EUGENE GARFIELDINSTITUTE FOH SCIENTIFIC lNFORMATlON@3501 MARKET ST, PHILADELPHIA, PA 19104

The Impact of Basic Research in GeneticRecosnbination-A PersonRl Account byJoshua Lederberg. Part 2. A Discovery

Recounted and Evaluated

Number 25 Iune 20, 1988

Last week we presented the first of twoinstallments of’ ‘Genetic recombination inbacteria: a discovery account” by JoshuaLederberg, president, The Rockefeller Uni-versity, New York. 1The article originallyappeared in the Annual Review of Geneticslast year.z With this second part, the nar-rative concludes.

In the first part, Lederberg described hisbeginnings as a scientist. He also introducedthe central research effort of his early career,starting in medical school: a search for evi-dence demonstrating the existence or ab-sence of sexurd recombination in bacteria.Bacteria, as Lederberg observed, could notbe adopted as models for genetic researchuntil there was some substantiation of theview that they had a genetic system likeother organisms. To that end, he applied inthe late summer of 1945 to work with bio-chemist Edward L. Tatum, who was settingup a microbiology lab at Yale University,New Haven, Comecticut.

Tatum generously offered laboratory fa-cilities, the wisdom of his experience, andhis collection of double-mutant strains ofEscherichia coli strain K-12. With these

strains Lederberg promptly demonstratedthe recombmtion process, and the result hasbeen that’ ‘K-12” has become a byword ingenetic research and biotechnology. For ex-ample, in 1987 there were 168 citations toa 1983 review of the genetic map of this or-ganism by Barbara J. Bachmarsn, Yale Uni-versity Medical Schools Her 1987 reprintof this review, with cumulative bibliogra-phy, cites 1,993 articles through 1982.’1

In 1958 the Nobel I%ze in physiology ormedicine was awarded to George W.Beadle, then at the California Institute ofTechnology, Pasadena, and TatUrnfor’ ‘thediscovery that the genes regulate statedchemical processes, ” and to Lederberg forhis “discoveries concerning genetic recom-bination and the genetic apparatus in bacte-ria. ”5

*****

My kinks to Chris~opher KiBg and PatTaylor for their help in the preparation ofthis essay.

~1988 [S!

1,

2.3.4.

5.

REFERENCES

Garffeld E. The impact of basic research in genetic recombination-a personal ruxnmrt byJoshua Lcderberg. Part 1. The search for a acxual stage in bacteria. Current Corrrerm(24):3-17, 13 Jmrc 198S.

Lederberg J. Genetic recombination in bacteria: a dkcovery accnmrt. Arrru. Rev, Gene?. 21:23-46, 1987,Bachmann B J. Linkage mapof Ercherichia cd K-12, edtion 7, ,44icmbioL Rev. 47:180-230, 19S3.-——--- Lirdrage map of .Ederichia di K-12, edition 7. (Neidhardt F C, cd.) Eschericfria coli and

Salmonella typhirrmrimn: cellular and molecular biology, Volume 2. Washington, DC: AmericanSnciesy for Microbiology, 1987. p. 807-76.

Royaf Swedfafr Academy of sciences. La PriJ Nobel en 1958 (The Nobel Prizes in 195g). Stockholm,Sweden: Nobel Fntmdatinn, 1959. p. 6.

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Reproduced, with permission. fmm the Amwd Reviewof Geneticf,Vol. 21. @ 1987 by #uumal Reticws Inc

GENETIC RECOMBINATION INBACTERIA: A DISCOVERY ACCOUNTJoshua Lederberg

The Rockefeller University, New York, New York 10021

Once I was at New Haven, my lab efforts were devoted to rechecking thestability of Tatum’s existing double-mutant strains, like 58-161 and 679-183(biotin-methionine and threonine-proline, respective y). Then, additionalmutations such as resistance to virus T 1 were also incorporated to allowsegregation of unselected markers among lhe prototrophs selected from themixed cultures on minimal agar medium. It took about six weeks from thetime the fiist serious efforts at crossing were set up in mid-April to establishwell-controlled, positive results. By mid-June, Tatum and I felt that the timewas ripe to announce them.

