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. 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