, in Philadelphia. He was bracketed by two broth- . We were impatient, however, and saw no

(;erwtic\ 144: 1-6 (September, l!)YC,) 2 J.W. Drake andJ.F. Crow

exploring this and related retroviruses. At Caltech he promptly engaged in a vigorous collaboration with HARRYRURTN. Their first success was the development of an RSV assay suggested by the then-recent results of MANAKERand GROWPI?(1956). This “focus” assay was a hybrid between a -induced plaque ancl a cell col- ony, in which RSV-transformed cells locallyout-prolifer- ate their contact-inhibited siblings (TEMINand RURIN 1958).Just as with the invention of the phage and the animal virus plaque assays, the focus assay brorlght all the analytical power ofquantitation to bear on the ques- tion of virus-induced cell transformation. 1950-1959 was a decade of great excitement in biol- ow.There was much discussion at Caltech about the still mysterious prophage state in lysogenic bacteria. By the middle1950s it had become clear that the prophage was so closely associated with the bacterial chromosome c that it behaved like a bacterial marker. Thus, HOWARD’S mind was soon steeped in the powerful concept of the chromosomal prophage, and he began to imagine “ly- sogeny” by an RSV “.” He later found an RSV variant that produced a variant morphology in trans- formed chicken cells, further strengthening theanalogy with bacterial lysogeny. In 1960 H0W44RDmoved to the University of M’iscon- sin, where he remained for the rest of his life. He con- tinued to probe the natureof the RSV-transformed cell c’ and began to advocate in print the notion that a provi- rus state could be achieved by an RNA tumor virus ( P.R., TEMIN1963, 1964a). He surmised that the RNA of the FI(;I’KEI.-How..\KI> in 197i with his favorite mode of virus would have to be copied into the DNA of the transportation in Madison. provirus and that progeny virus could result from tran- scription of this provirus. However, in addition to as- way to advance the field, the probes of the time often suming a different shape and becoming tumorigenic, being no better understood than their target tissues. most or all RSV-transformed cells continuously release After a while, as word of our frustrations spread, JIM virus without dying. This wasvery different from the

WATSONintervened. He was a visiting scientist at the prophage + phage transition so elegantly elucidated by time, fresh off DNA and anobject of considerable local ANDR~LWOFF a few years earlier in which an occasional curiosity. He decided thatwe might profitably work with cell suddenly bursts, releasing many phage particles. &NATO DULBECCO,a virologist who had recently in- In support of his hypothesis, HOWARD found that vented plaque assays for animal and was about blocking transcription with actinomycin D stopped vi- to apply the attack mode of the Phage School to these rus production (TEMIN1963), whereas transformed previously intractable viruses. DUI.REC<:Oagreed thatwe cells continued to make virus in the presence of inhibi- could work in hislab and introduced us to the members tors ofDNA replication such as fluorodeoxyuridine, of his group who were to become our daily mentors. aminopterin, and cytosine arabinoside (TEMIN1964b). These included MARGUERITE VOGT, whowas in charge Then, using hybridization techniques, he demonstrated of overall operations and was intimately familiar with sequence homology behveen RNA from the virus and the technical procedures involved in the plaque assays. DNA from transformed cells (TEMIN 1964~).However, Another was HARRY RURIN,a senior research fellow, an these studies are more persuasivein retrospect than adventuresome thinker, and a singer and guitar player they were when first described, in part because of com- much in demand at parties. plexities in some of the results and universal uncertain- Probably because of perceived parallels between de- ties inthe interpretation of inhibitor studies, but proba- velopmental processes and the cellular modifications bly more because of the novelty of the hypothesis. wrought by tumor viruses, HOWARDtook up Rous Sar- HOWARD’Sprophage model required thatviral single- coma Virus (RSV), which had been discovered by PEY- stranded RNA be copied into double-stranded DNA TON Rous in 1911 and was the canonical “cancer vi- and that the latter be integrated into the host DNA. rus.” HOWARDspent most of his subsequentcareer Early attempt5 to find an enzymatic activity to catalyze Perspectives 3