A remarkable opportunity was forthcoming at the international Cold SpringHarbor Symposium. This year, it was to be dedicated to genetics of microor-ganisms, signaling the postwar resumption of major research in a field thathad been invigorated by the new discoveries with Neurospora, phage, and therole of DNA in the pneumococcus transformation. Tatum was already sched-

uled to talk about his work on Neurospora. Happily, we were also granted alast minute insertion near the end of the program to permit a brief discussionof our new results (65). (I have found no written record of the precise date; theSymposium was scheduled for the week of July 4, 1946.)

The discussion was lively! The most reasoned criticism was Andre Lwoff’sconcern that the results might be explained by cross-feeding of nutrientsbetween the two strains without their having in fact exchanged geneticinformation. He was familiar (94) with nutritional symbiosis in Hemophilus(72). [1 did not think to counterargue that the apparent cross-feeding (94) wasactually a genetic exchange. Indeed,Hemophilus is now known to acceptDNA in a manner analogous to the transformation system in the pneumococ-cus (25). This counter hypothesis has not, however, been substantiated. ]

Having taken great pains to control the artifacts from cross-feeding, I feltthat the indirect evidence we had gathered, especially the segregation ofunselected markers, should be accepted as conclusive, and 1 spent moreargument than necessary on whether more direct proofs need be furnished thatthe purported recombinant were indeed pure strains. Fortunately, Dr. MaxZelle took me aside after the meeting and most generously offered to adviseand assist me in the direct isolation of single cells under the microscope, so asto lay such concerns to rest (100).

The Cold Spring Harbor meetings in 1946 (and again in 1947) were also amarvelous opportunity to benefit from new or renewed introductions tooutstanding figures in genetics. Many of the scientists were also extraordinari-ly supportive human beings, both in thoughtfully listening to the logic of my

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experiments, and in offering good advice (personal and technical) about howto respond to criticisms. Discussions with figures like Andre Lwoff, JacquesMonod, Guido Pontecorvo, Maclyn McCarty, Seymour Cohen, BernardDavis, Boris Ephrussi, Raymond Latarjet, Colin Pittendrigh, Curt Stem, C.B. van Niel, Ernst Caspari, J. F. Crow, M. Demerec, Alex Hollaender,Rollin Hotchkiss, Dan Mazia, Howard Newcombe, Elizabeth Russell, JackSchultz, Wolf Vishniac, M. J. D. White, Evelyn Witkin—and many others—helped to promote lifelong correspondence, and personal and scientific rela-tionships. I remember most vividly the warmth, interest, and friendshipoffered by Tracy Sonnebom, H. J. Muller, and Salvador E. Luria; later also

by Leo Szilard and J. B. S. Haldane, in discussing the work as it unfolded. Itis hard to overestimate the importance of these meetings in sustaining theinterpersonal network in science.

The most gratifying evidence of the acceptance of these claims by myscientific colleagues was the trickle (later a torrent) of requests for the culturesof E. cofi K-12 to enable others to repeat the experiments. The first significantconfirmatory publications bore the name of Luca Cavalli-Sforza (14), origi-nally from R. A. Fisher’s laboratory at Cambridge and later from Milan andPavia. This prompted the beginning of an extended transatlantic (and latercollegial) collaboration with Cavalli-Sforza that was most gratifying bothscientifically and personally.

The only studied holdout was Max Delbriick: he quite curtly expressed hisdisinterest in the phenomenon for the lack of a kinetic analysis. His admoni-tion was immaterial to the claims on the table; it was, however, the kernel ofthe methodology later used to such good effect by Wollman & Jacob (99) inshowing that fertilization was a progressive entry of the chromosome, takingabout 100 minutes for consummation. That story has now been well told inJacob’s personal memoir (36a).

