Honoring Avery, MacLeod, And McCarty: The Team That Transformed Genetics

Editor's Note: On Feb. 1, 1944, the Journal of Experimental Medicine published a scientific paper entitled "Studies on the chemical nature of the substance inducing transformation of pneumococcal types." Coauthored by Rockefeller Institute (now University) Hospital researchers Oswald Avery, Colin MacLeod, and Maclyn McCarty, the paper--preceding by a decade the Nobel Prize- winning revelations of James Watson and Francis Crick--described the discovery that genes are made of deoxyribonucleic acid, or DNA; prior to their studies, no biological assay was available to link genetic information with DNA. Immunologist and Nobelist Peter Medawar called the Avery team's isolation of genes in pure chemical form "the most interesting and portentous biological experiment of the 20th century." Another Nobelist, Joshua Lederberg--former president of Rockefeller University and now University Professor at that institution--lauds it as "the pivotal discovery of 20th-century biology." Lederberg, who received the 1958 Nobel in physiology or medicine for his contributions in genetics, was a young researcher at nearby Columbia University in New York during the period of momentous achievement at Avery's Rockefeller lab. In the following essay, Lederberg describes that achievement, recounts the controversy it spawned, and assesses its impact on subsequent investigations. A version of this essay appeared in a recent issue of the journal Genetics (136[2]:423-26, 1994) and is used here with permission. The publication 50 years ago this month in the Journal of Experimental Medicine of a research report by three Rockefeller University Hospital investigators--Oswald Avery, Colin MacLeod, and Maclyn Mc-Carty--marked the opening of the contemporary era of genetics, its molecular phase. The reverberations continue, now dominating large sectors of biomedical science and biotechnology, and have established the centrality of genetics in biological thought. The trio's findings can be dissected into the following observations, claims, and tacit extrapolations, which may be paraphrased as: Certain bacteria (pneumococci) have clonally inherited attributes, notably serospe-cific polysaccharide capsules. These are associated with virulence and can be selected for accordingly, by inoculation into mice or by serological reagents. The genetic anlage, or determinants, 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 succeeding clonal generations. The chemical structure of that transforming principle is DNA, to the exclusion of protein or other macromolecules. Founded on these claims, the radical ideas emerged that: Bacteria have discrete, autonomous genes analogous to those of higher life forms--namely, Drosophila. The gene is DNA; and the transformation phenomenon affords the first bioassay for genes extractable in vitro. Accordingly, bacteria might be favored subjects for genetic investigation and, eventually, for technological application of molecular genetic science. I recite these principles with some nostalgia: They are precisely how they came across to me as an undergraduate already working on neurospora at the Columbia University zoology department in New York City. here, I have noted how these principles vectored my own career aspirations into the pioneering of bacterial genetics (J. Lederberg, "Genetic recombination in bacteria: A discovery account," Annual Review of Genetics, 21:23-46, 1987). Studying in the academic archipelago called New York, I was uniquely well situated to observe and sometimes participate in the debate, since Rockefeller was just across town. Alfred Mirsky, a senior biochemist at Rockefeller, was a frequent visitor to my department at Columbia, where he was collaborating with Arthur Pollister. From 1942 on, I heard a good deal about the progress in Avery's laboratory, and reprints of the "Avery et al." article were circulated in the department. I borrowed one from Harriett Taylor (later Ephrussi-Taylor), a graduate student working on yeast budding kinetics, who would shortly join Avery's laboratory for her postdoctoral research. My personal exclamatory notes were "... unlimited in its implications...Direct demonstration of the multiplication of transforming factor...Viruses are gene-type compounds..." While Mirsky was the principal herald, he also was a dogged critic of the claim that DNA, alone, had proved to be the exclusive chemical substance of transforming activity (A.E. Mirsky, A.W. Pollister, "Chromosin, a desoxyribose nucleoprotein complex of the cell nucleus," Journal of General Physiology, 30:117-48, 1946). That was indeed a difficult proposition: Avogadro's number is a formidable protagonist in that contest. It meant that one microgram of a biopolymer might still have more than 10 trillion molecules. Even if 99.99 percent pure, that still allows for the possibility of a million contaminating molecules of other kinds. My stance was sympathetic to Mirsky's: I felt that so crucial a claim should not be impulsively engrafted into the corpus of science. More important than doctrinal conversion was that the issue was squarely on the table and could be settled--one way or another--by overwhelming experimental analysis. Previous fiascoes had darkened the history of biopolymers: for example, Wendell Stanley's initial assertion in 1935 that crystallized Tobacco Mosaic Virus is a pure protein. Avery himself was an epitome of caution, having had to weather similar skepticism that pneumococcal polysaccharide, devoid of protein, was a type-specific antigen. The main fruit of the debate was to stimulate a range of further inquiries: Erwin Chargaff on the base composition of DNA, for example, and my own on other modes of gene recombination in E. coli. Maclyn McCarty, later joined by Rollin Hotchkiss, added much to the repertoire of enzymatic and analytical refinements for the exclusion of protein from the DNA preparations. 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. The protein enthusiasm evoked by the successful crystallization of enzymes in the 1930s then dominated most biochemists' attention. In fact, the idea of DNA's bearing biological specificity was more popular at the beginning of the 20th century: "A tempting hypothesis...