The Tangled Tree

by David Quammen

  There was also a middle view, suggesting that the hereditary material might combine both protein and nucleic acid—the protein providing an alphabet of variation, the DNA serving some sort of supportive function. That might explain the abundance of DNA in chromosomes. But no one really knew. Opinion had fluctuated for decades. The tools didn’t yet exist, the methods didn’t yet exist—or, more accurately, the imagination didn’t yet exist—to settle the question. Into this arena of uncertainty stepped Oswald Avery.

  Avery was another of the eccentric, heroic paragons of early research in molecular biology. His story begins in Halifax, Nova Scotia, a city to which, like Urbana, this whole tale circles back and back. He was born there in 1877, second son of a zealous Englishman who had converted from Anglicanism to become an evangelical Baptist preacher and emigrated to Canada when a “strange impression” of holy duty seized him. Ten years later, another strange impression carried the family to New York’s Lower East Side, where Reverend Avery became pastor of the Mariner’s Temple, a mission in the Bowery. Oswald and his older brother took to music, got hold of cornets, and as young teenagers, helped the family enterprise, by standing outside the temple on Sundays and playing their brass horns to attract congregants. It sounds like a scene from Guys and Dolls: two kids on a street in the Bowery, blaring out “Follow, follow, before you take another swallow.” The older brother got sick and died when Oswald was fifteen, Reverend Avery died too—a bad year for the family—but Oswald himself, the steady middle boy, went off to prep school (somehow) and Colgate University and eventually drew the youngest brother, Roy Avery, into bacteriology on his coattails.

  Photos of Oswald, beginning from age six, show an angelic boy with big eyes and then a man with a great domed forehead, presumably full of brains, going gradually bald, stretching intelligence almost beyond the limits of the skull. Below that, always, a small, straight mouth. At Colgate, he played cornet in the band. From there he went to the College of Physicians and Surgeons at Columbia University, back in New York City, where he did well, except in bacteriology and pathology. His friends called him “Babe” because he still looked like a megacephalic little kid. With his medical degree, he went to a laboratory in Brooklyn, where he helped with administration and worked on topics such as the bacteriology of yogurt, but also a bit on influenza and tuberculosis. Six years later, Avery joined the Rockefeller Institute to do research on pneumonia, which by then had killed his mother.

  The Rockefeller Institute Hospital, where Avery had his lab, was America’s leading center of research on pneumococcal pneumonia, which Avery studied for much of the following two decades. But after the Griffith paper in 1928, and with some tugging by his junior colleagues, his focus changed gradually from the purely medical aspects to something broader. In summer 1934 a young man named Colin MacLeod, like Avery a Canadian, arrived. MacLeod had read Griffith’s paper as a medical student and wanted to work on transformation. Avery was gone on sick leave at the time, for a thyroid disease, and by the time he returned, MacLeod had taught himself the methods and gotten started. Avery supported the effort. He was still recuperating and weighed barely a hundred pounds, but he and MacLeod worked long hours and weekends.

  Avery seems to have sensed, with quietly increasing confidence over the next handful of years, that transformation in pneumococcus was more than just a medical issue—that it had huge implications for biology in general. He and his lab members began to speak of “the transforming principle,” their name for the magical pabulum, and to suspect that it achieved its effect by transferring genetic information. If that suspicion proved correct, their transforming principle was the hereditary material—and not just of pneumococcus pneumonia, maybe, but of all life. In other words, as they tried to identify the transforming principle, they were looking for the physical reality of the gene.

  Medical applications, though, were still their primary imperative at the Rockefeller Institute. The work on transformation flagged a bit in the late 1930s, as MacLeod struggled with the experimental challenges and as the first appearance of sulfa drugs—early antibiotics—promised the possibility of curing pneumococcus infections without need of distinguishing one type from another. If there was no need for typing, transformation of types might be moot—medically moot, anyway. MacLeod, needing some practical publications to advance his academic career, diverted his attention for a while to the sulfas. In the meantime, no one else in the scientific world seemed to share Avery’s strong sense that transformation was a large and ripe scientific question. After the hiatus, in autumn 1940 he and MacLeod returned to it.