By September 1946 I was scheduled to resume my medical studies at P & S,but this was obviously the most unpropitious time to interrupt the excitinginitial progress with crossing in E. coli. I was granted another year’s leavefrom P & S and a renewal of my fellowship from the Jane Coffin Childs Fund.The year enabled a consolidation of the preliminary reports and especially therecruitment of many additional genetic markers and the publication of the fmtlinkage map (44), The detailed physical mechanism of crossing was stillobscure; it was not, however, mediated by extracellular DNA, for it was quiteuninfluenced by the addition of deoxyribonuclease (generously provided byMaclyn McCarty) to the medium. [It would take later discoveries, especiallyof Hfr (High frequency of recombination) strains by Cavalli-Sforza (13) toopen up progress on mechanism.]

A persistent disappointment was the failure of efforts to demonstrate DNAtransfer in E. coli, which would have completed the paradigmatic aims of theexperiment. Boivin & Vendrely had reported such a finding (9), but none ofus, Boivin and his collaborators included, was able to reproduce it (R.Tulasne, personal communication; 8), perhaps owing to deterioration of therelevant strains.

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[

ZOOIOf&YDept.

1Columbia UniversityNew York, N.Y.

19 September 1945

Dr. E. L. Tatum,Dept. of Botany,Yale University,New Haven, Corm.

Dear Sir:

Your recent paper ‘X-Ray Induced Mutant Straineof EscherichiaColi’ has just come torny attention, andhas proven very fascinatingI should be very much obliged to you for reprinte of this paperand your preliminary one last summer. I ehall take the liberty ofwriting to you at this length in support of a request that I hopeyou will entertain.

After doing some work on adaptation (part of which ie nearlyready for publication) in Neurospora mutants, it occurred to methat no adequate investigation of a genetic nature had been madeto demonstrate the existence or abeence of sexual recombination inbacteria. Such things asthediatributionof somatic and flagellarantigens in the Salmonella group very strongly suggest that sucha process may occur, but no very successful attsmpt seems to havebeen made to determine the recombination of bacterial characters.The nutritional mutants described by yourself and Roepke et al.would seem to fill the bill.

I have not yet gone very far in the genetic tests I ❑entioned(explicitly) on these strains: themethionineless is quite Roughthersfore poesibly not so satisfactory. I had planned to do ess-entially what you have accomplished: prepare a double mutant bysubjecting the prolineless to the same eelective procedure usedobtaining methionineless, but that seems unnecessary now fora demonstration that independent (X–ray mutable) genes exist. It haseeemed to ❑e, however, that despite the apparent stability of thetypes I now have, and what is I hope adequate technique to eliminatecontamination that it would be highly desirable to have geneticallymarked strains befors any attempt was made to perform ths experiment.I should therefore be very much obliged to you for cultures of yourbiotindouble mutant series forthe purposes of this investigation.

If an investigation of thie sort has already occurred to you,pleaee let me know, as I ameure that you candoa much better joband have better facilities for it than I; on the other hand, if yourplane do not include work such as this I ehouldappreciats very❑uch the service I ask of you.

Very sincerely yours,

Joshua Lederberg, A.S. V-12 USNR.

Figure 1 Copy of 1945 letter to E. L. Tatum.

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Since 1946, E. coli K-12 has been the subject of innumerable furtherinvestigations, in hundreds if not thousands of laboratories (2). These havesubstantially revised and enriched our first simple models of the sexualbehavior and genetic structure of E. coli, though many questions remain open(29, 36a, 37,53,55, 56). Many technological applications of gene transfer inE. coli have, of course, also emerged. The detailed story of the fructificationof the initial discovery is an example of international cooperation and compe-tition that desewes a richer and better informed treatment at some future time.