undiscovered, RNA plasmids might have offered an even more compelling (albeit incorrect) hypothesis to explain thecarrier state. By standing firm, HOWARD later gained praise for his courage in the face of wide- spread disbelief of his thesis. HOWARDdid a great deal of virology during his de- cades in Madison, publishing some 276 research and review articles on the subject in addition to his Caltech output andtraining several-score graduate studentsand postdoctoral fellows. Starting even before Madison, he repeatedly wrote on the role of virusesin cancer, a vexing question before the discovery ofoncogenes, and he argued for the origins of retroviruses from cellular transposons. HOWARDbecame steadily more interested in the problem of viral variation. Even in the Caltech years it had been obvious that RNA viruses mutate rapidly, frustrating attempts to obtain stable genetic markers. From the middle 1970s onwards he worked on this problem. He showed that reverse transcriptases exhibit high error rates (MIZUTANIand TEMIN1976) and that FKX‘KE!?.-Ho\v..\Iw TI.MIS;~nd S1<\v.u.[. M’IW;IIT in an mi- viral mutations not only occur at high rates (ZARLING mated discussion in l!)80. and TEMIN1976; DOUG HER^ and TEMIN1986) but are sometimes bizarre (PATHAKand TEMIN1990a,b). He these steps in infected cells produced only negative re- also showed that the two retrovirus genomes that are sults. Only several years later did he find the requisite packaged together in the typical particle recombine at activity, , which turned out to be a high rate (Hu and TEMIN1990a,b), further enhancing sequestered in the virus particles themselves. The en- viral variability. This high rate of variation complicates zymewas simultaneously discovered by DAVIDBALTI- both the prevention and the treatment ofRNA-virus MORE (BALTIMORE1970; TEMINand MIZUTANI1970). infections. For this discovery, TEMIN andBALTIMORE received the The Wisconsin years (J.F.C.): I first noticed HOWARD 1975 Nobel Prize in Physiologyor Medicine (along with in seminars. Upon hisarrival in the fallof 1960, he DULBECCOfor his pioneering work with virulent animal immediately started his life-long custom of sampling viruses). widelyin several departments. His breadth of knowl- The power of the provirus hypothesis and its implied edge and depth of insight were apparent in the fre- biochemistry was attractive to some of HOWARD’Sassoci- quent questions he asked, always to the point. At about ates. However, the notion was less compelling to most the same time there began an active discussion group virologists, and the walk to consensus wasvery much devoted to peace. Most of those attending were moti- uphill. This is a period for which close scrutiny might vated to control nuclear weapons and prevent war, but contribute interestingly to the history ofscience, partic- HOWARDstood out. He had a detailed technical knowl- ularly to the problem of how scientists come to accept edge of arms control, weapons, and military strategies. a radical notion. Much \vas made of the apparent con- He more thanany other kept the group from degener- tradiction between the provirus hypothesis and CRICK’S ating into vacuous do-goodism. “Central Dogma” of the unidirectional flow of informa- My next notice of HOWARD,some months later, was tion from DNA (through RNA) to . The streak the increasing attention he was paying to my graduate of‘ antireligious humor that turned a paradigm into a student, RAYLA GREENBERG.They met in 1961, RAYLA dogma was unfortunate, for CRICK had laterto clarify having returned from a year spent in Edinburgh with that his dictum did not precludeinformation flow from CHARLOTTEAUERRACH. RAYLA and HOWARDmarried in RNA to DNA but stressed instead the impossibility of May of 1962. I didn’t lose RAYLA;I gained HOWARD. information flow from protein to nucleic acid. An at- HOWARDwas recognized for his knowledgeand intel- tractive but far less explicit alternative hypothesis for ligence. Yet, his provirus hypothesis was received with HOWARD’Sresults, and one undervigorous examination skepticism, if not downright hostility. His move up the in those days, was that of a “carrier” state, an ill-defined academic ranks was not rapid: four years as assistant association between viruses and infected cells. Workon professor, five as associate. Hispromotion to full profes- the carrierstate produced a bin full ofarcane phenome- sor came only a year before his discovery of reverse nology that could be interpreted in many ways without transcriptase in 1970 made him instantly famous. While embracing the TEMINhypothesis. Although then still serving as Acting Dean, I obtained some unexpected 4 J. W. Drake and J. F. Crow