  To identify the transforming principle, they first had to isolate it, whatever it was, in quantities that allowed for chemical analysis. So besides killing pneumococcus cells with heat treatment, they would break open the cells and derive an extract from the cell puree, then try to determine which component of the extract—a protein? a nucleic acid? some other kind of molecule?—carried the transforming power. MacLeod did most of the hands-on experimental work. In contrast to Avery, so precise and methodical, he was “much more impulsive and impatient,” according to one colleague, and that may explain his effort to scale up their operation using a cream separator as a centrifuge.

  They cultured their pneumococcus in beef broth and then had to centrifuge the culture—spin it, separating broth from cells—to get concentrated masses of bacteria. This took time, and the ordinary lab centrifuges could spin only a liter each, yielding just smidgens of bacteria. Scaling up would give them more cell puree, therefore greater quantities of extract, making the chemistry and the biological testing a bit easier. So MacLeod somehow got hold of an industrial cream separator, with a high-speed cylinder and separate outflow taps, that could process gallons of culture in a continuous cycle. The only problem was its tendency, when spinning at full throttle, to spray “an invisible aerosol laden with bacteria” throughout the room. This was more than inconvenient. A fine mist of low-fat milk might have been okay, even a fine mist of yogurt bacteria, but not a fine mist of virulent pneumococcus. To fix that, MacLeod found a technician from the Institute’s machine shop who helped him design a housing for the separator, a sort of containment vessel, with a gasket-sealed door that bolted safely shut and could be opened with a tire iron. Before opening, the interior of the vessel would be sterilized with a blast of steam. Then they would lug-wrench it open, scoop out their masses of bacteria, and the work continued.

  Colin MacLeod left in 1941 for a job elsewhere, and another young medical doctor with training in biochemistry, Maclyn McCarty, became Avery’s next chief collaborator in the search for the transforming principle. By this time, Avery’s nickname among his associates was no longer “Babe,” far from it, but “the Professor.” For short they called him “Fess.” His team, at work on the transforming principle, had nearly convinced themselves that the mystery stuff wasn’t a protein. McCarty devised a series of experiments aimed at narrowing the possibilities further, and by the summer of 1942, he and Fess had evidence suggesting it was probably DNA. That seemed counterintuitive, given DNA’s reputation as boring, repetitive, a “stupid molecule,” incapable of carrying hereditary information. “We were not unaware that this idea would be greeted with skepticism,” McCarty wrote later. “We had already been told by more than one person”—among them, a crotchety scientist with a lab two floors above them at the Rockefeller Institute, who did DNA-related work himself—“that the transforming principle could not possibly be deoxyribonucleic acid because ‘nucleic acids are all alike.’ ” Despite such discouragement, they trusted their evidence and wrote a paper, carefully limited but unambiguous in its assertions. They said: DNA causes transformation of pneumococcus. They didn’t say: DNA is the substance of genes.

  They didn’t say that publicly, anyway. But in May 1943, around the time their work was reaching a crescendo, Oswald Avery wrote a letter to his brother, Roy, by that time a professor of microbiology at Vanderbilt University in Nashville. It was a long letter,
discussing family matters and his impending retirement, but then he shifted into a detailed description of the work he had been doing with MacLeod and McCarty, and their discovery that the transforming principle was DNA. “Who could have guessed it?” He mentioned plans for one more batch of pneumococcus, one more phase of purification and testing to confirm their results, and then they would write up the work. “If we are right,” he told Roy, “& of course that’s not yet proven, then it means that nucleic acids are not merely structurally important but functionally active substances in determining the biochemical activities and specific characteristics of cells.” It meant that DNA could carry, from one cell to another, changes that are predictable and hereditary. It meant that DNA could take hold somehow in the second cell, and be passed along through multiple generations, then recovered in quantities far greater than the amount originally introduced. “Sounds like a virus—may be a gene,” Avery wrote. But he was judicious as ever, disinclined to overreach. “One step at a time—& the first is, what is the chemical nature of the transforming principle? Someone else can work out the rest.”