September 1947 was the next deadline of personal history: I was to return toNew York and continue my interrupted medical studies. Ryan also offered melaboratory facilities, and he and Tatum looked hard and partly successfully forsome financial support to make all that possible. Meanwhile, Tatum hadnegotiated with Yale my retroactive registration as a graduate student and hadobtained assent from other professors that I had de facto emolled in a numberof their lecture courses and seminars. The work of 1946-1947 became mydissertation, which I had already defended before an international panel ofexperts. A more serious personal obstacle was obligatory retroactive paymentof tuition to Yale University; but the happy result was to qu~ify for a PhDdegree that would, as it turned out, widen my career options. I spent thesummer of 1947 at Woods Hole (and the magnificent library of the MarineBiological Laboratory), completing the dissertation. The stacks gave a won-derful opportunity to explore the history of microbiology: how its pioneershad sought to cope with the perplexities of bacterial variability, totallyisolated from the intellectual apparatus of modern genetics.

In mid-August, days before the resumption of medical school, I learnedfrom Ed Tatum that the University of Wisconsin had contacted him about anopening in genetics. In a fashion revolutionary for the time, they were seekinga microbial geneticist! He had recommended my name, and as a Wisconsingraduate his word carried great weight there. 1 have since learned of thecontroversy that this proposal evoked. Understandably, the appointment of a22-year old as an assistant professor warranted close examination. Somereferees at Cal Tech were still skeptical about the E. coli research: a painstak-ing review by Ray Owen (at Cal Tech, but recently from Wisconsin) did muchto allay concerns in that sphere. Most troubling were allusions about characterand race—someone with far stronger suits of tact and polish than mine wouldhave been a more compelling nominee to be among the first Jewish professorsin a midwestem college of agriculture. (There have been some happy changesin this country over forty years. We still have many burdens of fairness inmeeting the cries for equity from other groups subject to discrimination.) Ithas been enormously gratifying to have learned in later years of the largeeffort and integrity of support that were offered by R. A. Brink and M. R.h-win (at Wisconsin) and by E. B. Sinnott (at Yale). It is a measure of theirstature that I was, in the event, offered the position; and when I did come toMadison I was given no inkling of what a struggle I had engendered.

The offer posed the deepest dilemma of my career. 1 was deeply committedto medical research. Two more years of clinical training (and to be meaning-

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ful another two or three of internship and residency) would have reinforcedthe medical credential, but been a grave (if not total) interruption of researchat its most exciting stage. The Wisconsin position was the only one visible forunmitigated support of research in bacterial genetics. That university wasfurthermore a seat of biochemistry (especially in the Enzyme Institute) andhad a long tradition of research in genetics and in microbiology (albeit quiteseparately up to that point). The Wisconsin Alumni Research Foundation,with income from professors’ patents, was a further resource in aiding pioneerresearch. All this was, however, seated in the College of Agriculture, notMedicine. The medical school at Wisconsin at that time, furthermore, gavelittle emphasis to research, except for the McArdle Institute for CancerResearch. In short order, I did of course go to Wisconsin, and have never hadsecond thoughts about the wisdom of the choice. Tbe agricultural researchcontext gave me a grounding in practical applications of biotechnology that 1have never regretted. I enjoyed a happy collegial association with Brink andh-win, and shortly thereafter with James F. Crow (whom I had met at the 1947Cold Spring Harbor symposium) and many other close friends and colleagues,that could be matched at no other time or place. in the long run, however,affiliation with a medical educational and research environment was to be amore compelling vocation. Together with Arthur Komberg’s concurrentmove, this was to be the principal attraction of Stanford University, when Iwas invited to join the new medical school effective February 1959. Thatopportunity to return to medically centered activities at Stanford, and later atThe Rockefeller University in 1978, has substantiated the advice 1 had fromTatum in 1947. Nevertheless, among my most cherished honorifics are theMD degrees (honoris causa) that I have received from Tufts and from theUniversity of Turin.