funds and used them to increase the salaries of a few He began to publish more, partly because of a plethora people who seemed particularly promising. One was of writing invitations. Nevertheless, paper after paper HOWARD, but my action led to a complaint from HAR- was full of new data. In the decade of the 1960s he OLD RUSCH, then Chairman of the Department of On- published 27 papers; by contrast, in the 1970s there cology. RUSCH had a detailed schedule of salaries for were 93 and in the 1980s, 104. his faculty and my move had thrown the plan out of He immediately set to work to discover whether re- balance. verse transcriptase (which he preferred to call RNA- Scientifically, the years from 1960 to 1970 were frus- dependent DNA polymerase) was a normal cell constit- trating ones for HOWARD.Although he was eager to uent and found that indeed it was. RNA transposons explain his ideas to anyone who was able to understand were not only present, but abundantlyso. Although he them, and didthis with vigor and patience, he was gen- contributed to numerous symposium volumes, he kept erally greeted with disbelief. MICHAELBISHOP (1995) up his laboratory work and with his students produced describes listening in on along discussion between a steady stream of important papers. In addition, he HOWARDand the skeptics at a Gordon Conference in became even more involved in political and social af- 1968. The argument was “strong, even vitriolic.” How- fairs. He was eager to lower the town-gown barrier, tak- ARD met all the arguments with reasoned answers, but ing time to talk with legislators and inviting the Gover- his was the lone voice of support among the many dis- nor to his lab. Yet, he was always in active contact with senters, and he failed to convince. his laboratory and was involved in the day-to-day work. In 1964 HOWARDgave me a manuscript reporting Increasingly he was drawn into the AIDS question, and that virus-infected cells contained DNA that was com- one of his last papers presented a novel strategy for plementary to RNA from the virus, whereas uninfected developing a vaccine (TEMIN 1993). cells did not.This seemed to HOWARD likethe evidence On the Wisconsin campus, HOWARDplayed an in- he was seeking and I communicated it to the PNAS creasingly influential role right up to the time of his (TEMIN1964~). I was innocent of any detailed technical death. When DONNASHALALA came to the University as knowledge, but greatly respected HOWARD’Sjudgment. Chancellor, she relied heavily on HOWARD’Sjudgment The paper waswidely regarded as unconvincing. Hy- about University policies, especially research policies. bridization techniques thenwere not what theyare now, This kind of behind-the-scenes, thoughtful, informal and the presence of other retroviral genomes, unsus- advice was HOWARD’Sstyle. His influence was felt in pected at the time, interfered. But HOWARDsteadfastly many ways, and no doubtwiser decisions emerged from continued to view this paper as an important part of the Chancellor’s office thanks to his input. When SHA- his argument. LALA moved to Washington as Secretary of Health, Edu- Is there a lesson to be learned from this? HOWARD’S cation and Welfare, she continued to rely on HOWARD critics found each experiment wanting in some im- for advice. It was on his suggestion thatshe invited portant respect, yet the several separate lines ofevi- HAROLD VARMUSto become Director of the National dence all pointed in the same direction. HOWARDac- Institutes of Health, an exemplary choice. In Washing- cepted this (DULBECCO1995), but the novelty of his ton as amember of the National Cancer Advisory hypothesis caused many to continue to doubt. In much Board, HOWARDwas a strong advocate of investigator- of biology, there is rarely a single definitive experimen- initiated research. He was an outspoken opponent of tal result. Evidence grows from a number of sources, the Vietnam war, using an occasion during the Stock- none perhaps singly conclusive, but impressive in their holm festivities to make his point publicly. He opposed cumulative impact. In retrospect, HOWARDwas right. I too much expansion of cancer research, citing other wonder how much farther along thebiology of retrovi- needs with higher priority and promise. He got involved ruses would be now if his work had gained acceptance in trying to define and cope with scientific dishonesty. in 1964 rather than only after reverse transcriptase was I remember a lawn party in which he conducted a poll, demonstrated chemically sixyears later. Would our systematically asking each of his guests for an opinion. ideas of an RNA basis of early life, our understanding Despite his heavy schedule of research and the innu- of retrotransposons, our concepts of HIV activity, and merable demands on his time, HOWARDwas somehow our biotechnology be six years farther along? able to take all this in his stride. He liked to