  He wrote the paper with McCarty in the following months, and they added MacLeod as a coauthor. It went to the Journal of Experimental Medicine in November and was published in February 1944. Maclyn McCarty, who was thirty-two at the time, sent a reprint to his mother, with a few scribbled words expressing his pride: “This is it, at long last.” Mrs. McCarty was no bacteriologist, unlike Avery’s brother, and what she made of that comment, or of the paper itself, is unrecorded by history. Does a mother back in Indiana sit down and read “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types,” or does she just put it on her coffee table and say, “That’s my boy”? Maybe the latter. Anyway, the international community of biologists concluded, gradually, as more evidence accrued, that this was it—that Avery, MacLeod and McCarty had discovered the physical nature of the gene.

  It’s a good story, a true story. But the point of telling it here is that they had done that and also something more.

  51

  Transformation, in the sense of that word as used by Fred Griffith and Oswald Avery, is one of the three cardinal mechanisms of horizontal gene transfer, the most counterintuitive phenomenon discovered by biologists in the past century. Griffith had shown that some mysterious pabulum could transform a nonvirulent type of bacteria into a virulent type. Avery’s group had shown that Griffith’s pabulum is DNA, the physical carrier of genes. And the Avery team had demonstrated that in its naked form—floating loose in the environment after having been liberated from a busted bacterial cell—DNA is capable of getting into another bacterium and causing heritable change. Avery and his colleagues had no inkling at the time that this sort of sideways passage can carry DNA not just across minor boundaries, type to type among Pneumococcus pneumoniae, but also across huge gaps—from one bacterial species to another, from one genus to another, even from one domain of life to another. The transformations that result from such horizontal transfer can be far more consequential than merely changing a pneumonia bug from mild to virulent.

  The two other primary mechanisms of such sideways genetics came to light in the decade after Avery’s team published its paper. One involved a sort of “sex” between bacteria, and was dubbed conjugation. The other involved viruses carrying foreign DNA into the cells they infect, and that was called transduction. Both discoveries came from the ambit of a brilliant young scientist named Joshua Lederberg.

  Lederberg was a twenty-one-year-old junior researcher in a laboratory at Yale University, with no doctorate, on temporary leave from medical school at Columbia, when he detected the phenomenon he named conjugation. He had requested bacterial cultures and guidance from Edward L. Tatum, a microbiologist whose specialty was bacterial genetics, in order to chase a question that interested them both: Did bacteria practice some sort of genetic exchange? If not, where did they get the diversity and plasticity that allowed them to evolve within changing environments? If they did exchange genes, how? Genetic exchange usually suggests sex, at least in multicellular creatures. Bacteria were thought to be asexual, reproducing by simple fission, when one cell splits into two. Where was the opportunity to get new genes, rearrange combinations of genes, and adapt to new circumstances? Lederberg was fascinated by Oswald Avery’s discovery: the uptake of naked DNA from a dead bacterium into a live one. Did something like that occur between living bacteria too?

  Within a year, working on the bacterium Escherichia coli under Tatum’s mentoring, with an ingenious experimental design he had concocted himself, Lederberg made his own discovery: yes, living bacteria trade genes. He didn’t see it happen, but he proved it by inference. Take a strain of E. coli with, say, a useful gene A and a disadvantageous gene B; put that strain together in culture with a strain carrying a disadvantageous version of gene A (call it a) and a useful version of gene B (call that b); then, as the bacteria make the best of their circumstances—some flourishing, some not—you would get a new strain, Lederberg found, carrying both the useful genes, A and b. He didn’t need to do gene transplantation by some kind of fancy technique. The bacteria did it themselves. The genes were traded sideways into a more adaptive combination.