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-substance," wrote E.B. Wilson (The Cell in Development and Inheritance, 2d ed., New York, Macmillan Publishing Co., 1906, page 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 (i.e., basichromatin). It is the expression of a morphological organization 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" (E.B. Wilson, The Cell in Development and Heredity, 3d ed., New York, Macmillan Publishing Co., 1925, page 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 (G. Clark, F.H. Kasten, History of Staining, Baltimore, Williams & Wilkins, 1983). The biological interpretation of the pneumococcus transformation was also fraught with uncertainty. Theodosius Dobzhansky and, later, Andre Boivin persisted in describing the phenomenon as a "directed mutation," and it was given overtones of "cytoplasmic inheritance" by Tracy Sonneborn; these were all rhetorical devices intended to seal off a vaguely understood phenomenon from the sureties of chromosomal inheritance. Nothing 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, like those of fruitflies and garden peas. For many years, the only marker studied had been the capsular polysaccharide. In that setting, even Harriett Ephrussi- Taylor in 1951, reporting from the Rockefeller Institute, remarked, "... no bridge can be seen leading over into classical genetics" and, in private correspondence, criticized my own efforts to do precisely that. Among early comments from geneticists, that of Hermann Muller's, in 1947, was closest to the mark: "[The] pneumococcus results then becomes that of [a] type of crossing over, though on a more minute scale....[involving] viable bacterial `chromosomes' or parts of chromosomes [penetrating] 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" (H.J. Muller, "The gene," Proceedings of the Royal Society of London, B134:1-37). In a retrospection over prior hypothetical interpretations of the Avery team's transforming principle, seven alternatives to it could be listed (J. Lederberg, "Genetic transduction," American Scientist, 44:264-80, 1956). It was a specific mutagen with a special ability to direct a particular gene to mutate in a definite direction. It was a polysaccharide autocatalyst (perhaps as a complex with DNA) that primed an enzymatic reaction for polysaccharide synthesis. It was a bacterial virus, which on infecting the bacteria provoked capsular synthesis as a host reaction. It was an autonomous cytoplasmic gene or a morphogenetic inducer. It might be acting at a distance without penetrating the bacterium. It was a fragment of the genetic make-up of the bacterium, the only one to have been tested to that time. It was a sui generis element 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 work using markers other than the capsule, and especially by the linkage of mannitol fermentation and streptomycin resistance. The notion also was bolstered by other phenomena of gene transfer, such as conjugal exchange in E. coli and virus-mediated transduction in Salmonella. Finally, the monopoly of the pneumococcus on transformation--and this was a notoriously difficult experimental system--was broken by Hattie Alexander and Grace Leidy's 1951 report on Hemophilus (H.E. Alexander, G. Leidy, "Determination of inherited traits of H. influenza by desoxyribonucleic acid fractions isolated from type-specific cells," Journal of Experimental Medicine, 93:345-59), so that a stream of other workers could provide mutual confirmation and reinforcement about the biological interpretations. Eventually, the debate about DNA chemistry petered out by sheer exhaustion of the critics and the conceptual plausibility of DNA as gene introduced in 1953 by James Watson and Frances Crick's double helix model. Alfred Hershey and Martha Chase's 1952 study of the injection of phage DNA into E. coli lent further support to the "DNA only" view; however this was quantitatively less rigorous than McCarty and Hotchkiss's 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--by Arthur Kornberg and Har Gobind Khorana--in the 1960s only with the in vitro synthesis of biologically active DNA. The 1944 Avery paper originally was 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 the research team's claims to the so-called prematurity of Mendel's ideas in the last third of the 19th century. Mendel was little known--and for the most part ignored--by his contemporaries. But I would argue that the critical reception initially given to the Avery et al. paper exemplifies the critical scientific meth-od at its most functional. Far from being ignored, the paper enjoyed almost 300 citations between 1945 and 1954, according to data from the Institute for Scientific Information. Indeed, it became so well known during that decade that it was often cited by indirection--that is, without specific reference. As for charges that the Avery team's findings were "premature," this might mean--as it does in any case in which "prematurity" is attributed--that (a) data did not exist at the time to explain all of the paradoxes and challenges of the new discovery; or that (b) the audience of the time was incapable of understanding the challenge implicitly presented. The touchstone is plainly in the operational reaction. For the Avery team--as it has been for others, such as Barbara McClintock--the operational reaction comprised open controversy and active inquiry. For Mendel, it meant oblivion, and a long delay before rediscovery. Fortunately, 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 revisions loom ahead even for our well-established dogmas. That Avery, MacLeod, and Mc-Carty failed to win the Nobel Prize has repeatedly been a subject of critical remark. In 1970, Wendell Stanley 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 fell 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 recombination, I thought it more important to acknowledge how genetics had been totally transformed by the Avery team's discoveries, and this acknowledgment was indeed embodied in my lecture (J. Lederberg, "A view of genetics," Les Prix Nobel en 1958, Stockholm, Imprimerie Royale P.A. Norstedt & Soner, 1959). Avery had consummated this research at the very end of his career, and died in 1955 before a full round of recognition could be fulfilled. The survivor of that team, Maclyn Mc-Carty, has written a vibrant memoir that is a model for expert and methodical tackling of very difficult technical problems (The Transforming Principle, New York, W.W. Norton & Co. Inc., 1985). It displays 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 20th-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. Genetics, especially as we explore the human genome, will be fraught with many more like opportunities and-- precisely because of their pervasive 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. In 1944, the Rockefeller Institute was perhaps the only site in the United States where that could have been possible. Joshua Lederberg is University Professor at Rockefeller University, New York.

Honoring Avery, MacLeod, And McCarty: The Team That Transformed Genetics

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