bicycle to In any case, the definitive evidence came in 1970. In work and did so when the Madison weather permitted; that summer Iwas in Japan. HOWARDmailed me a copy when it didn’t, hewalked. He took great interest in his of his paper along with a letter of thanks for my confi- garden and flowers, and kept detailed, computerized dence in him when the theory was in great disrepute. records. He met with his students every Friday after- He was finally vindicated. It was characteristic of How- noon at a conference known as the “confession ses- ARD to take the trouble to write a letter of thanks. sion.” He avoided having too many students and post- HOWARDbecame instantly famous. His standing on docs and was always acquainted in detailwith whateach the Wisconsin campus changed accordingly, and when was doing. He met for lunchMonday and Tuesday each the Nobel Prize came in 1975 nobody was surprised. week withlike-minded colleagues for an always-stimulat- Perspectives 5 ing discussion of science and public affairs. HOWARD pensable faculty member, was stricken by the very dis- liked to teach and felt it his responsibility to do so. For ease that he haddevoted his life to. TO addto the irony, morethan 30years he taught in the basicvirology the cancer started in the lung, although HOWARD had course that he had organized. Despite national and in- been a lifelong nonsmoker; however, hiswas an adeno- ternational commitments,he was in Madison and easily carcinoma, not associatedwith smoking. His treat- available most of the time. A call to his phone was usu- ments, many of them experimental, were devastating. ally answered by him personally. But he was determined and stoic. To the extent hu- HOWARD’S Jewishheritage meant a greatdeal to him, manly possible-superhumanly, it seemed-he carried and he regularly attended Sabbath services. Always an on with research conferences, writing, and advising pol- avid reader, he had anextensive library of Judaism. He icymakers. He published 15 papers in the short period, loved reading the Torah and often had a copy with a little over a year, between his diagnosis and his death. him. He enjoyed discussions with his Rabbi and was HOWARDhad always been avoracious reader, andin his especially interested in Jewish history and archeology. last days he read more thanever, especially biographies. HOWARDhad a strong institutional loyalty. He natu- One of my last contacts with HOWARDwas lending him rally received offers of other positions, but he preferred a biography of R. A. FISHER.His last public talk was in to stay in Madison. Part of the reason was that he and October, 1993 at a Chicago symposium celebrating the his family liked their home and their life. HOWARDwas fortieth anniversary of the WATSON-CRICKmodel of close to his wife, RAYLA,and their two daughters, SARAH DNA (TEMIN1995). His death came on , and MIRIAM.He spentmany hours with them, and their 1994 at the age of 59. interests took highest priority in his schedule. He was In planning the remainder of his life, HOWARDas- respectful of his daughters’ feminism and political activ- sisted in organizing a 60th birthday symposium featur- ism, and joinedthem in symbolically expressing sympa- ing his former students and postdocs. Alas, he did not thy for AIDS victims at a White House ceremony; the live to attend the symposium, which was held in Octo- occasion was HOWARD’Sreceipt of the National Medal ber, 1994. The resulting published book includes sev- of Science in 1992. eral of HOWARD’Skey papers along with contributions HOWARDliked to travel, and after each trip his friends from former studentsand associates, a fitting memorial were invited to his home to hear about it and see the (COOPERet al. 1995). numerous colored slides that he brought back. A trip HOWARDis survived by hiswife RAYLA GREENBERG for HOWARDwas often a working trip, reading books in TEMIN andtwo daughters, SARAHand MIRIAM.RAYLA advance and giving seminars and visitinglabs while carried out several studies on the concealed viability there. After touring China in 1977 as part of a trade and sterility loads in natural populations of Drosophila. mission, he wrote a report on the state of biological More recently, she has turned her attentionto the Segre- research (TEMIN1978). Most memorable was a trip to gation Distortionphenomenon and, among otherthings, Russiain 1976. He tookwith him several forbidden demonstrated the independentdistorting activity of the items, including Hebrew booksand recent scientificjour- Enhancerlocus, E( SD) . She is an unusually conscientious nals. Before leaving, he obtained lists of scientists, espe- teacher and regularly offers an introductory genetics cially