“Contrafactual history” is often derided. Nevertheless, if historical analysisis to go beyond the selection of narrative detail and to assert some theoreticaldepth, it ought ask “what if?” That is, it should make a plausible cause for“postdicting” alternative outcomes, given different hypothetical inputs. There

is. of course, no way to verify such speculations; but unless we indulge inthem, how can we speak of learning anything from history?

Without the serendipity of E. cofi K- 12, could sexual genetic recombina-

tion have remained undiscovered until this day? As we know in retrospect(47), the choice of E. cd strain K-12 was lucky; one in twenty randomlychosen strains of E. coli would have given positive results in experimentsdesigned according to our protocols. In particular, strain B, which Delbriickand Demerec had insisted upon as a canonical standard, would have beenstubbornly unfruitful. Tatum had acquired K-12 from the routine stock culturecollection in Stanford’s microbiology department when he sought an E. colistrain to use as a source of tryptophanase in work on tryptophane synthesis inNeurospora (92). The same strain was then in hand when he set out to makesingle, and then double, mutants in E. cofi (91). In 1946 I was very muchaware of strain specificities and was speculating about mating types (as inNeurospora). 1 have no way to say how many other strains would have been

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tried, or in how many combinations, had the June 1946 experiments notworked out so successful] y. K-12 has also been the source of the prototypicextrachromosomal elements, F and lambda.

The serendipitous advantages of K- 12 notwithstanding, E. ccdi recombina-tion might have been discovered eventually as a byproduct of studies on theinfectious transmission of drug resistance, which has become an importantpractical problem with many pathogenic bacteria. The development ofmolecular genetics along other paths would have eased the resistance toconjectures about a genetics of bacteria; it would have reduced the incentivesto topple the icons; above all it would have vastly multiplied the number ofpeople seeking their own creativity niches (78) in this general area of work. Itis hard to imagine that a bacterial conjugation system would not have beendiscovered at least by the 1950s or 1960s.

Let us stipulate nondiscovery and ask, only partly tongue-in-cheek, howmuch regret that resynchronization of history would have entailed. Withoutthe E. coli system, the optimistic hypothesis is that other paths would havereceived still more attention. (We cannot be sure that they would haveattracted a compensatory interest. ) Delbriick and Hershey had already dis-covered recombination in vimses (18, 30), The discove~ of sex in E. coli wasnot a prerequisite for the work of Hershey & Chase (31) on the role of DNA inthe virus life cycle, nor that of Watson& Crick (96) on the stmcture of DNA.Without the distractions of another genetic system like E. cofi, even moreattention might have been paid to the pneumococcal transformation and thesearch for more tractable systems like it.

A significant impediment would have been the lack of detailed geneticmaps of the bacterial chromosome; but they might well have been built uppiecemeal by other methods. Still more likely would have been less emphasison bacterial genetics and more on the viruses with their simpler structure. It isconceivable that this would have led to even deeper and more rapid advancesat the strictly molecular level, perhaps at the price of a scientific naturalhistory of bacteria, of correlating DNA research with classical genetics, andof some practical advances in biotechnology using bacterial hosts.

Other casualties of the reemphasis of bacteria might have been someaspects of phenomena like plasmids, Iysogeny, and Iysogenic conversion: theincorporation of vimses into the bacterial chromosome (30a, 43, 50, 54).These concepts have achieved some importance as models for oncogenes.Alternatively, some completely different and even more attractive ex-perimental models, unknown to us at the present time, might have emerged.