  “In order that various genes may have the opportunity to recombine,” he wrote in a short paper coauthored with Tatum, “a cell fusion would be required.” Recombine: meaning, rearrange or swap genes. Cell fusion: meaning, a temporary clinch. It might be brief—a quickie—but prolonged enough for genes to be transferred. Although this was a rare kind of event, “only one cell in a million” getting the recombined genome, Lederberg reproduced the effect on numerous tries. “These experiments imply the occurrence of a sexual process in the bacterium Escherichia coli.” He hadn’t yet reached his twenty-second birthday when the paper appeared in Nature. So he was well and early launched; but it would take him until age thirty-three to win his Nobel Prize.

  Lederberg grew up in New York City, son of a rabbi, oldest of three boys—a precocious kid who devoured books on science history and microbiology, received the textbook Introduction to Physiological Chemistry as a bar mitzvah present, and went off to Columbia College at age sixteen. After three years as an undergraduate, and despite some wartime work in clinical pathology at a US Navy hospital on Long Island, he was ready for med school. He started, and then came the interlude with Tatum in New Haven—a brief period but fruitful in that, on the strength of his discovery, Yale retroactively decided he was a graduate student and handed him, after modest additional work, a PhD. Before he could pack his bags to go back to Columbia and finish his MD, the University of Wisconsin offered him an assistant professorship in its Department of Genetics. Since boyhood, Lederberg had been pointed toward a career in medical research, solving urgent clinical mysteries in the tradition of Pasteur and Koch, but now he found himself a bacterial geneticist, paid to teach, to supervise graduate students, and to do basic research.

  Among his first grad students was Norton Zinder, another prodigious teenager from New York City who had steamed through Columbia in three years and then come to the Midwest. Zinder began his work in Madison following up on what Lederberg had done with Tatum, which was natural for a new doctoral student in the new lab of a new assistant professor. His assignment was to look for conjugation in a different bacterium, not Escherichia coli but Salmonella typhimurium, a bug in the same genus as those that cause typhoid fever and food poisoning. Zinder used penicillin to distinguish one mutant strain from another—a process that worked because penicillin, when introduced to a cell culture, killed only the mutant strains that were growing, not the mutant strains that were resting dormant. Separating mutant strains, as Lederberg had already shown, was a crucial step toward learning how those strains might exchange genes. But in his Salmonella cultures, Zinder found no sign of conjugation. Instead, he detected a different mode of genetic exchange. In this new mode, as far as Zinder could tell, only a small section of DNA was transferre
d, enough to account for a single genetic trait. And the donor bacterium never came into contact—not for a quickie, not even for a smooch—with the recipient bacterium. They remained separated like lovers on opposite balconies. Whatever carried the DNA was so small it could pass through a fine ceramic filter (that was the gap between balconies), too fine to allow passage of bacteria. Zinder recognized that the filter-passing agent was a virus—it had to be, since no other biological entity was so small—which evidently picked up some genetic material from one bacterium and carried it into another. This was so different from conjugation that Zinder and Lederberg, when they published the work, gave the process its own name: transduction.

  At almost the same time, Lederberg’s wife, Esther, who was also a bacterial geneticist, made another key discovery about sideways gene transfer. Through experiments on the old standby E. coli, she detected a system of “sexual compatibility” and incompatibility between various individual bacteria, which did or did not allow them to “mate”—that is, to exchange genetic material by conjugation. The evidence again was inferential. At first, Esther Lederberg could say only that compatibility was determined by a mysterious particle or factor of some sort, which she called F, for fertility. If one bacterium had F (a condition designated as F+) and the other did not (F-), then those two could do the deed, yes, with a passage of genes from one to the other. If both carried it (each of them F+), then too they could conjugate. If neither had F (a pair of F- virgins, clueless and pure), then no, they were incompatible for mating. It was a fresh insight on bacterial dynamics and the flow of genes through the invisible world. But there was more.