refuseniks and dissidents. He prepared for the trip course; she is held in great admirationand affection by by learning the Cyrillic alphabet and bringing a pocket her students. flashlight; these enabled him to find apartments at night. Wethank RAYLA TEMINand BILL SUCDENfor carefully reading In this way he was able surreptitiously to meet dissidents an early draft and offering thoughtful suggestions. RAYLA provided and refuseniks and to attend their clandestine “Sunday documents and shared personal memories that have enriched this Seminar.” Notably, he obtained and brought back a re- article. We thank RAY OWENfor his recollections and MATT MESELSON cording taped at the apartment of and FRANKSTAHI. for their critical comments. along with a message to the outside world from SAKHA- ROV himself. These evening expeditions were made in LITERATURE CITED considerable fear, both for himself and those he was BALTIMORE,D., 1970 Viral RNAdependent DNA polymerase in viri- visiting. The distressing details of the life of dissident ons of RNA tumor viruses. Nature 226: 1209-1211. Russian scientists are spelled out in a diary of his visit BISHOP,M., 1995 Viruses, genes, and cancer: a lineage of discovery, (TEMIN1977). HOWARDbrought back valuable informa- PP. 81-94 in The DNA Prouim: Howard Temin’s Sckti$c Legacy, edited by G. M. COOPER,R. G. TEMINand B. SUGDEN.American tion for such groups as Amnesty International. The Madi- Society for Microbiology, Washington. son evening when he told his guests about the trip was COOPER,G. M., R. G. TEMINand B. SUGDEN,1995 TheDNA Provirus: especially poignant for one attendee,my newly acquired Howard Temin’s Scientifc Legacy. American Society for Microbiol- ogy, Washington, DC. ALEXANDER graduate student, GIMELFARB, recentlya re- DOUGHERTY,J. P., and H. M. TEMIN,1986 High mutation rate of a fusenik himself. spleen necrosis virus-based retrovirus vector. Mol. Cell. Biol. 6: HOWARD’Sfinal illness, premature, unexpected, and 4387-4395. DULBECCO,R., 1995 Howard M. Temin (10 -9 Feb- ironic, was a body blow to the Wisconsin community. ruary 1994). Proc. Am. Philos. SOC.139: 452-462. This man, Wisconsin’s best known scientist, the indis- HU, W.S., and H. M. TEMIN,1990a Genetic consequences of packag- 6 J. W. Drake and J. F. Crow

ing two RNA genomes in one retroviral particle: pseudodiploidy TEMIN, H. M., 1963 The effects of actinomycin D on growth of Rous and high rate of genetic recombination. Proc. Natl. Acad. Sci. sarcoma virus in vitro. Virology 20: 577-582. USA 87: 1556-1560. TEMIN,H. M., 1964a Malignant transformation in cell culture. Hu, W.-S., and H. M. TEMIN,1990b Retrovirus recombination and Health Lab. Sci. 1: 79-83. reverse transcription. Science 250: 1227-1233. TEMIN,H. M., 1964b The participation of DNA in Rous sarcoma INGALIS,T. H., F. R. AWS,F. J. CURLEYand H. M. TEMIN,1953 Ge- virus production. Virology 23: 486-494. netic determinants of hypoxia-induced congenital anomalies. ,J. TEMIN,H. M., 1964c Homology behveen RNA from Rous sarcoma Hered. 44: 185-194. virus and DNA from -infected cells. Proc. Natl. MANAKER, R. A,, and V. GROUPE, 1956 Discrete foci of altered Acad. Sci. USA 52: 323-329. chicken embryo cells associated with Rous sarcoma virus in tissue TEMIN, H.M., 1977 Moscow diary. Sciences 17(4): 26-27. cultures. Virology 2: 838-840. TEMIN,H. M., 1978 China diary. Sciences 18(4): 27-28. MIZUTANI,S., and H. M. TEMIN,1976 Incorporation of noncomple- TEMIN,H. M., 1993 A proposal for a new approach to a preventive mentary nucleotides at high frequencies by ribodeoxyvirus DNA vaccine against human immunodeficiencyvirus type 1. Proc. Natl. polymerases and Escherichia coli DNA polymerase I. BiochemisLry Acad. Sci. USA 90: 4419-4420. 15: 1510-1516. TEMIN,H. M., 1995 Genetics of retroviruses. Ann. N.Y. Acad. Sci. PATHAK,V., andH. M. TEMIN,1990a Broad spectrum of in uivo 758: 161-165. forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after asingle replication cycle: substitu- TEMIN, H.M., and S. MIZUTANI, 1970 RNA-dependent DNA poly- tions, frameshifts, and hypermutations. Proc. Natl. Acad. Sci. merase in virions of Rous sarcoma virus. Nature 226: 121 1 - USA 87: 6019-6023. 1213. PATHAK,V., andH. M. TEMIN,1990b Broad spectrum of in uivo TEMIN,H. M., and H. RCTBIN,1958 Characteristics of an assay for forward mutations, hypermutations, and mutational hotspots in Rous sarcoma virus and Rous sarcoma cells in tissue culture. a retroviral shuttle vector after a single replication cycle: dele- Virology 6: 669-688. tions and deletions with insertions. Proc. Natl. Acad. Sci. USA ZARI.ING, D. A., and H. M. TEMLN,1976 High spontaneous mutation 87: 6024-6028. rate of an avian sarcoma virus. J. Virol. 17: 74-84.