Historians should ask about the likely consequences of other counteracts,stipulated in isolation. They are most provocative if directed at significantdiscontinuities in the history of science. Besides the further consequences, wecan also ask whether such discoveries, perhaps even manyfold, are fore-ordained in the contemporary milieu (74, 74a, 74b). What if Avery had notpursued the chemistry of the pneumococcus; if Beadle and Tatum hadnot thought of fungi for biochemical genetics; if Watson and Crick had notpursued the physical chemistry of DNA? Each of these questions has a

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different genre of answers; on some of them, we might even discover aconsensus. These fantasies point to the importance of how attention is focusedin the scientific community, a matter as important as, but coupled loosely to,

the specific knowledge that is passed on from generation to generation.Our historical and social understanding does not give us a predictive gauge

of the macroeconomics of scientific interest in given fields and the socialresources they will attract over periods of time. Every field of enquity has thelatent potential of enormous unforeseeable outcomes. One function of adiscovery is to lend credibility to a given avenue of pursuit, and to a newmomentum of effort there.

This memoir is dedicated to Francis J, Ryan and Edward L. Tatum. At atime when the public image of scientific fraternity is so problematical, theirlives reflect the survival of norms (74) and behavior exemplifying mutualrespect, helpfulness, consideration, and above all a regard for the advance-ment of knowledge.

Today’s popular portrayals of the scientific culture give short shrift to

anything but fraud and competition. What contrast to the idealizations by deKruif and others that inspired my generation! This emphasis may stem in partfrom the reluctance of scientists to speak out in literary vein, with a fewatypical exceptions: outstanding] y, June Goodfield in her books and televisionseries (23, 24), which are a renascence of the de Kruif tradition. Thecompetitive stresses on young scientists’ behavior today must be acknowl-edged. The role modeling and critical oversight of their scientific mentorshave also warranted celebration (39, 102). These are now complicated by thedisappearance of leisure in academic scientific life, the pressures for funding,and academic structures and a project grant system that give too little weightto the nurture and reassurance of the human resources of the scientificenterprise. The often contradictory demands on the scientific personality areill understood: antitheses such as imagination vs critical rigor; iconoclasm vsrespect for established truth; humility and generosity to colleagues vs arrogantaudacity to nature; efficient specialization vs broad interest; doing ex-periments vs reflection; ambition vs sharing of ideas and tools—all these andmore must be reconciled within the professional persona, not to mention otherdimensions of humanity (21, 75).

I have never encountered the extremities that Jim Watson painted in his

self-caricature of ruthless competition in The Double Helix (95), which ishardly to argue that they do not exist. Side by side with competition, scienceoffers a frame of personal friendships and institutionalized cooperation thatstill qualify it as a higher calling. The shared interests of scientists in thepursuit of a universal truth remain among the rare bonds that can transcend

bitter personal, national, ethnic, and sectarian rivalries.

ACKNOWLEOGMENTS

Anyone who knows them, and myself, will recognize how far I have beeninformed by the sociological insights of Robert K. Merton and Harriet A.

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Zuckerman. Our retrospection was launched in 1974 during a fellowship year

on the history and sociology of science at the Center for Advanced Studies inBehavioral Sciences in Stanford, California. My work in 1946 would not havebeen possible without the willingness of the Jane Coffin Childs Fund forMedical Research to support a calculated risk, in a way that had few parallelsthen and has had few since. I am indebted to many colleagues who havefurnished invaluable documentary sources. The relevant archival materialswill be deposited at the Rockefeller Archive Center, Pocantico Hills, NewYork, whose staff have also been most helpful throughout this effort. Thisarticle is taken from an autobiographical work in progress, many years shortof publication. I appreciate the editors’ assistance in selecting those elementsmost appropriate for the present readership.

BIBLIOGRAPHIC NOTE

A number of items of history, briefly touched upon here, have been reviewedmore fully: 45, 48-52, 54-56, 60a, 67.

NOTE ADDED IN PRCK)F An important encyclopedic overview of E. coligenetics has just been announced: Neidhardt, F. C., ed. 1987. Escherichiacoli and Salmonella typhimurium: CeUular and Molecular Biology. Vol. 1, 2.Washington, DC: Am. Sot. Microbiol.

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